o . A...Jttg...&4..t¢u .. .7; . ...: ...v. . ... .. ..c I. AM. ...... .. 2...... 22...... .22... ..- ,- .. .. .. _ L ...... an kc. .... .... w. . ...}...Eéurf .....i...t.......ufiéhfinnkfi.........v.....r............... .2 . 0. ...-v0. 0.... ....a v.3....:0....0.0‘\.).:.06 1 .0. 00000 «0.0.: . 00":0.3.0.0 ...0. _ ..g .. . . A0.|0nfi9010 U ‘0 .... 400... 0 ......L .“0W..00. .F... . — $0.“... ....) Inns. 1. v» u.“ .32 x30. . .53....on J ... .. We. . ..0... .80.. 0.... .. .. . .0! . 0... 0 3.0.1. 303.? if? ....0 0... : . .. . I . .... . . :80 0.3. .13 00...... 00. .o I . . . L . . . . 0... . ...... ...... .v . . .. 0n|3'.\0..ut‘.r..0...0Juof Jay??? 00.0.2). .. 0 0... . 0.. . | .. mhISCIOJIJ. . 010.0. 0 0.00.. . 0.4. .930VHP3un-“3uu0n .00 .9 .- ......0000...1.0.00.9 ....02..0,..V0.! 0.w0.0.0.2...0~u0.l!..00 ‘hffizg .7”? goétbfiu ha! a:é u a. ..L... r 3.0.. ' Lye-“.0nnu.u“Tqu...N‘r .30...0'0H..U’." 0000 0.0”.0u5..0000000..§ 3.3. , .. .i£{000.0..nnv3...ll.!\0u0..‘ .14.!..".0, .. . Us... .070. .0 5.0010 .0?‘ .o 80.0“ (otuix {.80.— ..‘-r, A. ‘ B ’0‘ l. O . - .0. . . . .. . 00 0.. i0 0.. . . . . . . . . . . OI.‘A.0 . ”4:“.- unmu 00?»: 00“.“..Uuuf...‘ ounudJ-nhnqhuuuw’fi.“suff 0-0 0 . . .3300- ‘00!“4”uu.0.0l00.1.dsdn0uruu+fi+'d,.‘\rdv.a.'ut .0.‘ 0- 00‘ a} ‘ .‘kln ”0““ . 0. .l 00-. 6.. 500,003.3160 IX. 39:00.12... ... 0.90. . . I91H'? ".0300- 00 '20 A Y. ... ..!£s.)._. . .. .. u I .... .0...0h'..0. 0" 0. .fi‘ 0 0 . 0.00.... 1000.00. 9‘, .m.‘.;..0£00“0. 000' 0." .0 .0..Q' 0000...‘ J.~0'\o 0‘ . 19.000.030.22... .0 . . 00 0 . 3... 3....0...~00 0.... .100. ...... :90» .v 000...}: ...Mu .. V~31 .10.“... . (.... 0 1.0304“. n t . . . ...8 0.. 3.00.7 “a 0... ...! Id. 3 . ...... .0 ...-a 30.03.... grg'nu Juu4o0c'e. r00!- 0 t 3.00.. 000'. 0.90.00?! Coo 3 00,000.0000” 00 I“. 0.... ......040....... 0.7%.. ‘ ... .0? . 021.40.“!I0. 0:. ’0. 9 r. I. . 6‘. 0 ..0.00.... A 0.0..05 .0838...- 32..J Jlknufltolgoc" 1’ 3.2.0.3 0.1.0.03... . s:..)..i.<~.0..0 00. ....0..ll 100. L903”: . .. 0 0 w. “urn... .00"..0.‘0 0.200011 0. '00.... 10.30....90. 0 0A 0"..00." 00.0.‘0.'..00.0 .. V . .00 . 3.00.0 ...-30.0.2. .....9...’ 000:. 6%,. 000. 0 . . (0.0!. 0.0.0 0.0.0. . c. 0 .. m1. . 0 . 10.5.70... .02! (..00 "3133040qu . ”Ur—0.0.0103r0fdua00ofll... 00.. .J. 0.0 0.. ...-9.... . 1‘27». €3.32. .0 .32. arc-.025... I..0.......8.... w.- .020“? 2.00.0 ..000'300 * .0. .5030? 0.00.0 ..0 . . l ’0.‘0..”000. .00. .... 0.0 100.0. 0.00.05.00.00? .00. 0 1C30-10.’ . 0.10,. .0. . .000...0.. ...”00 .6. . ..0..0.o €0.90... .2 1...... 0.0 01‘.- 0. . _ . :20 0.0.0.2.-..390... .0... ... 0. 00.0... 2.0 0. .0.— . 10.... o-.:.-".-... . ..., .‘0...’. . ..0 0000.0 .000. 0.0 . . Jun.” 3.... Junta”. a«..0....004. 0.3.. 07.9.1... . .90. «3.3.00.9... . 3900...... 10.20.03: 3.2.0.0... . -12.... 90., 000‘.3d.00300.10 .0....0.. .900 70.00.020.99?! .0000 . ..0 3.030;... 0013.000}??? 0.. .0... 0-23.. 0.0.!3000..00.0.0.l.. «.0 ...“.III-coi 30... 0.00 00030-000. .. 0.000.302.000V001030301. .9 .0 .009; .I 0.3.0.50... 0.0.8. 000. 0.0.0 ..0. 030.01.00.20: ...0. . .000. 00000.0 $0000.03... ....0... . . ”our...o.‘0000001.03.n .33.! ~o0.0.9.'.00.0 :0 ”0.0.00. . . . . - 0 . V . 0000 .. 0...! 0 . . 0000.. o. 3 . .000... .0..000.0.0.00.. 0 0. 0...!- . 0 . 0.0.. 01...... 0. .... 000 0. 9.030.930.2000I010 . . . . ...: .0 .0200... 0.. .000 79:!- 000. ..'0.0.0. 0.0.. . . . . . . . .. .00... .... . 5.00.22}... 1.02 : . .. . . “0411:: 0‘ '00 0 .... .030. 0.0000000 00 . ... . 0. 00"... .0! ‘0 .‘00 . 000.. ya‘l: .... 0 an. a... qW... \ ‘00}..0004... 00.0.30”. 0.. 190.... .0 3.800.000! ...!!15...t0.0 .30 0. .0000... .00... f0....07 .00... 0.000.030... o 0 3» I: t, I '00.! 0 .... -‘so. . .0: 02000“. ...)...0 .03... 00.0.00... 00000..”0 .00.. 0.20.0 2290.20.90.- fl. . . . . . r . .. .v .1 0.0.0....0...’ 0' 4. 0.5.0 0‘0‘0‘.) O Yv.‘ 00 ‘0‘. 9“ ‘ 0 . ._ . ’20... 0.. .0 0.10.1.0. . .0! .00 .4 .. ...0' .0023" .‘.§Uonh0‘3i3.0.b...0000000 :000300:‘ 00000..o£“.00 000.0 In»: 3.21.... 0.0.0.0 0_~.0.00..0.O0.0.....Y0. . r :72. 2.0‘!...’. ........=0. 2...... 0.1.. a .32...- 2.00:... . ..r... . .. SI. 0.... ~ 0. ..0 3» 3.30.505... 30.0.0. .002. .- .... .00... .3 .00.. . r 12:... {30100.00 .9tfigo..0..tf\=. .1 0.0 . .0000 (.00.. .03: 20......303003351300‘0001 .....003.0.00. ..000. in” .0. .603 .... .00.3.«a.0.l:..uur ‘ .. . I .0. 100.00. 70.0 . ...—8.0.103 ’00 00¢. .0 .0 .."00 0157.1... ..0.....0fifl_‘.§0130n0‘.3 . SJ! . . . 0 v. 41:... . .2. «0.00.0 .3‘0’3. .12.}..3... 0 ..rfiuo...” 0.... .0 .0 . o D 0 1:010... . 1:21... 0.. 0.. . . ... .53... r :0... . 0.2....IJ......0:. I . .. . . ......I . u . .l. I” 00.0300: .‘0 . 0.0000 .00 0. 0 000 0.0.. 0.00%00. ...r000..0. 0 . ll. .. ....l....!.’..’..0 0.3.8.0...01..0.‘0.t 1%.; D. 0. 2 0.. . U 00. o. 0.1 20- ...0...1..~.0...0.1.:6...... .00. .J00).00 I. ...“! ...-um. 0.00.10180-2..v....05.0.00...000 00. .0.- .900... r: . . . .1... 2.1.0.310... .0. J 0......08 .0 ...00‘. 0. 0.0.2.1.... . . . . a II: €000.33! .0030“ 0.0 0' 0 . J12... .....000.l.2. . .3..Jl!3’.~\.l. 0 . '...0..000 5.1.0.10024138 40..0.0.0o.0bv..0..00 .0... 0‘0!- 0.'..1‘.00..0.I .0. :0: .0. 0.0.... ......Y. ...... 3... ...?-.0...$..0. .. .10....0. 0...... 0.0...." .... .0 0. 01 . 000.. .00. 0. 1 0.01.. ... 20.0.. ..0 v |'0.0.0 . ... 0.0..0... ..0...’ . .0... .0 . .. ..E'Jifu v.0“..x.’ . . .. . . . 2...... ...I'..1....0 . ..0 . . : . ...... 50.31.20; 03.90. 00? 0.92.3.0. ... . . on... v. 0.1.... . .330... 70.04. ... 0.23.0.0... . 20.30.10 3.000.. A... .0000 ...-.00 0.00.... . . 00 ...0. 3.0.0:. l0... 1. .0 . 0‘... . . . a . . . .0... i. 0 0.d00 .. 00.00-04.0"00‘00. 09,0 00.1 00.0.500‘ 0 0.00 aw?” 3L9-"Wq’bflnnu.ucn 0000...“. ‘0..0.0.'00‘0.’ ... ...-0‘ 1.0 0.000 0 00. .000..-u.00.0.o .... .... . 0.....‘..3..£JI..1!>... .50....320.-. 2 .0. . ....00830...0... .. . . .... 3." ... .... ..0‘500'0....r.ao.0.f.o....(0l‘....: . ...-0.410.. 3.0.... ......00 . ..-. . . I 00 . 00.30.310.00: ...0 3.000.“. . 0|..gf...‘ _ 0 I?! .0... ’00.. 10.300.003.000 .0 . 00:02 ...... 0.0.0 00.0. Y..20.0\0.0.0n0.0.0. u a. . i ...0.09000. 0.0... .0.F.)'..9.0fi.'0 00“..‘l . 0.0 O! .. 0 04‘s.. 0. clu‘u") ..0-‘ ,A. 40.00.... ...-030 . '0. 0.. 00.0 0 0 . 0. . .. ....Q!’0v“..:.0'00'204' ' I. .0 .1301... 39000,... .0.000¢.0 0.0.0 00 £0.20]. 0.50.00 .100. C. .9 350.01.... . .00... ..I.... . . . . _ . 9.0.3.50"! .0. .10.. 0.3.0. 0- . 00‘0100 .'!p0.9.' . .. 0.3!: 5...... 00. 7'12?! .. 0.. . 0. 00... ..I. . .. . . . . ... .. ._. .00. ”'0. .... .. '40. II. 0.0. .0‘0‘!"‘..0‘Y v -. . . ...... ... 0.. 2 . . .o . .. . .. . .... ... ... . . . . Iris... . 0.. 8.0.0.1055... .... 09.0 00020.01... 0 .. ... ... 3.. .9... . . . . . .. .30.9..p..;"....0.0..:.:..£ (10.0.0.0...vw 0.90.0.0: 000 ... 0. .. . . . . . .. 00.0310 0 . 0.. ..s 010:0...lfv-13ttflu... Q! 0 .. 0.0.0 00.... 00. . l . 0 0 0.... 720.... ‘0. ..V... .... 0 . ...! .... .0 .. .00 “9.0.2."...4100"... 3.00.0 ItQ ......ta.I|..-;.0930.0.0 «5.0- .0 O 0'...‘“I.Qnu ”.90... . PM . 0.0.. 0.0. . 0.. .. ..c s. . 4:. ...).-.0 ... . .... .... 2060 ...-0.- 000 . . ...0.n.0..0..0000‘0..0. .... 0. ... 0.. . ...... 900..... .9. . 00 ...-....-. . . . . . . . . . ..0. 20... . . . .. . . . . . . . . . .. . 0.. 0.100.)! 100.0... 6.0210} 5."... . . 30. 0010.0.- 9702 01.0029 30.00.923.01. [90.80. .. 0.0.. ...0 .0... 0.0.000. ..0»' 00 0.... . ‘.00.0.'0.. . 0.5.00." Q00 '2 10.00'0110 Olllfi. I. 0.. . 0 0 .I0 3 00.03.20. 0 0.0 0.. 00.000 . 0.000.... 00.0.. 7.3.; $0.000 O. ’20....1 .. .. . 0.. 1.0. .. I00. .0: .....- 0.7.3.0.. .. . . . Q . . |!0.'0 0.0 0.0. Q‘QO’O3000’0 ... 0.0.0.000 0“.‘000r-V“00.0)0II§"I . _ . . . . . .. L .. . .0. .0. D .031..~c1........c. S... ...: .l....l.0oo..uu . 0 . .....00.u0..... “...?!(u? 0 r0. . . .0.. 0’. .. 0 n. . .... 3.1.02 . .... 10 ..0 ...-0.0050000. 30.00.. 0 ... ... o ...." . ‘00» I'l'..|2,|l“0.2700.!..0 ..l'.%.v§0.h .0000 3. 1000 . .00... 0.000 2.0.0.0380... .83030. . T0040 0’...) 0.0.1.0000“. 3002310..- .0. .. 2.30.3.0 0.0000...“ . .00. . 0.. I.!..00.0:c!.'..0..0.0 9. I fits-:10 Dill. 0.00 0. . (0...; 0.0.3.. 00.0.. 0.900035%... 00.. L .. .010 ...».0- w . . 0.0.“. 3 ”’90 10.0.9.3.cfi. ..2'..'. ..0! ‘0. .U. . .0.. . 0.. .0 v. 0 . ..X. ..0.. 0. .0- I .l”. . :40...0.I-.’. 0. ......V9’0 0000) u .. 10.. .1 ...... .00. ..00.10.I0.0.200.-....00.0.'0.9.\9 .“._.0.‘0.2:0.§0 . ’0 i .0..00.§.... .... 000.00.. '20.; 1' (00.0.0.3... 900.0. . 010 2.0.0.910 ._ 0 00a... 0.... .‘t’. '20... . . 0 0.0. .0000 .0‘003....0.0....:0J . .0.0'0...000 0 30.0.0321“: h. . .00 .000. . . . .00... 0 0. .0 f 0 0 0'0.- ... I 0.0. .000...’..: 0.0 000 -303... 0.0 0.0.0. .0 ..70.’ . ..05 .900. :0 . . . .. V . .... {0 1.20.0 . 320.010 0.20.0 I . 00. 0.40. .80... .loolz. . .. 4 0.0.000.0.0.¢.Q. ‘0. xtlf a... ... (... v ..0-. I-0...,. . V0.3... .. . .0...0 ..0.30.00.0.~.;1\._..0r. 0.0 .-0 0.0.0 r... 0 P0 . ...... ’00..0 .0. 00......00 0... . 0‘ 0— 0.0- 0.00000 0 0.. .. 10.0.. 0.00.. no ... 00.0. . .. . .a . 3:7... ...... £73.-.. 0 . . ..0. r. . .0 0.0 ..0 0.. 0 w. ..v .... .I.00 r. . . . a .0 0 00' 00 n. 0. .0.. . toy-.... 0.. 00.0". Y .00 0. . ... 5000000. .0... . . . 0 ...0 . 0.0 .009 00 0. .. 40:0 0 .0. . ..lll.0..0 .0 . . ... . . . II. .. .....X’... 0 .00... 10.90.. .-.... .. 0.. . . .-.... . .0 . .... .. . . 00.0 . 01 o ......0. . ...0 ... 0. . p . 'l‘o 0.0. ..0.....’0 V . .... ...- 00 .0 _ ..l . Dwin‘I 0.004 0 .0700... 0... ..0 . . . . 0 .0 .....0 I 0.0.. ... . . . 000.\.. 0 0...00.‘..0 . 0 0 0.0.0.. . . . .......0...¢.... . .0.‘ 0 0 0.0.0.000 0.0 0 ..0. . .... . 0. ..0.0....l.0 .. . .. 9.. 0 . 0 .003 0. ....r.......0....0.0....000 1.51.0.0" . . . .. a... ..00..?0 .... .... IO. 0 .4... s .00..0O.~.. .... '00....0 0... 0 . 000....12 . . ...00.J9.0.0.. .....V....0 0 . ...0.0.0..0 .000... 0...? .....000.‘00h .. ...- . 0. ..~ I. 0 .. . . _. 9 I. o... ...-1.. ...01..0.000 v. 00.0..0: 0.0.00 .0.\00.0..’00.'0.0.00§o ‘4 . -;‘v,f0‘.\|0.284 D.) 0 ..Oc' 0"}0..0 I _ 0- '0:- . .. ... {.90 0.. . . T... v-.. . . . 0 0 0..‘.Vl§0. . ...-.000v. .0006..00.. . ..0...Ov. 00. .. . 0. c .000 .0000 . ... 0 o _ . 004 _. . ‘0 . 0. . ......3. .... .... . 00.. . Q0. . . n . . 0 0......00...0 0..0000 .. . .. . Ol.0‘0.0!.‘.k ... 1.0.0.0.! . Il’..cb.'..0..' .. . . .. .. . 0.0 w. ... 0.. 0.....3.0.v... 00 0 . . . .00....0. 905101.000- ‘...v ... 0 _ 0 .. . .. .00 .- .0-..0 0 0 00..... '0 .. I .. .... ... O . . 0 . . 0 0 0.. 0.0 . .. .. . .. 0 .. . ........0. ......0..0 . . 0.... .... . . . . . b.r. ..0 .. 0 . .. . 0 .00 0.. a 0.0.0 00. . 0.. . . . . . V.-0‘0’0..§.210s. . . . . . .. a». . . . .. . I . u . . . . ....0 0...... ...u. ...... a. .. . ..o . . . . .I . . . 0 0 7.90.0.0 00: 0 u. .. . . 0 .. . .. .... 0 ... . . .‘0.I.0 . 620.1. .. 00-... 0. ...: .- ... 00.. A I. . . 0.0 ’0'000000.:.'00_ p -.. .210. l .. . .. a ..0......... .. . ,...0.0..0.0.00. 20...... ‘0‘? ...0.0.00.I.01100‘00...00.. ._ . .. . .. . . .. . v.. I . . . ... 0.0.00.3.9 ..0.000.!0p..0..00. ..0..?0.IO00.Q.0.O.0P0...I 0-0.0.0..l‘0 . . ... . ... a . .... 0.0...0. ... .0... 0.70. . . ltoll.‘0.0. 00000 ’00... 0 . ...0.00...0... 0 .. .... .0..... .. .. .......0. .0. v 00. . .0 . . . . n . .0 . .0.. ... .... . .. 0. 0. u .v. . . 0 . . . 00. . 0 I 40. u. . . . . ...0..00 . _ ... . 0 . . . .0 .0... . . .0 . . . ......Q .. .0; ...v. 3... ..0 .00... 0.000 . . ..0.. .0 00....0l00... .20. ... .. o o .0 00 a.‘ 0.. . . . 0 . ......0... 0 . 000 .0. . . .. . .. . . 0.0 . .. . . ...lu 0.......n.n.. _. .93.... .-7....a 0... 0.. 0.0. ...- ...... 00.0'.0 20...-..0 .. . 0......32 . .. . ..... .... . .00. t .. .. 0.00.70 0 O . \0. _. I 000.. v. 0. .00 0 0'.0.. . a -. . .... ...... .0. 0. ... ..0.....0‘0..0 ..0.0 ‘.. .0 .. . .... ...0 .0..00-0 ..~.c.. .0. . . 0 0. .0.0.. 0... 0.0.0..000...000.‘ -0. . 00.0.0 0 ...000000. . ... ..0 03040.. . .00..00 00- 00.0.0.0. 10.00aa. .0...0..0.0..0.~ 0 v. .. ...000000000‘. ... 0.. ..0.0 0.. 10.64... 0.01.0004.- .. .. . . r00 1.0.0 . 0-0.0.0 0 0y I 0 00 0 0‘59} I 0 ..A‘OO‘sztulQ. 0 J 0......00.’ 00.001 '0009000-0094J’.0..0'0|0 .. 00'0000 0.0 I. . 0.0.0.0000000070000. . .. ..00tn0. 0..100....0.0. .. ...0... 0.... a I. . c. I... . .n. 0 .0.0..0. .... .... ...f..0_... . .0 . 0.. .. u . .. ..00 .... ...-......00000 .0 .. _. - ...; .. . .. . . q . . . 00 . , . . . . . . . ... . . ... ‘ . . _ . .0 .100. .0 o 0 ... .. |.. . .0 . ........ |-.000‘0“..‘.. . . I b. . 0.. v .0. . .I.0 0 ' ... .0 a . 0 .00000 .vn 0-0.00.00 0...!0..0..I.. .. 000000000..- . . .. .... .0. u . c .0 ..0 0.0. .0. . . .00....0.....| .0 .. 0-00000000‘0 o. ... . 0 . . . .. . . ... ... . .... ..0....cuo. 0. 0... 0 0‘0- ..00. .. . 0 . ...... . . . .0. A. 0. ... ..0. 0 . 0.0 I. 0000. o. . .0 V0.01. 0000.00... .- . .0 0 . . «I o. .. 0.0 . . . . .. I u. . ...0 n..... 0. .000...0..0"...0.l . . . .. .. . . . . c ..0. 0.. A0 .0 0. 0 0 ... .0000 00.00.00'00090 w ... .. N. c . . ‘ . I. ..0. . .0. .0 ..0 .0 . 0 . . 0 r o 0 0200!... ... 0.. ... . ...Z o. .\.. .. n .. . . n . ... ... .. s. . . . . u. . . 0.. .. ..‘0.. .0 .00‘0Q0... 0 .o .. - ..0 . .. .0... .. 0 . 0 . u. ...... .0 . .. . - . . 0 . . . 0 0. . . . . . 0 ... 0 0 . . I . 0| 0 .. . .\ 0. .. q . .' ... .....3. . . .0. O 0. .00 . a v. 0... ..00 o 0 0 .. 0 | . .0... . . 0 0 00.0... .09.00.|.0I0.v10.0| . v . . _ .. .0 .... . .... _ u 4 o .... ..0 .. . .o . ... ...0 0 .. . 000 400 ...o. 01.00--- . .0. I .. .. .. . .. .. . . . o. c . . . . .. ..v. ....0 0 0.. ..0 ..0.0Q.._ .. ...0000..0.00.... . v o . 0 ..2. 0.. . I .00 0: . .0. ..0 . 0. . 1. ...00000..00... 000.0 0.. ..0. . I . . "0.. .100 .0 . I . . 0 . . ..0 .0 000.0 I .. .0000 .400..\0Q 0.00... .0I000s0 0!’.0|0|000lr00-.‘. o‘|.. .... . o. o 0 . . -0. . 1 .... .00 ......0 .00.0. . .0. . .. -..0 0.0.0 . 0. 04..0.3000ol' 1). 1000:00.tu.0...00 . .. 0 I Q 00.. 0 0 ... .. 0 . ... 0.. .s 0 a 000 . .0o.000. 000.....00-'0~0000 .u0\.00 1000 o. '00 00! 000.:- A. .-.. -. ... .0 .v 0... . 4 0 0 . .. 0 0 .0 ..0 0... {0.9.0000 Q0 0000 0-0...‘00.10.100.009.0 .. . 0|... .00. . .. 0. u . ... I00 0 . 0 .0 . O. 00 ..00.. 00.0000000'0 40'00... 0.- 0 0 00 0 00.0-000- ...... .. . . . . .. . .. 0 .o.. 0. . .. 1.0 0. .00 .. ..0... 00-0 0 .00.0.00“A.Ov 00.c00 .I.00|0 ..00 It. .. v. 0 0.. . Y . . . ..0 .. .0. .0 . 0.. .... .00 ..s.........0 0|... 0. 0.0.. ......l. A. 09' -000 . In 00.!0 . . .. . . .090 . ..0. ..v a. 0.. 0 o. 0 I. 0 ... 0 I..00.000'00t..?tcv0 0...0.00|‘o 0. 90 010000 . v u . . A . 0.0. 0 ..01 .... .0Iu ...00. 0.00.00. 0.13.00.00.00 00 o I 9 00| 0. 0 . 0 . 0.0 . 0 t 0. 00000 . ... ..I 0....000‘00l0.'000 ‘. .l00. I u. -0. . '00 I 0 0 0 . c . u l 0 .A 0 ..0. .a '0. 000. .... 0 0. 0' I00 .0 0 .0... Itolt.00.9r000000. . . .9 o . 0 o o . o. o .7 0. . . . . 0. .0. I0000I0.0 . ..00.0I0.. .0000 000. n .0 .0 0 . l . ._ 00. 00. . I .......o..o..0009v.0.\0.00.. 0 00 . 000.0,...00 0 . _. 0 0x 0. . .... 0 l... ~ o..."- 00» .. -u . . t .. .0 .. . o a. A . I O 0 .. ... I. ... 10"2..‘I0000 ‘0.00v.0 0- 00 20.000.0'000."00Q0 00 i0 .ldongLO'v 9.00.0. . u... ’4 0. 20.. I . 3.3.0.002 “a" m; . unfwyermflKLTWNR .... ...Knav .-. J . .. «6‘ I... 3‘. I 'l ' I'III .— .. J 540000.“. p‘vnflflmgwfl.w0’0 abut lax-l“... ol ..0..?' s 3r»; . ..-“...fimaw .50..” mf¢mfiémywfi$uufiq I- I l 0 ‘3 ..000 .000 3. ...unucomhAWUofla. .0 0 O ...-.f. . 90 IO . . 0‘00. 0 . 0 c o . . 0 o . ..00 O... .0 A00... 0 s. I .1 ...!»Q.. C 140. ..fiflo . a. 1.. . . I. v . I... 0! . 0 ... 0 .. v 63% . . .... r) .0 nit . H . . . . . . . . _ . ... a up“... n... 9...... .Huvfipg.22 ...?aa. ......hmwzfigba .. .. . ......«azz. .. . . . 3...... v 1...... 8.10.0.0. .0. . 03.9..00..0.:00.0030.000.0 qxjdf kl..\0.. 070.0 . . 04 . 1.. 0 0.00.0... . 30.. . I 0.," .LO»? ( . I 0 0 (2.: 1‘ IO 1— LIBRARY lMichigan State University This is to certify that the dissertation entitled LAYER-BY-LAYER ASSEMBLED MULTILAYERS AND POLYMERIC NANOPARTICLES FOR DRUG DELIVERY IN TISSUE ENGINEERING APPLICATIONS presented by SUMIT MEHROTRA has been accepted towards fulfillment of the requirements for the PhD. degree in Materials Science and Egclineering L". /\, ‘L~;%ur\; (44'1“ Major Professor’s Signature f/f’ifl/m Date MSU is an Affirmative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProj/Acc&Pres/ClRC/DateDue.indd LAYER-BY-LAYER ASSEMBLED MULTILAYERS AND POLYMERIC NANOPARTICLES FOR DRUG DELIVERY IN TISSUE ENGINEERING APPLICATIONS By SUMIT MEHROTRA A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Materials Science and Engineering 2010 ABSTRACT LAYER-BY-LAYER ASSEMBLED MULTILAYERs AND POLYMERIC NANOPARTICLES FOR DRUG DELIVERY IN TISSUE ENGINEERING APPLICATIONS By SUMIT MEHROTRA Tissues and organs in vivo are structured in three dimensional (3-D) ordered assemblies to maintain their metabolic functions. In the case of an injury, certain tissues lack the regenerative abilities without an external supportive environment. In order to regenerate the natural in vivo environment post-injury, there is a need to design three-dimensional (3-D) tissue engineered constructs of appropriate dimensions along with strategies that can deliver growth factors or drugs at a controlled rate from such constructs. This thesis focuses on the applications of hydrogen bonded (H-bonded) nanoscale layer-by-layer (LbL) assembled multilayers for time controlled drug delivery, fabrication of polymeric nanoparticles as drug delivery carriers, and engineering 3-D cellular constructs. Axonal regeneration in the central nervous system after spinal cord injury is often disorganized and random. To support linear axonal growth into spinal cord lesion sites, certain growth factors, such as brain-derived neurotrophic factor (BDNF), needs to be delivered at a controlled rate from an array of uniaxial channels patterned in a scaffold. In this study, we demonstrate for the first time that H-bonded LbL assembled degradable thin films prepared over agarose hydrogel, whereby the protein was loaded separately from the agarose fabrication, provided sustained release of protein under physiological conditions for more than four weeks. Further, patterned agarose scaffolds implanted at the site of a spinal cord injury forms a reactive cell layer of leptomeningeal fibroblasts in and around the scaffold. This limits the ability of axons to reinnervate the spinal cord. To address this challenge, we demonstrate the time controlled release of an anti-mitotic agent from agarose hydrogel to control the growth of the reactive cell layer of fibroblasts. Challenges in tissue engineering can also be addressed using gene therapy approaches. Certain growth factors in the body are known to inhibit axonal growth and nerve repair. Therefore, another possible method to promote axonal grth is to silence the genes to inhibit the production of such growth factors. Small interfering RNA (siRNA) is a powerful therapeutic tool which knocks-down the gene fimction. Gene therapy approaches to knock-down a gene in mammalian cells, requires optimal selection of a transfection carrier for the siRNA. In this study, 25 kDa linear polyethylenimine (LPEI) was shown as a promising transfection carrier for siRNA delivery in-vitro. LPEI-siRNA complex nanoparticles were Optimized for efficient siRNA delivery. Further, effort was made to fabricate LPEI particles of novel shapes, as particle shapes potentially have an impact on gene delivery efficiency. Finally, LbL assembled polyelectrolyte multilayers (PEMS) were engineered to tune surface properties to modulate the cell adhesion on a surface, to stamp and fabricate self- standing thin PEMs to create 3-D cellular constructs. COPYRIGHT BY SUMIT MEHROTRA 2010 DEDICATED TO MY FAMILY ACKNOWLEDGMENTS I would like to express my sincere gratitude to my advisors, Dr. Christina Chan and Dr. Ilsoon Lee, who always supported and guided me throughout my academic career at MSU. Their collaboration provided me wonderful opportunities to expand my work in the multidisciplinary fields and offered a unique position which broadened the future opportunities for me. I would also like to thank my committee members, Dr. Andre Lee and Dr. Gary Blanchard, for agreeing to reside on my committee and for their guidance and comments during the course of my PhD. I am also thankful to Dr. Jeff Sakamoto for providing a challenging project to work-on and his continuous motivation throughout the project. I would like to express my most sincere and deepest gratitude to my advisor Dr. Christina Chan, who consistently guided and mentored me during my stay at MSU. Dr. Chan was always there to look at my work and appreciate it, of-course with many criticisms, which provided me the motivation to continue work firrther even with more energy. Her insights were extremely important for me as it gradually improved my thinking and working abilities. To name a few, my writing skills were extremely poor (which are still poor, as you can judge while reading this acknowledgement), however, Dr. Chan’s critical review of my manuscripts helped me a lot in improving my writing skills. Her critical analysis of my presentations helped me a lot in making my speeches more professional. I always felt comfortable in discussing the work in detail with her. Besides our work, she has always vi been very helpful in other aspects too. It was a very much learning experience and pleasure working with her. I am really thankful to Dr. Lee as he always supported me, although I could not meet his expectations for providing the enough number of manuscripts. I enjoyed working with Dr. Lee. He always provided me the freedom to do work at my own pace, and I always felt very relaxed working with him. Because of the collaborative project, it could have been stressful for me to work with two advisors; however Dr. Lee provided me enough liberty to work in whatever style I wished to do. This made it comfortable for me to work in numerous collaborative projects with other professors also. I am also thankful to the past and present lab members of Cellular and Biomolecular lab, and Nanobiotechnology lab. I would like to thank Sri, Hemant, Linxia, Xuerui, Shireesh, Joe, Shengnan, Sachin Patil, Yifei, Amanda, Hyun Ju, Hirosha, and Li from Cellular and Biomolecular lab; and Devesh, Sachin Jadhav aka Jaddu, Sri, Neeraj, Troy, and Shaowen from Nanobiotechnology lab for their kind support to me in lab. I would like to extend my special thanks to my friends who were always with me during my stay at MSU: Devesh, Bandy, Amit, Leena, Deep C, Jaddu, Bhushan, Shishir, Tithi, Sutty, Tanmay. On top of all, I would like to thank my family — my parents, my brother (who has always been a continuous source of energy giving motivation to me), and my wife Deepika. Without their constant motivation and wishes, I would have not been able to complete my PhD. vii TABLE OF CONTENTS LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xxv CHAPTER 1 INTRODUCTION 1 1.1 OVERVIEW AND SIGNIFICANCE OF PROBLEM ............................................. 1 1.1.1 Tissue Engineering and Drug Delivery .............................................................. 1 1.1.2 Nucleic Acid based Drug Delivery - Gene Therapy ......................................... 5 1.2 BACKGROUND: METHODOLOGIES FOR DRUG DELIVERY ........................ 6 1.2.1 Layer-by-Layer (LbL) Assembled Multilayers as Drug Delivery Carriers ....... 6 1.2.2 Selection of a Transfection Carrier and Polymeric Nanoparticles for Drug Delivery in Gene Therapy ........................................................................................... 8 1.2.3 Microcontact Printing (uCP) for Patterned Drug Delivery ................................ 9 1.3 THESIS OUTLINE ................................................................................................. 11 CHAPTER 2 TIME CONTROLLED PROTEIN RELEASE FROM LAYER-BY- LAYER ASSEMBLED MULTILAYER FUNCTIONALIZED AGAROSE HYDROGELS 13 2.1 INTRODUCTION .................................................................................................. 13 2.2 MATERIALS AND METHODS ............................................................................ 17 2.2.1 Materials .......................................................................................................... 17 2.2.2 LbL Formation on Agarose .............................................................................. 18 2.2.3 Characterizations .............................................................................................. 20 2.3 RESULTS AND DISCUSSION ............................................................................. 22 2.3.1 Multilayer Growth on Agarose Substrate ........................................................ 24 2.3.2 Sustained Protein Release Profiles from LbL Coated Agarose Scaffolds ....... 33 2.3.2.] Effect of the Agarose Concentration on the Protein Release Profiles ...... 37 2.3.2.2 Effect of the Starting Polyelectrolyte on the Protein Release Profiles ..... 39 2.3.2.3 Effect of the Stacking Layer Configuration and Assembly Components on the Protein Release Profiles .................................................................................. 42 2.3.2.4 Lysozyme Release from LbL Coated Agarose in Cell Culture Medium.. 45 2.3.2.5 Lysozyme Release under Static Conditions, and Direct impregnation (Soaking) of Lysozyme in Agarose Hydrogels ..................................................... 46 2.3.2.6 Range of Protein Loading and Release from LbL coated Agarose Hydrogels .............................................................................................................. 51 2.3.2.7 LbL Formation at Higher pH and Lysozyme Release .............................. 52 2.3.3 Low Molecular Weight H-bonded Multilayer Disintegration on a Planar Substrate .................................................................................................................... 55 2.3.4 Cytophobicity of H-bonded FAA/PEG Multilayers ........................................ 57 2.4 CONCLUSIONS ..................................................................................................... 62 viii CHAPTER 3 TIME CONTROLLED RELEASE OF ARABINO- FURANOSYLCYTOSINE (Ara-C) FROM AGAROSE HYDROGELS USING LAYER-BY-LAYER ASSEMBLY 64 3.1 INTRODUCTION .................................................................................................. 64 3.2 MATERIALS AND METHODS ............................................................................ 68 3.2.1 Materials .......................................................................................................... 68 3.2.2 LbL Formation over Agarose ........................................................................... 69 3.2.3 Cell Culture and Imaging ................................................................................. 70 3.2.3.1 Cell Culture and Ara-C Release from Agarose Hydrogels ........................... 70 3.2.4 Ara-C Release Measurements .......................................................................... 71 3.3 RESULTS AND DISCUSSION ............................................................................. 72 3.3.1 AraC Incorporation into Agarose during LbL Multilayer Assembly .............. 74 3.3.2 Qualitative Evaluation of the Effect of Agarose Released Ara-C on Fibroblast Growth ......................................................................................... 77 3.3.2.1 Dose Response of Pure Ara-C on Fibroblast ............................................ 81 3.3.3 Quantitative Evaluation of Free Ara-C ............................................................ 84 3.3.3.1 Quantitative Evaluation of Free Ara-C in a Mixture of PAA-PEG-Ara-C84 3.3.3.2 Quantitative Evaluation of Agarose Released Ara-C ............................... 85 3.4 CONCLUSIONS ..................................................................................................... 90 CHAPTER 4 MULTILAYER MEDIATED FORWARD AND PATTERNED SIRNA TRAN SFECTION USING LINEAR-PEI AT EXTENDED N/P RATIOS.. 91 4.1 INTRODUCTION -- ....................................... .. -- - - - .91 4.2 MATERIALS AND METHODS ............................................................................ 96 4.2.1 Materials .......................................................................................................... 96 4.2.2 Cell Culture ...................................................................................................... 97 4.2.3 Degradable Layer-by-Layer (LbL) Multilayer Fabrication ............................. 97 4.2.4 PDMS Preparation ........................................................................................... 98 4.2.5 PEI-siRNA Nanoparticle Formation ................................................................ 99 4.2.5.1 N/P Ratio Calculation ............................................................................... 99 4.2.6 Nanoparticle Stamping (Microcontact Printing) onto Multilayers ................ 100 4.2.7 Normal Forward Transfection (N FT) and Multilayer mediated Forward Transfection (MFT) ................................................................................................ 101 4.2.8 Characterization ............................................................................................. 101 4.2.8.1 Agarose Gel Electrophoresis Assay ........................................................ 101 4.2.8.2 Ultraviolet Visible (UV/vis) Absorbance ............................................... 102 4.2.8.3 Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AF M) ................................................................................................................. 102 4.2.8.4 Dynamic Light Scattering (DLS) ............................................................ 103 4.2.8.5 Zeta (Q-potential ..................................................................................... 104 4.2.8.6 Fluorescence Analysis of Patterned Delivery ......................................... 104 4.2.8.7 Real-Time Quantitative Reverse Transcriptase- Polymerase Chain Reaction (qRT-PCR) ........................................................................................... 104 4.2.8.8 Cytotoxicity Tests ................................................................................... 105 4.2.9 Statistical Analysis ......................................................................................... 105 4.3 RESULTS AND DISCUSSION ........................................................................... 106 ix 4.3.1 Physical Property Evaluation of LPEI-siRNA Nanoparticles at Different N/P Ratios ...................................................................................................................... 106 4.3.1.1 Agarose Gel Electrophoresis ................................................................... 106 4.3.1.2 Ultraviolet-Visible (UV/vis) Spectroscopy ............................................. 107 4.3.1.3 Particle Size Analysis ............................................................................. 110 4.3.1.4 Zeta (Q-potential Analysis ...................................................................... 113 4.3.2 Multilayer mediated Forward Transfection (MF T) for Patterned siRNA Delivery, and Effect of the Number of Bilayers ..................................................... 115 4.3.3 Evaluation of Transfection Efficiency: Real-time Quantitative Reverse Transcriptase—Polymerase Chain Reaction (qRT-PCR) Analysis ........................... 121 4.3.4 Cytotoxicity Evaluation: Transfection Reagent and N/P Ratio Optimization 124 4.3.5 Relationships between Degree of siRNA Incorporation, C—potential, Size and Transfection Efficiencies of LPEI-siRNA nanoparticles ........................................ 126 4.3.6 Hypothesis for number of 25kDa LPEI molecules per siRNA molecule ...... 128 4.4 CONCLUSIONS ................................................................................................... 132 CHAPTER 5 FABRICATION OF LINEAR-PEI NAN OPARTICLES USING HIGH SHEAR RATE MIXER 134 5.1 INTRODUCTION ................................................................................................ 134 5.2 MATERIALS AND METHODS .......................................................................... 136 5.2.1 Materials ........................................................................................................ 136 5.2.2 LPEI-IPC Micro and Nano-Particles Formation ............................................ 137 5.2.3 Scanning and Transmission Electron Microscopy (SEM and TEM) ............. 138 5.3 RESULTS AND DISCUSSION ........................................................................... 139 5.3.1 High Shear Rate Mixer .................................................................................. 139 5.3.2 Fabrication of LPEI-IPC Micro and Nano-particles under High Shear Rate 5.3.2.1 Effect of High Viscosity and High Shear Rate Mixing on Particle Shape ............................................................................................................................. 147 5.3.2.2 Effect of Polymers, High Viscosity and High Shear Rate Mixing on Particle Shape ...................................................................................................... 152 5.3.2.3 Effect of Vacuum and High Shear Rate Mixing on Particle Shape ........ 153 5.3.2.4 Effect of High Viscosity, Non evaporating and Miscible Solvent, Vacuum and High Shear Rate Mixing on Particle Shape .................................................. 164 5.3.3 Hypothesis for LPEI particle Aggregation at High Shear Rate Mixing ........ 169 5.3.4 Hypothesis to Reduce Nanoparticle Aggregation .......................................... 169 5.4 CONCLUSIONS ................................................................................................... 171 CHAPTER 6 CELL ADHESION RESPONSE OF THIN POLYELECTROLYTE MULTILAYERS - - - -- 173 6.1 INTRODUCTION ................................................................................................ 173 6.2 MATERIALS AND METHODS .......................................................................... 178 6.2.1 Materials ........................................................................................................ 178 6.2.2 Polyelectrolyte Multilayer (PEM) Fabrication .............................................. 178 6.2.3 Cell Cultures .................................................................................................. 179 6.2.3.1 Bone Marrow MSCs Isolation and Culture ............................................ 180 6.2.3.2 Fibroblasts Culture .................................................................................. 180 6.2.3.3 Primary Hepatocytes Isolation and Culture ............................................ 181 6.2.4 Cell Immunostaining ...................................................................................... 181 6.2.5 Characterizations ............................................................................................ 182 6.3 RESULTS AND DISCUSSION ........................................................................... 183 6.3.1 Thickness and Roughness of Poly(diallydimethylammonium) chloride/(Polystyrene sulfonate, sodium salt) (PDAC/SPS) Multilayers ............... 183 6.3.2 Adhesion of Mesenchymal Stem Cells (MSCs) and Fibroblasts on PDAC/SPS Multilayers .............................................................................................................. 185 6.4 CONCLUSIONS ................................................................................................... 192 CHAPTER 7 POLYELECTROLYTE MULTILAYER STAMPING IN AQUEOUS PHASE AND NON-CONTACT MODE 194 7.1 INTRODUCTION ................................................................................................ 194 7.2 MATERIALS AND METHODS .......................................................................... 196 7.2.1 Materials ........................................................................................................ 196 7.2.2 Stamps for NAM Transfer ............................................................................. 196 7.2.3 Polyelectrolyte Multilayer Fabrication for non-contact aqueous-phase multilayer (NAM) transfer ...................................... , ................................................ 1 97 7.2.4 NAM Transfer process ................................................................................... 198 7.2.5 Cell Culture .................................................................................................... 199 7.2.5.1 Primary Hepatocytes Isolation and Culture ............................................ 199 7.2.6 Cell Immunostaining ...................................................................................... 200 7.2.7 Optical Microscopy ........................................................................................ 200 7.3 RESULTS AND DISCUSSION ........................................................................... 201 7.3.1 Non-Contact Aqueous-Phase Multilayer (NAM) Transfer ............................ 201 7.3.2 NAM Transfer Characterization .................................................................... 203 7.3.2.1 NAM Transfer Characterization: Optical and Atomic Force Microscopy ............................................................................................................................. 203 7.3.2.2 NAM Transfer Characterization: NAM transfer over a monolayer of cells ............................................................................................................................. 209 7.3.3 Partial NAM transfer ...................................................................................... 217 7.4 CONCLUSIONS ................................................................................................... 219 CHAPTER 8 FABRICATION AND CHARACTERIZATION OF THIN SELF- STANDING COMPOSITE-POLYELECTROLYTE MULTILAYERS ................ 220 8.1 INTRODUCTION ................................................................................................ 220 8.2 MATERIALS AND METHODS .......................................................................... 222 8.2.1 Materials ........................................................................................................ 222 8.2.2 Self Standing Composite-Polyelectrolyte Multilayer (SSC-PEM) Fabrication ................................................................................................................................. 223 8.2.3 Characterizations ............................................................................................ 224 8.2.3.1 Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) .................................................................................................................. 224 8.2.3.2 Optical Microscopy ................................................................................. 225 8.2.4 Cell Culture .................................................................................................... 225 xi 8.2.4.1 Three-dimensional (3-D) Cell Culture using SSC-PEM ........................ 226 8.2.4.2 Cell Immunostaining ............................................................................... 226 8.3 RESULTS AND DISCUSSION ........................................................................... 227 8.3.1 Self Standing Composite-Polyelectrolyte Multilayer (SSC-PEM) Fabrication ................................................................................................................................. 228 8.3.1.1 Critical thicknesses for SSC-PEM .......................................................... 230 8.3.1.2 Effect of Molecular Weight of PAA and PEG ........................................ 231 8.3.2 Thickness and Surface Morphology of SSC-PEM: AFM, SEM, and Optical Microscopy ............................................................................................................. 232 8.3.2.1 Surface Morphology and Thickness of the Composite Film .................. 232 8.3.2.2 Surface Morphology of the SSC-PEM ................................................... 237 8.3.3 SSC-PEM Composition: XPS and FTIR ....................................................... 239 8.3.4 SSC-PEM in Cell culture Applications .......................................................... 245 8.3.4.1 SSC-PEM for 3-D Cell Culture .............................................................. 245 8.3.4.2 SSC-PEM for Cell Sheet Culture ............................................................ 250 8.4 CONCLUSIONS ................................................................................................... 251 CHAPTER 9 CONCLUSIONS AND FUTURE DIRECTIONS 253 9.1 FUTURE DIRECTIONS ...................................................................................... 254 APPENDIX A: AGAROSE HYDROGEL STAMPS FOR TIME AND SPACE CONTROLLED DRUG DELIVERY 258 APPENDIX B: LIST OF PUBLICATIONS 260 BIBLIOGRAPHY 261 xii LIST OF TABLES Table 4.1 Average dimensions of an atelocollagen, a 25kDa LPEI and a siRNA molecule. ......................................................................................................................... 130 Table 5.1 Parameters and conditions obtained during and after high shear rate mixing when LPEI(CHC13) was injected to vessel afier F 68(H20) reached at maximum temperature in mixer. All the temperatures shown are with-in the error limits of 3:20C. Total run time was 15 min, and coolant temperature was maintained at 4°C ................. 144 Table 5.2 Parameters and conditions obtained during and after high shear rate mixing of LPEI(CHCI3)-F68(100%Glycerol). All the temperatures Shown are with-in the error limits of $200 Coolant temperature was maintained at 40C. ........................................ 148 Table 5.3 LPEI(CHCI3)-F68(H20); Run time = 2 min; Vacuum applied after run of 1min in each case. Chiller temperature was maintained at 40C. All the temperatures were with- in the error limits of :tZOC. .............................................................................................. 164 Table 8.1 Critical number of bilayers of (PAA/PEG) and (PDAC/SPS) to obtain SSC- PEM from PDMS substrate. ........................................................................................... 231 Table 8.2 Atomic concentrations obtained from the XPS analysis of top component of (PAA/PEG)20,5(PDAC/SPS)30,5 SSC-PEM i.e. PDAC/SPS multilayers as the surface analyzed (corresponding to Figure 8.6a). ....................................................................... 243 Table 8.3 Atomic concentrations obtained from the XPS analysis of bottom component of (PAA/PEG)20,5(PDAC/SPS)30.5 SSC-PEM i.e. PAA/PEG multilayers as the surface analyzed (corresponding to Figure 8.6b). ....................................................................... 243 xiii LIST OF FIGURES Images in this thesis/dissertation are presented in color Figure 2.1 Diagram showing the three- and two-components LbL assembly fabricated onto native agarose as the substrate. Templated agarose scaffolds (as shown in this Figure) were used to characterize film growth, and agarose-filled TCPS plates were used to characterize protein releases. Three component assemblies consisted of PAA, PEG and protein as the multilayer constituents, and two component assemblies consisted of PEG or PAA and protein as the multilayer constituents. BPEI, LPEI, or protein (lysozyme, denoted as Lyso) was used as the LbL initiating polymer in the different cases shown in a-f. Curved lines in a-f represent the agarose, and BL indicates the bilayers. .................. 24 Figure 2.2 (a) Fluorescence intensity measurements of T RITC conjugated to amine terminated PEG (PEG-Amine-TRITC) showed increased adsorption of PEG into the agarose structure during the LbL deposition of BSA/(PEG-Amine-TRITC) multilayers on agarose. (b) UV/vis absorbance measurements at 280nm showed increased adsorption of bovine serum albumin (BSA) protein onto the agarose structure during the LbL deposition of BSA/(PEG) multilayers. The absorbance values are shown with respect to (w.r.t.) the absorbance of bare agarose i.e. the difference between the absorbance of LbL coated agarose and the absorbance of bare (non-coated) agarose. Five precursor bilayers of (PAA/PEG) with LPEI as the LbL initiating polymer were built over agarose in each case. BLs denote the number of bilayers (0) Corresponding decrease in lysozyme concentration in the bulk solution from the initial concentration as a function of the number of bilayers. ........................................................................................................... 26 Figure 2.3 Confocal and scanning electron microscopy images showing the formation of LbL assembled multilayer thin films on agarose substrate. (a) Confocal microscopy images and associated fluorescence intensity profiles of a section below the top surface of agarose scaffolds coated with 30 bilayers of PAA and PEG followed by three bilayers by PEG and TRITC conjugated BSA (lefi image), and of agarose scaffolds coated with only 3 bilayers by PEG and TRITC conjugated BSA (right image). (b) Confocal microscopy acquired z-section images of agarose scaffolds coated with 30 bilayers of PAA and PEG followed by three bilayers by PEG and TRITC conjugated BSA. Fluorescence intensity profiles of the topmost section (top panel: leftmost image) and a middle section (bottom panel: rightmost image) are shown. (c) Scanning electron microscopy images of the scaffolds LbL coated with 30 bilayers of PAA and PEG. Images in red boxes are of the non-coated scaffolds. BPEI was used as the LbL initiating polyelectrolyte in all images. ...... .- 31 Figure 2.4 (a) Cumulative lysozyme release up to 4 weeks triggered by physiological pH, from LbL multilayer (as shown in Figure 2.1a; BPEI initiating) coated 3% agarose gel. (b) Comparison between the total and enzymatically active lysozyme released per day, corresponding to the protein released in Figure 2.43. Active concentrations were calculated from a standard curve obtained from pure lysozyme used to determine the degree of lysis of Micrococcus Iysodeikticus by lysozyme. ............................................. 36 xiv Figure 2.5 (a,b) Cumulative lysozyme release over time triggered by physiological pH from agarose hydrogel of varying concentrations, coated with LbL multilayer assembly (as shown in Figure 2.13; BPEI initiating). (a) Comparison between 1%, 2% and 3% agarose. (b) Comparison between 3% and 4% agarose. (c) Total surface area per unit volume of pure agarose hydrogel as a function of hydrogel concentration determined by BET. .................................................................................................................................. 38 Figure 2.6 The effect of LbL initiating polymer on lysozyme release triggered by physiological pH. BPEI, LPEI, lysozyme or PAA was used as the LbL initiating polymer as shown in Figure 2.1a and 2.1b ...................................................................................... 41 Figure 2.7 Cumulative lysozyme release from 3% agarose gel, triggered by physiological pH, (a) with varying stacking order of the polymers within a multilayer but the same number of cumulative bilayers (as shown in Figure 2.1d, 2.10 and 2.1b), and (b) with two-component assembly of lysozyme and PAA, two-component assembly of lysozyme and PEG, and three component assembly of PAA, PEG and lysozyme (as shown in Figure 2.1f, 2.1e and 2.1b respectively) ............................................................................ 44 Figure 2.8 Optical densities of the bacteria in cell culture medium (without lysozyme), and bacterial solutions containing active lysozyme released from LbL coated agarose incubated in fibroblast medium for 1-4 days. Cell culture medium was replaced each day. ........................................................................................................................................... 46 Figure 2.9 (a-e) Static lysozyme release, triggered by physiological pH, corresponding to the Figures 2.4-2.7. (f) Dynamic lysozyme release, triggered by physiological pH, showing release from soaked lysozyme (i.e. direct impregnation of lysozyme) sample. 48 Figure 2.10 (a,b) Dynamic and Static lysozyme releases, triggered by physiological pH, with lysozyme pH at 4.75 in acetate buffer during LbL fabrication on agarose. (0) Dynamic and Static lysozyme releases, triggered by physiological pH, with pH of all solutions at 3.0 during LbL fabrication on agarose. ......................................................... 54 Figure 2.11 SEM images of H-bonded films composed of 25 bilayers of 10kDa PEG and 15kDa PAA formed on a planar substrate and exposed to deionized water (DI) (pH 5.6- 6.3) for the time durations indicated. Top panel: SEM images of films after fabrication. Middle panel: SEM images of films after immersion in DI water for five days. Bottom panel: SEM images of films after immersion in DI water for ten days. Same spot on the films before and after degradation were imaged for comparative analysis. Columns 1, 2 and 3 show three different spots on the film. .................................................................... 57 Figure 2.12 (a) Phase contrast microscopy images demonstrating the cytophobicity of the 30.5 bilayers of PAA/PEG multilayers over time (days). Top panel: NIH-3T3 fibroblasts cells on TCPS plates coated with 30.5 bilayers of PAA/PEG. Bottom panel: Fibroblasts on bare TCPS plates (control). (b) Phase contrast microscopy images demonstrating the cyt0phobicity of the 5.5 bilayers of PAA/PEG multilayers over time (days). (c) XV Cytotoxicity levels of fibroblast cells after 4 days of culturing on (PAA/PEG)5PAA multilayers built onto TCPS (denoted here as 5.5BLs), (PAA/PEG)30PAA multilayers built onto TCPS (denoted here as 30.5 BLs), and bare TCPS plates. The absorbance values corresponds to the amount of lactate dehydrogenase (LDH) released into the culture supernatant. A higher absorbance value indicates higher LDH release and thus higher toxicity. Serum in the cell culture medium contains a small amount of LDH, which is shown as “background media”. ..................................................................................... 61 Figure 3.1 Diagrams showing the growth of leptomeningeal fibroblasts along the inner periphery of a single channel of templated agarose scaffold (top image), and at the distal end ofchannel (bottom image). 65 Figure 3.2 (a) Chemical structure of 1-B-D-arabino-furanosylcytosine (Ara-C). (b) Multilayer assembly of PAA, PEG and Ara-C over agarose. Curved line represents the agarose. ............................................................................................................................. 74 Figure 3.3 (a) UV-vis spectrum of agarose loaded with multilayers of (PAA/PEG)5(AraC/PEG)2(PAA/PEG)5(AraC/PEG)[(PAA/PEG)5(AraC/PEG)5]n. n represents the number of repetitions of sequence [(PAA/PEG)5(AraC/PEG)5]. In the plot, P represents (PAA/PEG) and A represents (AraC/PEG) multilayers and the 5 represent the number of bilayers. P5A5P5Al is the base multilayer for each case in the UV-vis measurements. (b) Plot of AraC absorbance at 272 nm as a function of increasing number of AraC layers. .................................................................................................................. 76 Figure 3.4 Fibroblasts cultured on a cover-slip in contact with LbL multilayer ([(PAA/PEG)5(Ara-C/PEG)5]3PAA) coated agarose. Prior to exposure to the cells, the LbL coated agarose discs were incubated in cell culture media (without any cells) for 24 hrs, with fresh culture medium replaced at 30 min, 16 hrs, and 24 hrs. (a) Cells were monitored for 3 days (indicated as Day 1 (48 hrs), Day 2 (72 hrs), and Day 3 (96 hrs)), (b) The number of cells cultured on the covers-slips in contact with LbL multilayer coated and non-coated agarose. Day 0 implies fresh cells, i.e. just before placing the cells in contact with LbL coated agarose. (c) After 3 days, cells on agarose were replaced with fresh cells cultured on new substrate and were again monitored for next 3 days (indicated as Day 4 (120hrs), Day 5 (144 hrs), and Day 6 (168 hrs)). Scale bar represents lOOum. Time in parenthesis represent the age of the LbL coated agarose, i.e. it denotes the time since the start of the multilayer degradation in culture media, whereby the initial 24 hrs of degradation was performed in the absence of cells. ......................................................... 78 Figure 3.5 Fibroblasts cultured on a cover—slip in contact with LbL multilayer ((PAA/PEG)15_5) coated agarose, without Ara-C. Cells were monitored for 3 days (indicated as Day 1 (48 hrs), Day 2 (72 hrs), and Day 3 (96 hrs)). Scale bar represents 100nm. Time in parenthesis represent the age of the LbL coated agarose, i.e. it denotes the time since the start of multilayer degradation in culture media, where the initial 24 hrs of degradation was performed in the absence of cells (with the medium without cells replaced at 30 min, 16 hrs, and 24 hrs). ............................................................................ 80 xvi Figure 3.6 (a) The effect on fibroblast growth of directly adding pure Ara-C. Varying concentrations of Ara-C were added once, for the first 24 hrs and subsequently replaced with non-Ara-C containing media. The fibroblasts were monitored for up to 3 days. The effectiveness of the Ara-C decreased with concentration. Scale bar represents 100nm. (b) The number of cells as a fimction of time. Day 0 implies fresh cells, i.e. just before adding the AraC to cultured cells. The growth rate of the control cells (data not plotted) was similar to those plotted in Figure 3.4b. ...................................................................... 83 Figure 3.7 RP-HPLC measurements of the amount of free Ara-C in the solution mixtures of PAA, PEG and Ara-C as a function of increasing PAA and PEG amounts (no LbL). X- axis indicates the amount of both PAA and PEG, individually, in the solution e. g. 100 ug on x-axis indicates that 100 pg of PAA and 100 ug of PEG was added to the solution. Total amount of pure Ara-C added to each solution was 6.25 pg. ................................... 85 Figure 3.8 Non-cumulative and cumulative free Ara-C concentrations released from: (a,b) LbL coated agarose, and (c,d) non-coated Ara-C soaked agarose, both exposed to 1X PBS at physiological pH. ............................................................................................ 87 Figure 3.9 Fibroblasts cultured on a cover-slip in contact with LbL multilayer ([(PAA/PEG)5(Ara-C/PEG)5]3PAA) coated agarose, when the bulk concentration of AraC at the beginning of LbL formation was 500 pg/ml. Prior to exposure to the cells, the LbL coated agarose discs were incubated in cell culture media (without any cells) for 24 hrs, with fresh culture medium replaced at 30 min, 16 hrs, and 24 hrs. Cells were monitored for 3 days (indicated as Day 1 (48 hrs), Day 2 (72 hrs), and Day 3 (96 hrs)). Scale bar represents 100nm. Time in parenthesis denotes the time since the start of the multilayer degradation in culture media, whereby the initial 24 hrs of degradation was performed in the absence of cells ...................................................................................... 89 Figure 4.1 Diagram illustrating the multilayer mediated forward transfection (MFT) of cationic vector complexed siRNA for patterned delivery ................................................. 94 Figure 4.2 Agarose gel electrophoresis of LPEI-siRNA nanoparticles at various N/P ratios with 200nM siRNA concentration. Numbers indicate the N/P ratios. (0 N/P ratio indicates the naked or free siRN A). ................................................................................ 107 Figure 4.3 Ultraviolet-visible (UV/vis) absorption spectra of LPEI suspended at varying concentrations in: (a) DI water, or (b) OptiMEM buffer, without siRNA, showed maximum peaks at 240nm and 244nm, respectively. (“LPEI concentration (E N/P ratio)” corresponds to the concentration of LPEI for a given N/P ratio calculated for siRNA final concentration of 750nM). Same peak positions were observed for all the other LPEI concentrations used in the range for nanoparticle fabrication. (c) UV/vis absorption spectra of LPEI-siRNA nanoparticles Show a blue shift with increasing N/P ratios. (Inset: Maximum UV/vis peaks of siRNA and LPEI in OptiMEM are 260nm and 244nm, respectively). ((1) Plot of the wavelengths corresponding to the absorbance peak of LPEI- siRN A nanoparticles as a function of the N/P ratio. ....................................................... 109 xvii Figure 4.4 Variation in the size of LPEI-siRNA nanoparticles as a function of N/P ratio. (a) Scanning electron microscopy (SEM) images of the nanoparticles and their sizes as a function of N/P ratio. Scale bar represents 500 nm, and the error bars indicate the standard deviations of at least ten measurements performed on three different scanned areas per sample. (b) Top images — Atomic Force microscopy (AF M) images of LPEI- siRNA nanoparticles at N/P ratios of 30 and 90 (using 240pmol of siRNA). Middle images — Particle size distribution at these N/P ratios. Bottom images — Section analysis of three representative particles of different sizes within a given image. (c) Histograms of DLS determined nanoparticle sizes for N/P ratios of 5, 45, 60 and 75 show a decrease in the average particle size with increasing N/P ratio. ........................................................ 111 Figure 4.5 Zeta-potential values of LPEI-siRNA nanoparticles as a function of N/P ratio, immediately after they were formed (Direct Complexes) and released upon multilayer degradation (Multilayer Released Complexes) in deionized water, measured at 250C. ”LPEI (N/P90) only” corresponds to the amount of LPEI for N/P ratio of 90 without siRNA. Error bars indicate the standard deviation of three measurements of ten runs per sample. ............................................................................................................................ 115 Figure 4.6 Fluorescent images demonstrating patterned siRNA delivery to HeLa cells with multilayer mediated forward transfection (MFT) using (PAA/PEG)6.5 multilayer assembly, fluorescent dsRNA oligomers (100pmol) and Lipofectamine 2000 (LF 2k, Sug). Nanoparticles and HeLa cell patterns transfected with: (a) Alexa Fluor SSS-labeled oligomers, (b) Fluorescein and Alexa Fluor SSS-labeled oligomers (overlaid images). Top panel- CLSM images of LF2k-fluorescent oligomer nanoparticles arrayed onto multilayer. Middle and bottom panels- HeLa cell patterns transfected with fluorescent oligomers and their corresponding phase contrast images acquired using CLSM (middle panel) and conventional fluorescence microscopy (bottom panel). Scale bar represents 500 um. ........................................................................................................................... 117 Figure 4.7 Conventional fluorescence microscopy images demonstrating square patterned siRNA delivery to HeLa cells with MFT using (PAA/PEG)30_5 multilayer assembly, fluorescent dsRNA oligomers (40pmol) and Lipofectamine 2000 (LF2k, 2ug). Top Image- Fluorescence image of LF2k—fluorescein labeled oligomer nanoparticles arrayed onto multilayer. Middle and Bottom images- HeLa cell patterns transfected with fluorescein labeled oligomer; and their corresponding phase contrast images. Scale bar represents 100 um. .......................................................................................................... 118 Figure 4.8 LPEI-siRNA nanoparticles stamped on a plasma-treated quartz did not release from the substrate to the cell culture medium. These nanoparticles remained intact on the quartz even after 48 hrs. There was no transfection observed with the cells. (a) Conventional fluorescence microscopy image of the intact patterns of LPEI-fluorescein labeled dsRNA oligomer nanoparticles, imaged on the quartz substrate, were stamped onto plasma-treated quartz and placed onto the cultured cells for 48 hrs. The edges of the intact patterns on the quartz are clearly visible. Scale bar represents 200 um. (b) Conventional fluorescence microscopy image of HeLa cells (corresponding to image a) after contact with the stamped quartz substrate for 48 hrs showed no appreciable xviii transfection. No patterns of transfected cells observed in image b. Guidelines were created manually for areas (A1 and A2), representing where the patterns should have transferred to the cells had the MFT been successful. Very few cells, if any, show fluorescence in image b. (c) Phase contrast images of the untransfected HeLa cells corresponding to image b. (d) qRT-PCR of PKR gene expression levels in HeLa cells, 48 hr post-contact with LPEI-siRNA nanoparticles stamped on a plasma-treated quartz, indicating no transfection. MFT and NFT efficiencies for N/P ratio 45 are shown for comparison. ..................................................................................................................... 120 Figure 4.9 qRT-PCR quantified expression levels of PKR gene knockdown in HeLa cells 48 hr post-transfection of siRNA delivery using 25kDa LPEI or 25kDa BPEI at different N/P ratios, or Lipofectamine 2000 (LF2k, Zug) with: (a) normal forward transfection (NFT) and (b) multilayer mediated forward transfection (MF T). Amount of siRNA was 240pmol (E 200nM final concentration in NFT) and transfection reagent was LPEI, unless specified otherwise. Numbers in parenthesis denote the amount of LPEI corresponding to the noted N/P ratio of 45, but without siRN A. BLs denote the number of bilayers. Error bars indicate the standard deviations of RT-PCR reactions on three independent samples. #, #p<0.01 as compared with “cells only” (i.e. cells without any transfection reagent or siRNA). ###p<0.05 as compared with “cells only”. *p<0.05 compared with “N/P 5 in Figure 4.9(a)”. ........................................................................ 123 Figure 4.10 Cytotoxicity levels in HeLa cells 48 hr post-NFT of siRNA delivery using 25kDa LPEI or 25kDa BPEI at different N/P ratios, or Lipofectamine 2000 (LF 2k, Zpg). Concentration of siRNA was 200nM and transfection reagent was LPEI, unless specified otherwise. Numbers in parenthesis denote the amount of LPEI or BPEI corresponding to the amount of PEI at the noted N/P ratio but without siRNA. Error Bars indicate the standard deviations of %LDH release of three independent samples. *p<0.01 compared with “N/P 45” and “BPEI-N/P 10”. ................................................................................ 126 Figure 5.1 Micrographs showing: (a) mixing vessel of the high shear rate mixer and (b) different turbines designs available (T.K.FILMICS®, PRIMIX Corporation, Japan)... 140 Figure 5.2 Structural formulae of linear polyethylenimine (LPEI) and Pluronic F68. .. 142 Figure 5.3 Micrographs showing particle suspension obtained after homogenization of LPEI (in CHCl3) and Pluronic F68 (in H20) solutions at the mixing speeds of 10m/s, 20m/s, 30m/s, 40m/s and SOm/s (from left to right, respectively) without any subsequent solvent diffusion or evaporation. LPEI(CHC13) was injected to vessel after F 68(H20) reached at maximum temperature in mixer. Phase separation was observed for the speeds of 10 m/s, 20m/s, and 30 m/s. ......................................................................................... 144 Figure 5.4 SEM images of the particles obtained after homogenization of LPEI (in CHCI3) and Pluronic F68 (in H20) solutions at the mixing speeds of lOm/s, 20m/s, 40m/s and SOm/s, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. LPEI(CHC13) was injected to vessel after F68(H20) reached at maximum temperature in mixer. The meshed structure observed in images is the xix aluminum oxide filter membrane (sample substrate) through which the particles were filtered and dried. ............................................................................................................ 146 Figure 5.5 Micrographs showing particle suspension obtained after homogenization of LPEI (CHC13) and Pluronic F68 (100%Glycerol) solutions at the mixing speeds of lOm/s, 30m/S, and 50m/s (from left to right, respectively) without any subsequent solvent diffusion, evaporation or dialysis. Phase separation was observed for the speed of 10 m/s. ......................................................................................................................................... 149 Figure 5.6 SEM images of the particles obtained after homogenization of LPEI (CHC13) and Pluronic F68 (100%Glycerol) solutions at the mixing speeds of lOm/s, 30m/s and SOm/s after subsequent dialysis of the emulsion mixture in water. The meshed structure observed in images is the aluminum oxide filter membrane (sample substrate) through which the particles were filtered and dried. .................................................................... 151 Figure 5.7 SEM images of the particles obtained after homogenization of PLA (in ethyl acetate) and 2.5kDa LPEI (in 50%glycerol-water) solutions at the mixing speeds of 50m/s and after subsequent dialysis of the emulsion mixture in water. ......................... 153 Figure 5.8 SEM images of the particles obtained after homogenization of LPEI (in CHC13) and Pluronic F68 (in H20) solutions at the mixing speeds of lOm/s, 20m/s, 40m/s and 50m/s under the vacuum (applied during run after 60 sec of mixing), and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. The meshed structure observed in images is the aluminum oxide filter membrane (sample substrate) through which the particles were filtered and dried. ...................................... 156 Figure 5.9 Low magnification SEM images of the particles obtained after homogenization of LPEI (CHC13) and Pluronic F 68 (H20) solutions at the mixing speeds of lOm/s, 20m/s, 40m/s and 50m/s (clockwise from top-left, respectively) under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. ......................................................................................................................... 157 Figure 5.10 SEM images of the particles obtained after homogenization of LPEI (in CHCl3) and Pluronic F68 (in H20) solutions at the mixing speed of 30m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. ......................................................................................................................... 159 Figure 5.11 TEM images of the particles (corresponding to Figure 5.10) obtained after homogenization of LPEI (in CHC13) and Pluronic F 68 (in H20) solutions at the mixing speed of 30m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. ......................................................................... 160 Figure 5.12 SEM images of the particles Obtained after homogenization of LPEI (in CHC13) and Pluronic F68 (in H20) solutions at the mixing speed of 40m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. ......................................................................................................................... 161 XX Figure 5.13 TEM images of the particles (corresponding to Figure 5.12) obtained after homogenization of LPEI (in CHC13) and Pluronic F68 (in H20) solutions at the mixing speed of 40m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. ......................................................................... 162 Figure 5.14 TEM images of the particles obtained after homogenization of LPEI (in CHC13) and Pluronic F68 (in H20) solutions at the mixing speed of 50m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. ......................................................................................................................... 163 Figure 5.15 SEM images of the particles obtained after homogenization of LPEI (in ethanol) and 66 v/v% glycerol at the mixing speed of 50rn/s under vacuum and after subsequent dialysis of the emulsion mixture in water. Coolant temperature was set at 20 C ................................................................................................................................. 167 Figure 5.16 SEM images of the particles obtained after homogenization of LPEI (in ethanol) and 100 v/v% glycerol at the mixing speed of 50m/s under vacuum applied before run (left column) and under vacuum applied 30 see after run (right column), and after subsequent dialysis of the emulsion mixture in water. Coolant temperature was set at 20 C ................................................................................................................................. 168 Figure 6.1 Diagram showing multilayers composed of linearly growing strong polyelectrolytes i.e. PDAC and SPS, fabricated at a deposition ionic strength of 0.1M NaCl, exhibit increased cytophobicity as the number of bilayers increases, as shown in images (A) to (E). Bands with violet and blue colors represents positively charged PDAC and negatively charged SPS polyelectrolyte chains, respectively; and one set of violet/purple colored band represents ten bilayers of PDAC/SPS. Red, green and blue colors inside the cell structure represent actin filaments, focal adhesion contacts and nucleus of the cell, respectively. Image (F) illustrates a previous study152 with a higher deposition ionic strength, the multilayers exhibit more cytophobicity due to swelling and hydration within the multilayer structure. The thickness band in image (F) represents a more loopy configuration of the polyelectrolytes with enhanced swelling and hydration within the multilayer152 as compared to those in images (A-E) ...................................... 176 Figure 6.2 Ellipsometric thickness of dried (PDAC/SP8)n multilayer film deposited at an ionic strength of 0.1M NaCl, as a function of the number of bilayers n. The thickness errors are reported (significantly small) as the standard deviation of the measurements from at least three different areas on three different samples. The dashed line is a linear fit to the reported data points. ......................................................................................... 184 Figure 6.3 AFM topographic images of the morphology of (a) 30 bilayer and (b) 50 bilayer dried PDAC/SPS films deposited at an ionic strength of 0.1M NaCl. Images are 10pm x 10pm, and the z-scales are shown. .................................................................... 185 Figure 6.4 Confocal laser scanning and phase contrast microscopy images of (a) bone marrow mesenchymal stem cells (MSCs) and (b) NIH3T3 fibroblasts, cultured on (PDAC/SPS)n multilayers. Green, red (only fibroblasts), and blue channels Show the focal adhesion sites mapped by rabbit anti-paxicillin primary antibody and Alexa Fluor 488 goat anti-rabbit IgG secondary antibody, actin filaments mapped by Texas Red-X phalloidin, and nuclei mapped by DAPI, respectively. Images were immunolabeled 48 hrs post cell seeding. Phase contrast images were obtained just prior to immunostaining. (c) Phase contrast microscopy images of primary rat hepatocytes cultured on (PDAC/SPS)n multilayer coated TCPS substrates (scale bar = 100 um). n represents the number of multilayer bilayers (BLs), as indicated on the images. Non-coated TCPS or glass served as control surfaces. ..................................................................................... 188 Figure 6.5 Confocal laser scanning of bone marrow mesenchymal stem cells (MCSs) cultured on (PDAC/SPS)n multilayers in the presence of serum (top panel) and absence of serum for 48 hrs (bottom panel). n represents the number of PDAC/SP8 bilayers (BLs). Images are control, 10 BLs and 30 BLs starting from left-to-right. Non-coated TCPS or glass served as control surfaces. Green and blue channels show the focal adhesion sites mapped by rabbit anti-paxicillin primary antibody and Alexa F luor 488 goat anti-rabbit IgG secondary antibody, and nuclei mapped by DAPI, respectively. CLSM images were acquired at 40X magnification. Images were immunolabeled 48 hrs post cell seeding. 192 Figure 7.1 (a) Scheme showing non-contact aqueous-phase multilayer (NAM) transfer in aqueous medium. 0n the stamp, the first two initial layers (green and orange layers) represent degradable H-bonded (PAA/PEG)10,5 multilayers, and next subsequent four layers (dark blue and dark red) represent (PDAC/SPS)30,5 (PDAC as topmost layer) multilayers. 0n the substrate before NAM transfer (left image), the two layers (light red and light blue) represent (PDAC/SP8)”, (SPS as topmost layer) multilayers. During the NAM transfer, the (PDAC/SPS)30_5 multilayers gets released from stamp and adhere onto (PDAC/SP3)“, base multilayers on the substrate, as shown in right image. The curved lines in surroundings represent liquid medium at physiological pH. (b) NAM transfer onto a layer of cells cultured on a base polyelectrolyte multilayer (PEM) ..................... 203 Figure 7.2 (a - d) Stamps before and after NAM transfer of BPEI(PAA/PEG)10,5(PDAC/SPS)30,5 on top of (PDAC/SPS)10. Top panel: Fluorescent images of 6-CF stained PDMS stamp before and after NAM transfer. Middle panel: Dark field optical images of PDMS stamp before and after NAM transfer. Bottom panel: (e, f) Bright field optical images of glass stamps before and after NAM transfer of BPEI(PAA/PEG)10.5(PDAC/SPS)30,5 on top of (PDAC/SPS)10. ..................................... 205 Figure 7.3 Substrates before (left column) and after (right column) NAM transfer of BPEI(PAA/PEG)10.5(PDAC/SPS)30,5 on top of (PDAC/BPS)“; as illustrated by: (a, b) Fluorescent images of negatively charged 6-CF dye staining of substrates, and (c, (1) Optical images of negatively charged 3 pm carboxylated polystyrene latex particle addition to substrates ....................................................................................................... 207 xxii Figure 7.4 Surfaces afier mdPEG stamping and 6-CF staining on: (a) PAH coated glass, (b) substrate after NAM transfer of BPEI(PAA/PEG)1o_5(PDAC/SPS)30_5 on top of (PDAC/SPS)10. ................................................................................................................ 208 Figure 7.5 Polyelectrolyte multilayer transfer of PAH(SPS/Rh-PDAC)4,5 over (PDAC/SPS)10,5 using patterned PDMS stamp in (A) dry conditions, (B) aqueous conditions (scale bar = lOOum). ..................................................................................... 209 Figure 7.6 Fluorescent (confocal and conventional microscopy) images and phase contrast images of primary hepatocytes and fibroblast co-cultured in 3-D fashion using NAM multilayer transfer process. Successful staining of top layer of cells i.e. primary hepatocytes, and no staining of bottom layer of cells i.e. fibroblasts suggests that the (PDAC/SPS)30.5 multilayers were transferred during the NAM transfer process. Thick (PDAC/SPS)30,5 films inhibited the diffusion of staining dyes to the bottom layer of fibroblasts ........................................................................................................................ 213 Figure 7.7 Primary hepatocytes cell attachment on the PEMs as an evidence of (SPS/PDAC)4_5 transfer in NAM transfer process giving SPS as the topmost layer, using PAH(SPS/PDAC)4,5 multilayer on stamp. Figures (a) and (b) shows the primary hepatocytes cultured on the multilayer of (PDAC/SPS)10,5 (PDAC as topmost surface) at Day 1 and Day 3. Figures (0) and (C!) shows the primary hepatocytes cultured after NAM transfer of PAH(SPS/PDAC)4,5 on the multilayer of (PDAC/SPS)10,5 at Day 1 and Day 3. (e) and (f) shows the cells on control TCPS substrate at Day 1 and Day 3. Scale bar represents 100nm. ........................................................................................................... 216 Figure 7.8 AFM analysis showing fiactal transfer of multilayers after NAM transfer under partially dried conditions. ..................................................................................... 218 Figure 8.1 Schematic showing assembly of rough and thick (PAA/PEG) multilayers formed followed by smooth and thin (PDAC/SPS) multilayers on hydrophobic PDMS substrate. ......................................................................................................................... 229 Figure 8.2 Macroscopic images of SSC-PEM (PAA/PEG)20,5(PDAC/SPS)30,5: (a) half peeled film from PDMS and PDMS block are shown (b) peeled off film and PDMS blocks are shown. ............................................................................................................ 229 Figure 8.3 AFM surface analysis of (a) (PAA/PEG)20.5 films formed on hydrophobic PDMS, (b) (PAA/PEG)20_5(PDAC/SPS)n multilayers formed on hydrophobic PDMS i.e. composite films before peeling them off from PDMS. 11 = 20, 60 and 80 for image bl, b2 and b3, respectively. ....................................................................................................... 234 Figure 8.4 Optical micrographs (leftmost column), AF M signal images (middle column), and AFM height analysis (rightmost column) of the edges of (PAA/PEG)20_5(PDAC/SPS)n composite films assembled on PDMS. n = 20, 60 and 80. Scale bar in optical micrographs is lOOum ..................................................................... 236 xxiii Figure 8.5 (a) SEM images of SSC-PEM (PAA/PEG)20,5(PDAC/SPS)30,5 (i.e. composite films alter peeling-off from PDMS) with (PDAC/SPS) multilayers as the scanning surface, (b) optical micrographs of SSC-PEM (PAA/PEG)20_5(PDAC/SPS)30_5 with (PDAC/SPS) multilayers as the imaging surface ............................................................ 238 Figure 8.6 XPS spectra of SSC-PEM (PAA/PEG)20_5(PDAC/SPS)80,5: (a) top component of SSC-PEM i.e. PDAC/SPS multilayers as the surface analyzed, and (b) bottom component of SSC-PEM i.e. PAA/PEG multilayers as the surface analyzed. ............... 241 Figure 8.7 Transmission FTIR spectrum of (PAA/PEG)20,5(PDAC/SPS)30,5 SSC-PEM. ......................................................................................................................................... 244 Figure 8.8 Schematic showing 3-D cell co-culture with an intermediate layer of SSC- PEM between two cell types. First layer of cells was cultured over a charged substrate, and SSC-PEM with bottom component as (PAA/PEG) multilayers was adhered over the cultured cells. Second layer of cells was cultured on top of (PDAC/SPS) component of SSC-PEM. ....................................................................................................................... 246 Figure 8.9 (a) Three-dimensional cell co-culture showing two layers of fibroblasts with an intermediate layer of (PAA/PEG)20_5(PDAC/SPS)80,5 SSC-PEM which is adhered to the cells and substrate at bottom. Lower panel of images shows contrast modified images corresponding to the images in top panel. Red and green highlighted areas shows cells cultured above and below SSC-PEM, respectively (scale bar = 100 um). (b) High magnification image showing viable cells cultured below a (PAA/PEG)20,5(PDAC/SPS)30.5 SSC-PEM which is adhered to those cells and the exposed substrate. ........................................................................................................... 247 Figure 8.10 3-D cell co-culture of fibroblasts and HeLa cells using (PAA/PEG)20_5(PDAC/SPS)30.5 SSC-PEM as an intermediate layer, in the sequence of fibroblast, SSC-PEM, and HeLa cells from bottom to top. Confocal microscopy z-series analysis is shown ............................................................................................................. 249 Figure 8.11 Schematic showing SSC-PEM as a cell sheet. Cells can be cultured over the (PDAC/SPS) component of a floating SSC-PEM to create a floating cell sheet ............ 251 Figure A.l Micro-pattemed agarose hydrogel stamps. .................................................. 258 Figure A.2 Fibroblast cells stamped on a glass surface using microcontact printing (uCP) process and agarose hydrogel stamps. ............................................................................ 259 xxiv LIST OF ABBREVIATIONS AraC or Ara-C: l-B-D-Arabino-firranosylcytosine BDNF: Brain derived neurotrophic factor BPEI: Branched poly(ethylenimine) BSA: Bovine serum albumin CHCl3: Chloroform DNA: Deoxyribonucleic acid F 68: Pluronic F68 H—bonded: Hydrogen-bonded IPEC: Inter-polyelectrolyte complex IPC: Inter-polymer complex LbL: Layer-by-layer LF2k: Lipofectamine 2000 LPEI: Linear poly(ethylenimine) Lyso: Lysozyme uCP: Micro-contact printing MFT: Multilayer mediated forward transfection MSCs: Mesenchymal stem cells NAM: Non-contact aqueous-phase multilayer NF T: Normal forward transfection NGF : Nerve growth factor N /P : Nitrogen/Phosphate XXV PEM: Polyelectrolyte multilayer PDAC: Poly(diallyldimethylammonium chloride) PDMS: Polydimethylsiloxane PAA: Poly(acrylic acid) PAH: Poly(allylamine hydrochloride) PEG: Poly(ethylene glycol) qRT-PCR: Quantitative reverse transcriptase-polymerase chain reaction RCL: Reactive cell layer RNA: Ribonucleic acid RNAi: RNA interference RP-HPLC: Reverse phase high pressure liquid chromatography siRNA: small interfering RNA SPS: Sulfonated poly(styrene), sodium salt SSC-PEM: Self-standing composite polyelectrolyte multilayer TRITC: Tetramethyl isothiocynate UV/vis: Ultraviolet/visible (O-potential: Zeta-potential 3-D: Three—dimensional xxvi CHAPTER 1 INTRODUCTION 1.1 OVERVIEW AND SIGNIFICANCE OF PROBLEM 1.1.1 Tissue Engineering and Drug Delivery Certain tissues in mammalian body, such as damaged nerves in central or peripheral nervous system (CNS or PNS), lack the regenerative abilities without the support of an external biological construct. Tissue engineering brings in the concept where such damaged tissue sections can be regenerated successfully upon the implantation of biocompatible matrices designed specifically in labs. Tissue engineering, as introduced and defined by Langer and Vacanti, is “an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function“. Choice of the suitable materials for tissue engineered constructs and their appropriate designs to regenerate the natural in viva three dimensional (3-D) cellular architectures; drug delivery at the controlled rate from the tissue engineered constructs; and optimization of the surface characteristics to achieve a controlled or desired level of cell adhesion under physiological conditions, are among the major challenges in the field of tissue engineering. Tissue engineering approaches to the repairs in central nervous system are attractive because they allow for the strategies that can promote native organization of axons after spinal cord injury, failing to which can result in a permanent functional loss and painful consequences for injured patients. Axons of the adult central nervous system exhibit an extremely limited ability to regenerate after spinal cord injuryz. While axonal regeneration can be induced experimentally, the resulting patterns of axon growth are typically disorganized and randomly oriented without the supportive enviommentz‘ 3. Support of linear axonal grth into spinal cord lesion sites has been demonstrated using tissue engineered biological constructs fabricated with array of channels along the length of scaffold (i.e. bridges or templated scaffolds) to guide nerve fibers along the scaffold, thus mimicking the nerve architecturez' 4'9. Different materials such as poly(l-lactic acid) (PLL)6, poly(lactic-co-glycolic) (PLGA)4' 9, Schwann-cell-seeded Matrigel10 (a commercial preparation of basal lamina components), alginate-based scaffoldss, laminin- fibronectin coated collagen tubes”, PEG hydrogels6, agarosez’ 7’ 8 have been employed in preparing the templated scaffolds. Depending on the material properties, different biomaterials show their own advantages or disadvantages for the nerve tissue recovery. Materials selected for the bridge or templated scaffolds should offer ease in processing techniques to generate linearly channeled structures of optimum dimensions, as channel dimensions and intrinsic porosity of the scaffolds influence the extent of axonal growth and affect the glial cell’s (non-neuronal cells, supportive or non-supportive for regeneration) infiltration at the site of injurf. Other important factors for material selection are that the materials should be biocompatible, match with the mechanical properties and integrate well with the host tissue, be able to provide sustained release of drugs (growth factors, plasmid DNA, small interfering RNA) for extended times at the implant site, and not trigger the inflammatory responses post-implantation. Recently, agarose hydrogels have been shown as a suitable material as nerve guidance scaffolds because they can be designed to match the mechanical properties of the spinal cord, are biocompatible and bioinert, and more importantly, they are stable for the extended period that is required for regenerating organized axonsz. Rapidly degrading scaffolds may fail to maintain adequate orientation for regenerating axonslz’ 13 . Support of linear axonal growth into spinal cord lesion sites has been demonstrated using arrays of uniaxial channels, templated with agarose hydrogelz. Neurotrophins such as nerve growth factor (N GF ), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (N T-3) are known to promote the neuron outgrowth and axonal regenerationz’ 6' 7. In-spite-of the templated agarose shown being a promising material for nerve repair“ 7’ 8, fabricated agarose scaffolds cannot adsorb protein or growth factors readily and can only provide short-term growth factor release at the site of injury”. To overcome this limitation, genetically engineered cells that secrete BDNFZ, a growth factor that enhances the axonal growth, were immobilized within templated agarose hydrogel and improved axonal regeneration was shownz. However, immobilizing neurotrophic factors secreting cells within a scaffold is relatively cumbersome, and raises the possibility of immune response if non-autologous cells are used. Therefore, alternative strategies are needed to provide sustained release of growth factors, such as BDNF, from templated agarose scaffolds. Existing methods of loading the drug 15-21 or protein into hydrogels through entanglements or physical interactions with the drugs16’22'26, cannot provide sustained release from templated agarose hydrogels. There is a need for a method to provide sustained drug release over a number of days from an agarose hydrogel, whereby the drug is loaded separately from the fabrication of the agarose. Further, micro-environment of the scaffold is infiltrated with glial cells such as macrophages, microglia, astrocytes, which are non-permissive for axonal regenerationé. Although the permissive or non-pennissive role of leptomeningeal fibroblasts as a glial cell is not clear in axonal regeneration, these cells appear to limit the ability of axons to regenerate by reducing the available space in channel and blocking the distal end of scaffold27. Strategies are needed which can control the undesired grth of such fibroblasts forming reactive cell layer. Controlled release of an anti-mitotic agent at low concentrations from templated agarose scaffolds is one possibility to address this challenge. Organs and tissues generally exhibit three dimensional (3-D) cellular arrangements in- vivo. Thus, there is a requirement to generate more and more tissue sections that exhibit layered cellular structures to gain similarity with in vivo tissues and organs”. Tuning of cell adhesion on a surface and subsequent polymeric deposition on the surface of cells has been shown as one possible method to create 3-D cellular architectureszg'”. However, there is a need to innovate the new methods for modifying the cell surface without compromising the health state of the cell; and also the surface characteristics further needs to be optimized to achieve a controlled or desired level of cell adhesion under physiological conditions. 1.1.2 Nucleic Acid based Drug Delivery — Gene Therapy Challenges in tissue engineering can also be addressed using gene therapy approaches. For prolonged delivery of growth factors in spinal cord repair, an altemative way could be the delivery of a nucleic acid, such as plasmid DNA (genetic modification of cells for protein over expression), to the mammalian cells at the site of injury so that cells can over express the growth factor of interest. Further, there are certain growth factors in the body that are known to be associated with stimulation of inhibitory components of glial scar (cellular environment of macrophages, microglia, astrocytes) which inhibit the axonal growth and nerve repair". Therefore, another possible method to address the nerve repair problem could be to silence the genes which make those growth factors. Small interfering RNAs (siRNAs)3 1’ 32, which are 19-22 nucleotide dsRNA molecules, serves as one of the powerful therapeutic drug agent”, 34 in gene therapy which knocks down the gene function via a sequence-specific post-transcriptional gene silencing process called RNA interference (RNAi)35, and thus inhibits the production of malfunctioning protein. Therefore, once the axonal growth inhibiting growth factors are identified, RNAi based gene therapy can be a promising method to inhibit the production of such factors to promote nerve repair. The main challenge to deliver a nucleic acid to inside the mammalian cells (transfection process) is the selection of an optimized drug delivery carrier (i.e. transfection reagent or vector). Transfection reagents or vectors are required because of the poor interaction of negatively charged nucleic acids with cells and their inefficient cellular uptake in their native form. A non-viral vector (henceforth referred as vector in this study) is usually a lipid, a positively charged polymer or a combination thereof; so that it can form complex with the negatively charged DNA or siRNA, interact with the negative cell membrane and thus initiate the cellular uptake process35'37. Polyelectrolyte complex mixtures of nucleic acid and transfection reagent (vector) are prepared and then exposed to cells for the uptake of these complexes. Cellular uptake follow the process where these complex 7 35'3 , and mixtures are engulfed by the cells (usually by a process termed endocytosis) then nucleic acid is released from the vector inside the cells to perform it’s firnction, i.e. plasmid DNA to over-express a protein of interest or siRNA to inhibit the production of undesired protein from a malfunctioning gene. However, there are various challenges starting from the selection of an appropriate transfection reagent, selection of optimal concentrations of reagent and nucleic acid, mixing time and order of mixing of polyelectrolyte complex mixtures, the time of complex mixture exposure to cells and the cell culture conditions (like cell confluency, forward or reverse transfection, etc). Gene therapy success depends on the choice of vectors that allow efficient transfection with minimal cytotoxicityss. Providing an efficient transfection reagent (high transfection efficiency and low cytotoxicity) is one of the major challenges in the field of gene therapy. In this study, linear polyethylenimine (LPEI) was selected as the transfection reagent and optimized for its improved performance for siRNA transfection. 1.2 BACKGROUND: METHODOLOGIES FOR DRUG DELIVERY 1.2.1 Layer-by-Layer (LbL) Assembled Multilayers as Drug Delivery Carriers The LbL assembly method, introduced by Decher and co-workers”' 40 in early 90’s, is based on the sequential assembly of oppositely charged ionic species held together in the alternate fashion, by the electrostatic forces of attraction to give nano-scale polyelectrolyte multilayer (PEM) structures. Polymer layers are deposited via alternate substrate dipping process, where charge over-compensation takes place at each dipping step and the charge reversal takes place at each subsequent step”. LbL technique is not limited only to fabricate PEMs, but has been extended to yield other nano—scale multilayer structures involving various polymeric species, such as proteins, DNA, RNA, viruses, and various drugs; held together by the virtue of other secondary forces of attraction, such as hydrogen bonding (H-bonding), hydrophobic interaction, or Vander Waals force. This deposition technique is largely independent of the nature, size and topology of the substrate. Spray deposition of polyelectrolytes is another technique employed for the fast fabrication of PEMS41’42. LbL thin film formation is an attractive approach for the controlled release of biomolecules from the surfaces43' 44 . LbL thin films provide flexibility in terms of their choice of substrate and constituent components, fabrication conditions, tunable structural properties and surface patterning techniques”. Other advantages include their case of preparation and cost-effectiveness. LbL multilayers can be tuned to incorporate varying amounts of drugs or proteins as well as provide sustained release under specific conditions of pH, salt or temperature‘m’s. Multiple drugs can be delivered from a single LbL multilayer system. Use of LbL films increases the range for the size of complex drug molecules that can be incorporated and released into the body. In this study, we show LbL multilayers as a method to incorporate and tune the release of protein under physiological conditions, from the agarose hydrogels in a concentration and time dependent fashion to address the aforementioned problems in nerve repair tissue engineering”. 1.2.2 Selection of a Transfection Carrier and Polymeric Nanoparticles for Drug Delivery in Gene Therapy Among the polymeric vectors, PEI is the gold standard for DNA delivery, but its high transfection efficiency is often associated with high cytotoxicity46' 47. The use of PEI”57 as a polymeric DNA transfection reagent has been studied extensively, with PEI-DNA complexes analyzed for a broad range of compositions (nitrogen/phosphate (N/P) ratios)48’5°’ 54'56‘ 58. DNA and siRNA posses distinct characteristics33’ 51' 59 with respect to their optimal delivery formulation with transfection reagents”, and thus their delivery vehicles and transfection conditions must be designed and optimized to cater to their individual requirements for efficient delivery. A less toxic form of PEI i.e. linear PEI (LPEI) has been shown as a good in vitro transfection reagent for DNA”, but has been reported not to provide in vitro siRNA transfection”: Thus, there is a need for a less toxic form of PEI to be evaluated for siRNA silencing. In this study, 25kDa LPEI was selected non-viral siRNA delivery vector and transfection process was evaluated for a suitable range of LPEI-siRN A formulations. Polyelectrolyte complex mixtures of nucleic acid and transfection reagent (vector) gives nan0particles of different sizes, charge, composition; depending on the choice of transfection reagent and nucleic acid i.e. DNA or siRNA, their relative concentrations, mixing ratio, time and order of mixing. All of these parameters affect the efficiency of transfection; where smaller sizes, higher charge and complete encapsulation of nucleic acid by transfection reagent provide improved transfection performances”. Therefore, gene therapy success depends on the optimal formulation of the vector—nucleic acid nanoparticles. In this study, LPEI-siRNA nanoparticles were optimized for their improved transfection performance. In addition to size, the other properties of nanoparticles such as their shape60 also plays an important role for efficient cellular uptake“; but the effect of nanoparticle shape has not been investigated for the nucleic acid (DNA/siRNA) delivery. One of the limitations in particle shape investigations of nanoparticles is the lack of commonly applicable fabrication techniques for creating particles of different shapes and uniform sizes. Therefore, there is a need for the techniques to fabricate nanoparticles of novel shapes to investigate the particle shape effect for a more efficient siRNA delivery. In this study, effort was made to fabricate LPEI particles of novel shapes, so that they can be complexed with siRNA and evaluated for improved siRNA delivery. 1.2.3 Microcontact Printing (pCP) for Patterned Drug Delivery Space controlled drug delivery can help in the formation of organized tissue formation by generating space controlled gene expression patterns in a tissue. Gene therapy approaches can be used to manipulate the location of transfected cells giving spatially controlled gene expression patterns via surface-mediated gene delivery62’ 63 . However, gene delivery from a surface depends on the properties of vector—nucleic acid complex that is bound to the surface. Strategies are needed to deliver nucleic acid from the surface in patterns, avoiding the issues of vector-nucleic acid complex and surface interactions, and thus providing the space controlled drug delivery. Different patterning techniques can be employed to conjugate biomolecules, such as nucleic acids, to multilayer structures. Soft-lithographic microcontact printing (uCP)64' 65 is one such technique, which has emerged as a platform of choice for biochips and drug delivery applications“. Microcontact printing (uCP) is an easy and efficient way to produce micro-pattems of polymers on a substrate giving multiple fimctionalities on the surface. This approach was developed by Whitesides and co-workers, and was first employed for formation of self assembled monolayer (SAM) patterns of alkanethiol over gold as the substrate“. Recently, this approach has been extended to incorporate broad range of substrates and stamping “inks” containing biological and non-biological polymer chains. Various inks, including, proteins, DNA, RNA, and polyelectrolytes have been used in uCP to pattern surfaces without the need for dust-free environments and harsh chemical treatments“. The elastomeric stamp made from polydimethylsiloxane (PDMS) is generally used in the Stamping process and it transfers the soaked ink to the substrate upon contact printing. There could be different methods to coat ink on the PDMS stamp, few of which are as: (i) Spin-ink method, (ii) cotton-swab method, and (iii) dip-ink method”. Quality of resulting patterns onto substrate depends on concentration of ink used, procedure of inking the 10 stamp, and surface chemistry of the substrate"). Contact printing time and pressure applied during printing are also among the major factors which influence the efficiency of uCP. The transfer efficiency of the whole process largely depends on the relative strength of the interaction between stamp coated polymer and the substrate”. uCP has significant advantages over conventional lithographic techniques, such as no limitations for curved and non-planar surfaces. Further, it has no significant requirement for the dust free environment. This technique is advantageous because it is simple and easy to use even without complicated tools and facilities. Overall, this is a versatile method for making patterns over a surface, with controlled molecular chemistry”. In this study, we Show a method to deliver patterns of siRNA, where pH responsive LbL assembled multilayers were used as the delivery platform and uCP was used to pattern nanoparticles of transfection reagent-siRNA complexes onto degradable multilayers”. 1.3 THESIS OUTLINE This thesis focuses on the applications of hydrogen bonded (H-bonded) LbL multilayers for drug delivery from agarose hydrogels in nerve repair tissue engineering applications. Another aspect of this thesis focuses on the fabrication and characterization of polymeric nanoparticles for the drug delivery to mammalian cells as a gene therapy approach to tissue engineering. At last, this thesis describe about the engineering of PEMs (fabrication of self-standing multilayers, and PEM stamping in non-contact mode) to create 3-D tissue engineered constructs. Chapter 2 describes an LbL multilayer based ll technique for the controlled release of protein from agarose hydrogels which can eventually be extended to provide controlled release of BDNF growth factor for nerve repair tissue engineering. Chapter 3 describes the controlled release of an anti-mitotic agent from agarose hydrogels to address the problem of fibroblast reactive cell layer formation observed in templated scaffold based nerve repair tissue engineering. Chapter 4 describes the selection and optimization of LPEI as a transfection reagent for siRNA delivery, and also describes the surface-mediated patterned delivery of siRNA to cells using LbL multilayers and uCP. Chapter 5 describes the fabrication of novel shaped LPEI polymer micro-particles under high shear rate mixing conditions. Chapter 6 describes the cell adhesion response on ultrathin nanometer scale PEMs as a function of varying film thickness. Chapter 7 describes the PEM stamping on a substrate under aqueous conditions in non-contact mode. Chapter 8 describes the fabrication of thin self standing composite-polyelectrolyte multilayers and their application in creating 3-D cellular cO-culture. Finally, Chapter 9 concludes the thesis and suggests some future work. 12 CHAPTER 2 TIME CONTROLLED PROTEIN RELEASE FROM LAYER-BY- LAYER ASSEMBLED MULTILAYER F UN CTIONALIZED AGAROSE HYDROGELS 2.1 INTRODUCTION Experimentally-induced axonal regeneration in the central nervous system (CNS) after spinal cord injuryz’ 3 is often disorganized and random, lacking the organization of long linear tracts that normally project through the intact nervous systemz. To support linear axonal growth into spinal cord lesion sites, arrays of uniaxial channels of uniform diameter, wall thickness and physical texture similar to normal spinal cord have been patterned with agarose hydrogelz. These templated nerve guidance agarose scaffolds were shown to exhibit excellent integration with host tissue. Growth-promoting neurotrophic factors have been shown to promote axonal regeneration into the sites of spinal cord injury3'71'73. Indeed, loading scaffolds with genetically engineered cells that secrete brain- derived neurotrophic factor (BDNF) were shown to significantly promote linear penetrating axons through their channels in vivo for spinal cord injuryz. However, immobilizing neurotrOphic factor secreting cells within a scaffold is cumbersome, and raises the possibility of immune response if non-autologous cells are used. The objective of this study was to provide an alternative strategy to deliver sustained release of growth factors or proteins to enhance axonal regeneration from templated agarose scaffolds. Ag arose hydrogels were chosen as nerve guidance scaffolds because they can be designed to match the mechanical properties of the spinal cord, are biocompatible and bioinert, and more importantly, they are stable for the extended period that is required for 13 2. Rapidly degrading scaffolds may fail to maintain regenerating organized axons adequate orientation for regenerating axonslz’ 13. Agarose is a weakly ionic hydrogel” 75 and the pore morphology and porosity are both strongly dependent on agarose concentration” 77. Agarose hydrogels can have a broad range of pore diameters, of less than lnm up to greater than 500nm74'79. The absence of strong interactions between the proteins and agarose and the relatively large average pore size of the agarose as compared to the size of the protein (e.g. the size Of lysozyme is ~ 3 x 3 x 4.5m”), preclude direct impregnation (soaking) of proteins into the agarose hydrogels for sustained release over long periods (see Figure 2.9). This is in contrast to other hydrogels that can achieve sustained release through entanglements or physical interactions with the drugs“’ 22'26. Current methods of incorporating drugs into hydrogel structures during the fabrication process”19 are not compatible with the templated agarose scaffold fabrication processz. The latter exposes proteins to harsh organic solvents that are used to selectively etch patterning constituents during the templated scaffold fabrication process, which can denature the proteins. Therefore, a strategy is needed that can load the drug apart from the scaffold fabrication. Layer-by-layer (LbL) assembled multilayers, introduced by Decher”, offer promise in the field of controlled drug delivery, due to their tunable film properties, flexibility in choice of assembly components and ease of processing“ 44. LbL multilayers can be tuned to incorporate varying amounts of drugs or proteins as well as provide sustained release under specific conditions of pH, salt or temperature‘m’s. Initial strategies that used the LbL multilayers to control drug release were based on non-degradable polyelectrolyte l4 multilayer capsule formation technology” 8" 82. However these capsules trigger drug release at non-physiological conditions of pH or ionic concentrations45 and thereby restrict their in viva applications. Loading the drug either before or after fabrication of non-degradable multilayers and their subsequent mechanism of release depends on the permeability properties of the film, degree of film swelling, interaction of drug molecules with the polyelectrolytes, i.e. the charge and size of the drug molecule, and the type of multilayer assembly. Furthermore, it is limited to small (<5kDa) molecules that can diffuse under physiological conditions45' 83‘” , thus restricting the type of drug that can be used with non-degradable LbL assemblies for controlled release applications. The LbL methodology has also been shown to provide controlled drug or protein release from synthetic hydrogels; however, these processes again loaded the drug during the hydrogel 20, 21 fabrication process which is not an option with the fabrication process of templated agarose scaffolds that involves harsh organic solvents. In contrast, degradable multilayer assemblies, based on sequential embedding of drugs during the fabrication, can incorporate any drug independent of the molecular weight of the drug90’94. Fabrication of hydrogen bond (H-bond)-based LbL multilayer films was initially reported by Rubner95 and Zhang96. Subsequently, Sukhishvili and Granick demonstrated the pH controlled assembly and degradation of poly(carboxylic acid)-based H-bonded LbL films97’ 98, which was followed by numerous studies involving these multilayers for different applications, e.g. to generate self-standing floating films, solid 38, 41, 99 polymer electrolyte films, or for patterned delivery of nucleic acids to cells Degradable H-bonded films have been used for sustained release of charged compounds, 15 albeit limited to a few hours of controlled releaseloo. However, a recent study demonstrated prolonged drug release up to a couple of weeks using a carboxylic acid- based cross-linked H-bonded LbL assembly with a hydrophobic drug contained in amphiphilic block copolymer micellesgo. Further, formation of degradable101 or non- degradablezo' 45 multilayers over protein impregnated agarose is also not a feasible option since the protein is not easily retained within the agarose gel. The protein leaches from the agarose during the multilayer fabrication, making it difficult to control the amount of protein loaded, and likely results in minimal, if any, protein encapsulation. Here, we present a simple approach for controlled delivery of proteins from agarose gels, where the proteins are incorporated within the degradable LbL multilayer coatings formed over the agarose. Carboxylic acid (-COOH)-based weak polyelectrolytes form H- bond interactions at low pH (e.g., pH < 3.5 in the case of poly(acrylic acid (PAA)) and deprotonates to carboxylate ions (-COO') at high pH, which degrades the H-bonded multilayer assembly”. H-bonded PAA/poly(ethylene oxide) (PEO) multilayer films when built on a planar substrate are known to degrade in about 30 min upon exposure to a pH of 3.5 or higher”. However, here we show that the H—bonded films when prepared over agarose as the substrate provided sustained release of the incorporated protein under physiological conditions for a period of more than four weeks. Since nervous system growth factors such as BDNF are rather expensive, a more practical analog, lysozyme, was evaluated because of its similarity in size and isoelectric point to BDNFm. Multilayers were formed with either a three component assembly of poly(ethylene glycol) (PEG), PAA and protein, or a two component biocompatible assembly of PEG l6 and protein, under acidic conditions (pH _<_ 3.0). The protein was loaded subsequent to the agarose fabrication rather than pre-loaded directly into the agarose hydrogel, avoiding the caustic conditions used in the templated agarose scaffold fabrication. It was determined that there is a close relationship between the weight percent of agarose hydrogel and the amount of released protein. This relationship is believed to be a result of increasing total surface area per unit volume of agarose hydrogel. A variety of drugs or proteins of varying nature, size, and amounts can be explored for controlled release using this LbL approach without the concern or constraints that may be imposed by potential interactions between the drug and hydrogel. Moreover, this approach does not require any specific chemical alterations to the LbL forming polymers, such as copolymerization or covalent bonding. 2.2 MATERIALS AND METHODS 2.2.1 Materials Ultrapure agarose (Sigrna-Aldrich, USA) was dissolved at a required concentration in 18.2 MQ-cm resistivity deionized (DI) water GVIilliQ, Millipore) at near 100°C. For preparing gels for Brunauer-Emmer-Teller (BET) analysis, the solutions were heated in a closed vial. Templated agarose scaffolds were prepared by a phase inversion process using polymer fiber templates supplied by Paradigm Optics Inc. (Vancouver, WA) as described previouslyz. For all the protein release experiments, 3ml of hot agarose solution was poured into 12-well tissue culture polystyrene (TCPS) plates (Costar, Corning, NY). For fluorescence and ultraviolet/visible (UV /vis) absorbance experiments, the hot agarose solution was filled into a 96-well polystyrene plate (Evergreen Scientific, USA). Agarose l7 was allowed to gel at room temperature. Poly(acrylic acid, sodium salt) solution (PAA, Mw 15,000; catalog no. 416037), poly(ethylene glycol) (PEG, Mw 10,000; catalog no. P6667), poly(ethylene glycol) bis(3-aminopropyl) terminated (PEG-Amine) (Mw 1,500; catalog no. 452572), lysozyme (Lyso) from chicken egg white (catalog no. L6876), tetramethylrhodamine isothiocynate (TRITC) (catalog no. 87918) were purchased from Sigma-Aldrich. Cytotoxicity detection kit for LDH release measurements was purchased from Roche Applied Science, Indianapolis, IN. 2.2.2 LbL Formation on Agarose Lysozyme protein was used for all the protein release experiments, whereas bovine serum albumin (BSA) protein (US Biological, MA) was used for all the LbL growth characterizations (fluorescence, UV/vis absorbance and confocal microscopy). PAA and PEG polymer solutions used to fabricate multilayer assemblies were prepared in DI water to final concentrations of 1 mg/ml, and their pH was adjusted to 2.0. Protein (lysozyme or BSA) was dissolved in 1X phosphate buffer saline (PBS) (Invitrogen, USA) at a concentration of lmg/ml and solution pH was adjusted to 3.0. Different numbers of bilayers of PAA, PEG and protein were prepared as shown in Figure 2.1(a-f) and as explained in the Results and Discussion sections. DI water adjusted to pH 2.0 was used as the wash solution during assembly fabrication. Agarose filled polystyrene plates were immersed in LbL starting polymer (1mg/ml 25kDa branched polyethylenimine (BPEI) (Sigma-Alrich) at pH 10.5; or 1mg/ml 25kDa linear polyethylenimine (LPEI) (Polysciences Inc.) at pH 7.2-7.4; or protein in PBS at pH 3.0) for 30 min and then rinsed in wash solution for 10 min with agitation. The substrates were then dipped into PAA or 18 PEG solution (depending on multilayer assembly, as shown in Figure 2.1(a-f)) for 30 min followed by 10 min in wash solution with agitation to create one bilayer. The dipping cycle was repeated to form multilayer films. A Carl Zeiss slide stainer was used to form multilayers. Multilayers were fabricated in different bilayer arrangements as shown in Figure 2.1(a-f). PAA was kept as the terminating layer in each case. In some cases, an additional set of five bilayers of (PAA/PEG) was added before terminating the assembly with PAA, however these additional layers was found to have no significant effect on the protein release profiles. To characterize the LbL adsorption of PEG, PEG-Amine was conjugated to the TRITC dye for fluorescence measurements in 96-well plate. 300 mg of PEG-Amine was dissolved in 300ml of IX PBS buffer (pH adjusted to ~90) and ~15mg of TRITC dye (dissolved in DMSO) was added. The mixture was stirred continuously at 4°C for 24hrs and the mixture pH was subsequently reduced to 2.0. Since the molar ratio of dye to PEG-Amine was much less than one, no dialysis or column purification was performed. The mixture was directly used to form multilayers with the BSA protein (pH 3.0) using the slide stainer with extensive intermediate washes (pH 2.0) during LbL. To study the effect of the molecular weight of the polymer on the multilayer degradation behavior, 25 bilayers of PAA/PEG multilayers were prepared onto clean gold substrates, which were coated with mercaptoundecanoic acid (Sigma-Aldrich) followed by BPEI and PAA/PEG deposition. Films, when abbreviated as n.5, denote the PAA/PEG multilayer where n is the number of PAA and PEG bilayers and the “.5” indicates an additional, single terminating layer of PAA. For the fibroblast cytophobicity tests, PAA/PEG multilayers were prepared on 02 plasma-treated (Harrick Plasma Cleaner) and LPEI- coated TCPS plates. NIH-3T3 fibroblasts were purchased from American Type Culture 19 Collection (Rockville, MD), and all cell cultures were maintained in DMEM with 10% FBS and 100 U/ml penicillin plus lOOug/ml streptomycin (P/S) at 37°C and 5% C02. 2.2.3 Characterizations Emission spectra of TRITC in 96-well plate were acquired using a SPECTRA-MAX GEMINI-EM fluorescence plate reader (Molecular Devices, CA) at an excitation wavelength of 529nm with bottom-read option. UV/vis peaks in 96-well plate were measured at 25°C at 280nm using SPECTRAmax Plus 384 (Molecular Devices). Confocal laser scanning microscopy (CLSM) images of the scaffolds were Obtained using Zeiss Pascal laser scanning confocal microscope. Excitation wavelength used for TRITC dye for confocal microscopy was 543nm, and emission spectrum was obtained at wavelengths greater than 560nm using a long pass filter. Gain and offset settings were kept the same during sample and control imaging. Phase contrast optical microscopy images were collected using Leica DM IL inverted microscope (Bannockbum, IL) equipped with SPOT RT color camera (Diagnostics Instruments, MI). For scanning electron microscopy (SEM) imaging of the scaffolds, LbL-coated and non- coated scaffolds were dehydrated through a series of 30 min immersions in ethanol solutions of 25%, 50%, 75%, 95% and 100% concentration; supercritically dried using C02 (Balzers CPD, Lichtenstein) followed by a 7-10nm gold sputter coating (EMSCOPE SCSOO Sputter coater, Ashford, Great Britain). SEM images were obtained with field emission JEOL 6400 electron microscope. The series of dehydration before SEM imaging likely caused the defects in the coatings, as observed in Figure 2.3c. In order to 20 visualize the coating formation on the agarose surface, particular areas of defects were selectively identified for imaging. For BET experiments, hydrogels were dehydrated using pure ethanol (at 10 times the volume of the gel) and the ethanol was replaced twice, each time immersing for 24 hrs. This was followed by C02 supercritical drying103 and stored in a desiccator. BET surface area characterization was performed with a nitrogen- adsorption testing apparatus (Micromeretics ASAP 2020), using helium to measure the free space and nitrogen to measure the surface area. Each sample was fractured into small pieces using a scalpel in order to fit into the test chamber and reduce the amount of time spent waiting for diffusion of the analysis gas. Before analysis, samples were degassed under vacuum at 90°C for eight hours followed by a nitrogen backfill, run through a short analysis to obtain the free space of the test chamber, then degassed again under the same conditions for 24 hours with no backfill prior to the actual analysis. This second degassing step allows the microporosity to be measured accurately by removing any previously adsorbed gasses. Unless otherwise specified, for all protein release experiments, LbL coated agarose in 12- well plates were incubated in lml of 1X-PBS(pH 7.4)/well at 37°C, the buffer was replaced with lml of fresh PBS after each sampling interval (to obtain the cumulative release profiles; here-in this release referred as aynamic release), and the collected buffer was stored either at -20°C (or 4°C for short term) until further analysis. Additionally, lysozyme releases were also characterized by incubating the LbL coated agarose in 1 ml of fibroblast cell culture medium at 37°C. Concentrations of the lysozyme released from the LbL coated agarose were measured by the BCA or micro-BCA protein assay (Pierce, 21 Rockford, USA) according to the manufacturer's instruction. The releases obtained at each sampling interval were added to obtain the cumulative release profiles. Lysozymal enzymatic activity was determined spectrophotometrically by measuring the degree of lysis of Micrococcus lysodeikticus bacteria (Sigma-Aldrich)’°4. Active lysozyme damages the cell wall of the bacteria and thus its activity is characterized by the decrease in the amount of live, intact bacteria in the solution. A 3mg/ml M. Iysodeikticus suspension was prepared in an assay buffer containing 50mM phosphate buffer (pH adjusted to 7.4), 0.1% sodium azide (Sigma-Aldrich) and 1mg/ml BSA. An aliquot of 200p.1 of collected sample was added to 1.8m] of assay buffer with M. Iysodeikticus at a final concentration of 300ug/m1 (1:10 dilution), and incubated at 37°C for 2 h. Change in turbidity was monitored at a wavelength of 450nm with the assay buffer as a blank. A standard curve for the lysozyme activity was obtained using pure lysozyme dissolved in 1X PBS to calculate the active lysozyme concentration released from the agarose. All data shown as mean :t SD are from at least three independent sets of experiments. 2.3 RESULTS AND DISCUSSION Agarose is a linear polysaccharide consisting of repeat units of agarobiose (1,3-linked ,B- D-galactopyranose and 1,4-linked 3,6-anhydro-oI-L-galactopyranose)74‘ 75. Upon cooling the hot solution of agarose in water, it forms a physically cross-linked double-helix 3-D gel network of polymer chains interconnected by H-bonding and hydrophobic interactions” 75. We capitalized upon the H-bonding and hydrophobic interactions in building the LbL assemblies onto the agarose scaffold. Polymers, such as polyamines and proteins, can provide varying degree of H-bonding and/or hydrophobic interactions with 22 agarose and their varying degree of bonding with agarose can affect the degradation kinetics of the multilayer assembly. Here, branched poly(ethylenimine) (BPEI), linear poly(ethylenimine) (LPEI), and protein were used as the LbL initiating polymers and examined for their affect on the protein release profiles from multilayer-coated agarose. LbL initiated with BPEI, LPEI or protein was followed by formation of H-bond multilayer films with the protein as one of the multilayer constituents. Two component- and three component-multilayer assemblies consisting of PAA, PEG and protein were formed, as shown in Figure 2.1(a-f). The loading of the polymers and the layered structure formation during LbL assembly over agarose was characterized with fluorescence measurements, UV/vis spectroscopy, confocal microscopy, and SEM. The protein release profiles were evaluated for different LbL compositions, varying agarose porosity, initiating LbL polymer, and the number of components in the assembly. 23 Figure 2.1 Diagram showing the three- and two-components LbL assembly fabricated onto native agarose as the substrate. Templated agarose scaffolds (as shown in this Figure) were used to characterize film growth, and agarose-filled TCPS plates were used to characterize protein releases. Three component assemblies consisted of PAA, PEG and protein as the multilayer constituents, and two component assemblies consisted of PEG or PAA and protein as the multilayer constituents. BPEI, LPEI, or protein (lysozyme, denoted as Lyso) was used as the LbL initiating polymer in the different cases shown in a-f. Curved lines in a-f represent the agarose, and BL indicates the bilayers. Templated Agarose Scaffold —> AA/PEG AA/PEG pH 5 3.0 AA/PEG Lyso[5,5]3 T c d I PAA PAA BPEI[ 513/ I .513 ‘—" AA/PEG (PAA/PEGlisel. (Protein/PEGliset AA/PEG Lyso[15,15] Lyso[10,10,5,5] e i f i (Protein/PEGllsBL (Protein/PAAlisat ILYSOIPEGII5 [Lyso/PAA]15 2.3.1 Multilayer Growth on Agarose Substrate In order to assess the LbL growth of the polymers on agarose, we measured the fluorescence intensity of a LbL polymer component (PEG conjugated with a dye) as the number of bilayers increased. PEG terminated with primary amines at both ends (PEG- Amine) was conjugated to the TRITC dye, which was used to form the LbL assembly with the bovine serum albumin (BSA) protein on agarose. Figure 2.2a shows the 24 adsorption of PEG during intermediate steps of LbL formation on the agarose. The measured fluorescence intensity of the emission spectrum of TRITC continuously increased as more layers of PEG-Amine-TRITC were adsorbed onto the agarose during the film formation (Figure 2.2a). Further, in order to Show the adsorption of the second LbL component, BSA, we measured the absorbance of the protein at 280nm as the number of bilayers increased (Figure 2.2b). The measured absorbance of the BSA protein continuously increased as more layers of BSA were adsorbed onto the agarose. Therefore, Figure 2.23 along with Figure 2.2b show the increasing deposition of both PEG and protein, respectively, during intermediate steps of the LbL assembly onto agarose. Correspondingly, a continuous decrease in the protein concentration of the loading bulk solution (solution from which the multilayers were formed) was observed (Figure 2.20). There was no decrease in the protein concentration in the bulk solution for the case where the agarose was soaked in the protein solution without LbL assembly (see Figure 2.9), as discussed below. However, these measurements do not provide information on the multilayered structure deposited onto the agarose by the LbL process. 25 Figure 2.2 (a) Fluorescence intensity measurements of TRITC conjugated to amine terminated PEG (PEG-Amine-TRITC) showed increased adsorption of PEG into the agarose structure during the LbL deposition of BSA/(PEG-Amine-TRITC) multilayers on agarose. (b) UV/vis absorbance measurements at 280nm showed increased adsorption of bovine serum albumin (BSA) protein onto the agarose structure during the LbL deposition of BSA/(PEG) multilayers. The absorbance values are shown with respect to (w.r.t.) the absorbance of bare agarose i.e. the difference between the absorbance of LbL coated agarose and the absorbance of bare (non-coated) agarose. Five precursor bilayers of (PAA/PEG) with LPEI as the LbL initiating polymer were built over agarose in each case. BLs denote the number of bilayers (c) Corresponding decrease in lysozyme concentration in the bulk solution from the initial concentration as a function of the number of bilayers. (a) 10000 _ .F—a— 5.5 BLs I+ 10.5 BLs l+ 15.5 BLs l—x—20.5 BLs a: on O O O O O O I I Fluorescence intensity (arbitrary units) 560 570 580 590 600 610 620 630 640 650 Wavelength (nm) 26 Figure 2.2 continued 0)) 0.70 a 999 888 0.30 ~ ”I 0.20 - 0.10 ~ 0.00 l I T? I F fl f j SBLs 7BLs 1OBLs 153Ls ZOBLs 3OBLS 4OBLS SOBLs Number of (BSA/PEG) bilayers A (Absorbance w.r.t Bare Agarose) (C) w.r.t lnltlal conc. (mg/ml) P P P P P P 8 3 3 ‘6’ 8 8 A (Decrease) In stock BSA cone. f if T I T I 7BLs 1OBLs 158Ls 208Ls 308Ls 4OBLs 5OBLS Stock BSA solution collected after indicated nurrber of bilayers Next, we characterized the thin film formation of 30 bilayers of PAA/PEG onto 3 wt% templated agarose scaffolds composed of uniaxial channels2 (as shown in Figure 2.1). 27 The nominal film thickness of 30 bilayers of PAA/PEG multilayer film (PAA/PEG)30 is about 600nm41 (discussed further in section 2.3.3), and the average pore diameter of 3 wt% agarose was determined to be about 20nm (discussed further in section 2.3.2.1). It is expected that during the initial phase of multilayer growth the film formation would occur in the intrinsic micropores of the agarose scaffold. As subsequent layers are deposited, larger and larger pores will fill in to the point where the majority of the intrinsic porosity (all pores small to large) is filled and the films eventually deposit on the superficial surface of the agarose hydrogel scaffolds. To test this hypothesis, 30.5 bilayers of PAA/PEG and subsequently three additional bilayers of PEG and TRITC conjugated BSA, i.e. ((PAA/PEG)3o-(TRITC-BSA/PEG)3), were built onto the templated scaffolds. A confocal image section captured below the top surface of the ((PAA/PEG)30- (TRITC-BSA/PEG)3) coated scaffold showed a clear fluorescent ring formed from the (TRITC-BSA/PEG)3 deposited after (PAA/PEG)”, along the inner periphery of the scaffold channels (Figure 2.3a, left image). In another case, three bilayers of PEG and TRITC conjugated BSA, i.e. (TRITC-BSA/PEG)3, were coated onto the templated scaffolds. A confocal image section captured below the top surface of the (TRITC- BSA/PEG)3 coated scaffold showed the fluorescence in this sample was diffused throughout the agarose structure (Figure 2.3a, right image). Corresponding intensity profile (Figure 2.3a, left spectrum) for the ((PAA/PEG)3o-(TRITC-BSA/PEG)3) sample shows a sharp increase in fluorescence along the inner walls of the channels and minimal fluorescence inside the agarose structure. This suggests that the (TRITC-BSA/PEG)3 films formed on the outer surface of the agarose channel wall, contributing to the fluorescent ring formation in the presence of (PAA/PEG)30 layers. 0n the other hand, the 28 fluorescence intensity profile (Figure 2.3a, right spectrum) for the (TRITC-BSA/PEG)3 sample is more diffused and does not show the sharp edges along the channel wall. The fluorescence which concentrated along the inner periphery of the ((PAA/PEG)30-(TRITC- BSA/PEG)3) scaffold (with an average intensity of 250 units) channel was more distributed throughout the structure of the (TRITC-BSA/PEG)3 scaffold (reducing the average intensity to 100 units). The three bilayers of (TRITC-BSA/PEG)3 in the absence of the (PAA/PEG)3o layers did not form the ring, suggesting that the initial three bilayers penetrated uniformly throughout the internal pores of the agarose. Comparing these two samples suggests that during the initial phase of multilayer formation (at least up to three bilayers), the fihn formation was occurring within the intrinsic pores of the agarose. Then, at some stage during the process of multilayer formation (between three and 30 bilayers), the intrinsic pores filled in, causing subsequent layers to be formed predominantly on the outer surface of the agarose. Figure 2.3b illustrates the z-series images captured for the ((PAA/PEG)3o-(TRITC- BSA/PEG)3) sample, traversing from the top surface down the structure of the agarose channel. Corresponding intensity profiles of the topmost section (0.0um) and a middle section (211nm) are shown (Figure 2.3b). A constant ring of fluorescence along the inner wall of the channels and a decrease in the fluorescence intensity through the structure of the scaffold are observed. Therefore, in addition to the discussion of Figure 2.3a above, this constant ring of fluorescence along the inner wall of the channels further suggests coating on the outer surface of the agarose after formation of the 30 bilayers. The gradual decrease in the intensity profile along the channel of the agarose structure suggests the 29 presence of a diffusion gradient that reduces the polymer deposition within the pores of the agarose down the channel as the building of the bilayers increases on the surface of the agarose. Overall, these results suggest that the LbL assembly process resulted in formation of a thin multilayer structure on the surface of the agarose, if sufficient (~ 30) bilayers are built. Figure 2.3c shows the SEM images of an uncoated scaffold (outlined by red boxes) and scaffolds coated with 30 bilayers of PAA/PEG, the latter shows a film has formed on the agarose surface. It is important to note that although there this is clear evidence that bilayers formed on the outer surface of the scaffolds, the majority of the proteins loaded in the gel are immobilized in the agarose hydrogel intrinsic pores, as explained further in section 2.3.2.1. 3O Figure 2.3 Confocal and scanning electron microscopy images showing the formation of LbL assembled multilayer thin films on agarose substrate. (a) Confocal microscopy images and associated fluorescence intensity profiles of a section below the top surface of agarose scaffolds coated with 30 bilayers of PAA and PEG followed by three bilayers by PEG and TRITC conjugated BSA (left image), and of agarose scaffolds coated with only 3 bilayers by PEG and TRITC conjugated BSA (right image). (b) Confocal microscopy acquired z-section images of agarose scaffolds coated with 30 bilayers of PAA and PEG followed by three bilayers by PEG and TRITC conjugated BSA. F luorescencc intensity profiles of the topmost section (top panel: leftmost image) and a middle section (bottom panel: rightmost image) are shown. (c) Scanning electron microscopy images of the scaffolds LbL coated with 30 bilayers of PAA and PEG. Images in red boxes are of the non-coated scaffolds. BPEI was used as the LbL initiating polyelectrolyte in all images. (a) Intensity Intensity IT 200 l 200i 100 I 100. . , l 0 _HA. AH: , fi_ —r' ma-‘.l... ,1_ —-¥ 0 ......J ...... .a. “.-.“..- . .. .. .. 0 l 00 200 300 400 0 l 00 200 300 400 Distance (um) Distance (um) 31 Figure 2.3 continued 0)) I'IIIIIII IIIIIIIIII , .# IFI'I‘IIIII If‘l‘xlllll Illll lllll Intensity 200 0.0pm I, 100 1' A ‘ .. Distance (um) Intensity 32 III” lllll Hill Inn 211.0 llm \-l.-I Illll :1 I ll _llIll ‘._. ... A. 400 Distance (um) lllll _lllll Illll Illl‘l 800 Figure 2.3 continued 2.3.2 Sustained Protein Release Profiles from LbL Coated Agarose Scaffolds 3 wt% agarose concentration was found to be suitable for stable templated scaffold preparation and better axonal growth in repairing nerve injuriesz. Figure 2.4a shows a cumulative profile of the lysozyme released from 3 wt% agarose coated with multilayers in the configuration depicted in Figure 2.1a, with BPEI as the LbL initiating polymer. BPEI can provide a sufficient degree of H-bonding due to its multivalent amines, as well as hydrophobic interactions with agarose, and thus was selected as a LbL initiating polyelectrolyte. Multilayer assembly with BPEI as the initiating layer was followed by five bilayers of PAA/PEG, and five bilayers of lysozyme/PEG. These sets of five bilayers of PAA/PEG and lysozyme/PEG were repeated two more times, followed by PAA as the terminating layer (the multilayer assembly is denoted as BPEI[5,5]3). Sustained release was monitored over a period of four weeks, until the concentration of protein released per 33 day was within jig/ml. Daily releases are expected to continue further in the concentration range of ng/ml, which are not reported here. This amount of protein released from the agarose hydrogel was within the range required for neurite outgrowth and axonal regeneration'os, as discussed below. The effect of using other polymers, such as LPEI or lysozyme, as the LbL initiating polymers were also evaluated (discussed in section 2.3.2.2). In addition, we formed multilayer assemblies with different arrangements and combinations of PEG, PAA and protein, resulting in either three component or two component assemblies (Figure 2.1(a-f)) and analyzed their protein release profiles (discussed below). The enzymatic activity of the lysozyme released from the multilayers on the agarose was assessed by a bacterial assay (see Materials and Methods section for further details), and found to be active during the earlier time points, with decreasing activity at later time points. Figure 2.4b compares the concentration of total lysozyme vs. the concentration of active lysozyme released per day, for up to 15 days. The total as well as active lysozyme released were both in the jig/ml range for up to a week. Active lysozyme continued to be released after a week in the sub-ug/ml and subsequently in the ng/ml range. Sukhishvilli and co-workers showed lysozyme forms stable multilayers with carboxylic acid-based weak polyelectrolytes under low pH conditions due to H-bond interactions, whereas the multilayers are disrupted if formed under high pH conditions106 . They reported that it is likely after dissociation of the H-bonds under physiological conditions and release of lysozyme from the multilayers, the lysozyme (isoelectric point, p1 ~ 11) can interact electrostatically with the ionized PAA. Moreover, these interactions depend on the 34 concentrations of the salts, PAA and lysozyme present in the solutionm’ 107. Further, upon release from the multilayers, lysozyme could remain bonded with PEG through H- bonding. Although, the interaction between the released lysozyme with PAA or PEG is possible, it did not appear to have caused an adverse effect on the controlled release of the lysozyme from the agarose (Figure 2.4a). However, interactions between the released lysozyme and PAA or PEG cannot be precluded. This interaction may sequester the released lysozyme by forming inter-polyelectrolyte complexes in the solution, and thereby lowering the activity level of the released lysozyme over time (Figure 2.4b). 35 Figure 2.4 (a) Cumulative lysozyme release up to 4 weeks triggered by physiological pH, from LbL multilayer (as shown in Figure 2.1a; BPEI initiating) coated 3% agarose gel. (b) Comparison between the total and enzymatically active lysozyme released per day, corresponding to the protein released in Figure 2.4a. Active concentrations were calculated fi'om a standard curve obtained from pure lysozyme used to determine the degree of lysis of Micrococcus Iysodeikticus by lysozyme. (a) 900- @800- 3700- %600~ g soo~ 4.34004 n: 3001 %200- 355100- 0 a . fen. I . . s.T see 3 0 “~ara°~°ee=eceeseeeececane a iiiiiiiiisasarraasaarrssassr 0’) =550— «140:5 3’ 45 - lActive Release/Day (Left axis) r 120 B) 3:40“ " . . a. a. 35 _ EITotaI Release] Day (RightaXIs) _ 100 ; a 30~ +80 8 \ 25“ \ 0 g 20- ““60 § %15‘ «40 2 10i o :54 ””lIIIIllllII” .2 oi *0 g ‘5 a < swevcekecekwevc A-x-x-x-x-x-xs-XPNNNKN 0'” o” 0'" 6" 0° 0° 6" 0” o” 06‘ of on" 65" 0:5" 06“ 36 2.3.2.1 Effect of the Agarose Concentration on the Protein Release Profiles Multilayers with BPEI as the LbL initiating polymer (Figure 2.1a) was formed on different concentrations of agarose. Figure 2.5a compares the protein release profiles of 1%, 2%, and 3% agarose over five days, and Figure 2.5b compares the release profiles of 3% and 4% agarose over an extended period of 15 days. The profiles of the protein released were similar across the different agarose concentrations but the amount of proteins released increased with time and agarose concentrations. The amount of protein released is governed by the wt% of the agarose as shown in Figure 2.5. To understand the observed increase in protein release from the multilayer-coated agarose with increasing agarose concentration, we measured the total surface area per unit volume available from the intrinsic pores of the agarose. As mentioned above, it is important to note that the agarose hydrogels likely consist of a broad range of pore diameters ranging from <1nm to several hundred nanometers in diameter74'79. However, most of the surface area normalized by the superficial volume of the gels is derived from the high concentration of pores in the tens of nm range or less. Thus, it is believed that the greater the concentration of micropores, the higher the internal surface area for the films to deposit on per unit volume of agarose hydrogel. More surface area available would thus suggest more protein can be loaded into the pores of the hydrogel during LbL assembly. Here, the total surface area of the agarose (prior to multilayer formation) per unit volume of gel was measured using the Brunauer-Emmer-Teller (BET) model on 103 d supercritically drie agarose hydrogels. An increase in the weight percent of the hydrogel resulted in an increase in the BET surface area per unit volume of gel (Figure 37 2.5c). This is likely due to the creation of more gel network branches rather than increased branch thickness in agarose. Therefore, at higher concentrations of agarose, more protein would be expected to be loaded into the intrinsic pores of the agarose, resulting in higher protein release. Figure 2.5 (a,b) Cumulative lysozyme release over time triggered by physiological pH from agarose hydrogel of varying concentrations, coated with LbL multilayer assembly (as shown in Figure 2.1a; BPEI initiating). (a) Comparison between 1%, 2% and 3% agarose. (b) Comparison between 3% and 4% agarose. (c) Total surface area per unit volume of pure agarose hydrogel as a function of hydrogel concentration determined by BET. (a) §§ i N 8 200 r '3‘ .100 ° 0| 0 O l Cumulative Release (pg/ml) 38 Figure 2.5 continued (b) . 1200 - '5- +4% E 1000 ~ +3% E 800 ~ 3 600 . a? 0 400 - ..>. 5 200 - E 0 1 . 3 0 Day 1 Day 2 Day 3 (C) 7330- 3 . £25“ a20~ 2 “'15- 8 £10“ 0) 5' E O I- 0% —i ..4 -—4 Days ”an Day 5 o W-.--.____.__.__--. -.. _ , .11 —4 —4 q —< _l eseeeeeze %DDfiggA ~00 °°°oooooo ,w... _m. ”A--. ._ ,_-__ ,_.___-_T_,__ ,,__ ____ 3% 4% 5% 6% Agarose Concentration (wt%) 2.3.2.2 Effect of the Starting Polyelectrolyte on the Protein Release Profiles Three component BPEI[5,5]3 or LPEI[5,5]3 multilayer assemblies (Figure 2.1a) were built on agarose with either BPEI or LPEI as the LbL initiating polymer, respectively. To 39 avoid using non-biocompatible BPEI or LPEI, similar multilayer assemblies starting with lysozyme (Figure 2.1b) and PAA were also prepared. Figure 2.6 shows the protein release profiles with BPEI, LPEI, protein or PAA as the initiating LbL, fabricated on 3% agarose scaffolds. BPEI or LPEI as the initiating LbL was also fabricated onto 1% agarose. The profiles of the protein release were similar in all cases, however, there were significant differences in the actual amount of protein released at a given time for the different cases. The amount of protein released with BPEI as the initiating LbL was lower than with LPEI as the initiating LbL, both for the 1% and 3% agarose. Further, the amount of protein released with PAA as the initiating LbL was lower than with BPEI as the initiating LbL. In contrast, the amount of protein released from the multilayers was similar with lysozyme or LPEI as the initiating polymer. The difference in the amounts of protein released as a function of the initiating LbL polymer (i.e., BPEI, LPEI, protein or PAA) may be due to their degree of binding with the agarose. LPEI, BPEI, lysozyme (protein) and PAA, all are weak polyelectrolytes and their degree of ionization is dependent on the pH. During the LbL assembly, BPEI and LPEI were kept at a high pH, whereas lysozyme and PAA were kept at a low pH (see Materials and Methods section). BPEI contains primary, secondary, and tertiary amines and is a hydrophobic polymer, whereas LPEI which contains only secondary amines is a hydrophilic polymer. Owing to its hydrophobicity, BPEI has been shown to preferentially adsorb onto hydIOphobic regions on surfacesmg. Furthermore, due to the presence of the multivalent amines, BPEI can also form more H-bonds with the hydroxyl groups and oxygen on the agarose than LPEI. PAA is hydrophobic at low pH conditions where it is 40 in protonated form, and can form H-bonds. Therefore, PAA can also form H-bonds and hydrophobic interactions with agarose, possibly stronger than BPEI. Depending on the isoelectric point and pH, proteins also can form hydrophobic and H-bonds with agarose. The increased amount of protein released with the lysozyme initiating LbLs in comparison to BPEI as the initiating LbL suggests that at pH of 3.0, lysozyme (p1 ~ 11) binds less strongly than BPEI to the agarose surface, perhaps due to more protonation of the amine groups on lysozyme at low pH, making lysozyme less hydrophobic than BPEI. Therefore, the BPEI initiating LbL would likely bind more strongly to agarose than LPEI or lysozyme initiating LbLs. This would affect the overall degradation kinetics of the multilayer, and consequently the protein released profiles. Figure 2.6 The effect of LbL initiating polymer on lysozyme release triggered by physiological pH. BPEI, LPEI, lysozyme or PAA was used as the LbL initiating polymer as shown in Figure 2.1a and 2.1b. 500 —B—LPEI[5,5]3-3% 4 i +Lyso[5,5]3-3% _., 400 — +BPE|[5,5]3-3% ../ 350 — +PAA[5,5]3-3% 300 a +LPEI[5,5]3-1% + BPEI[5,5]3 - 1v /' Cumulative Release (pg/ml) N O! O 41 2.3.2.3 Effect of the Stacking Layer Configuration and Assembly Components on the Protein Release Profiles The previous sections were based on forming [5,513 sandwiched multilayers with five bilayers of a three component assembly (Figure 2.1a or 2.1b). This type of assembly arrangement was chosen initially because the degradation of PAA/PEG multilayers reported by Ono and Decher showed that fewer than seven bilayers of PAA/PEG did not release the upper films as self-standing, floating films; whereas more than seven bilayers allowed the upper films to be released within 30 min“. Therefore, we hypothesize that stacking of two components with greater than five bilayers at a time (e.g., Lyso[10,10,5,5] or Lyso[15,15] in Figure 2.10 or 2.1d) could degrade faster and release more protein at a given time than a five bilayer arrangement of three component assembly (Lyso[5,5]3 in Figure 2.1a or 2.1b). We formed multilayers using PAA, PEG, and protein, with two different arrangements starting with lysozyme as the LbL initiating polymer (Figure 2.1c and 2.1d). The total number of layers for each assembly was kept the same as in Figure 2.1b. Figure 2.7a shows the profiles of protein release over five days for the three different arrangements, Lyso[5,5]3 (Figure 2.1b), Lyso[10,10,5,5] (Figure 2.1c) and Lyso[15,15] (Figure 2.1d). NO significant difference was observed for the three arrangements at earlier time points, however, the release profiles diverged at later time points. The amount of cumulative protein released was less for the Lyso[5,5]3 than the Lyso[10,10,5,5] assembly and was highest for the Lyso[15,15] assembly. This suggests that a higher number of bilayers stacked together degraded faster and released more protein than the stacking assembly with fewer bilayers. This is in accordance with the previous finding by Ono and Decher41 that suggested fewer (less than seven) bilayers 42 degraded slower than more bilayers of PAA/PEG. Thus, using fewer bilayers in the stacking assembly may provide sustained release and more control of the amount of protein released over a longer period. Some studies has reported PAA as a biocompatible and bioinert polymer, however cytotoxicity has not been explicitly evaluated” 90. In addition, PAA is not a FDA approved polymer and thus may not preclude the possibility of being toxic under certain conditions. Nevertheless, PAA toxicity would depend also on the amount of PAA supplied in vitro or in viva. Therefore, we also evaluated a completely biocompatible multilayer arrangement on agarose consisting of a two component H-bonded assembly of lysozyme and PEG ([Lyso/PEG]15 in Figure 2.1e) built at low pH. For comparison, a multilayer assembly of lysozyme and PAA ([Lyso/PAA]15 in Figure 2.10 was also prepared over agarose. In both cases, the total number of layers for each component was kept the same as in the arrangements shown in Figures 2.1(a-d). Figure 2.7b shows the protein release followed similar trends as with the three component Lyso[5,5]3 (Figure 2.1a) multilayers over agarose (Figure 2.7a), but released higher amounts of protein. The amount of protein released from [Lyso/PAA]15 was the highest, followed by [Lyso/PEG]15 and than the three component assembly, Lyso[5,5]3. This difference observed could be due to a stronger H-bonding of the oxygen on PEG with the carboxylic acids and amino groups on the protein than the interactions between the carboxylic acids on PAA and the amino groups on the protein, thereby slowing the degradation of [Lyso/PEG]15 over the [Lyso/PAA]15 assembly. 43 Figure 2.7 Cumulative lysozyme release from 3% agarose gel, triggered by physiological pH, (a) with varying stacking order of the polymers within a multilayer but the same number of cumulative bilayers (as shown in Figure 2.1d, 2.1c and 2.1b), and (b) with two-component assembly of lysozyme and PAA, two-component assembly of lysozyme and PEG, and three component assembly of PAA, PEG and lysozyme (as shown in Figure 2.1f, 2.1e and 2.1b respectively). (a) E 80° ‘ +Lysol1s.1sr‘— E + Lyso[10,1 0.5.513 f; 600 — +Lyso[5,5]3 3 2 g 400 ~ Q .2 g 200 ~ '5 :5. 0 0 w . j I .1." £3 2‘ 1’ 5° 5° 5° 5° 6" (b) ‘5 1600 ‘ +[Lyso/PAA115 E: 1400 “ +[LfioIPEG]15 ;’ 1200 “ +LYSOI5.5I3 3 1000 -« .2 IE 800 r g 600 ~ 3 400 - E 200 ~ 8 o l l I l? 2' 32’ 3’. 1’ 6" o" o‘" o" 45" 44 2.3.2.4 Lysozyme Release from LbL Coated Agarose in Cell Culture Medium Lysozyme release from LbL coated agarose in DMEM supplemented with 10% Fetal Bovine Serum (F BS) and 2% penicillin/streptomycin i.e. in the presence of a competitive environment of external proteins and amino acids was also evaluated. Figures 2.4 — 2.7 show the controlled release of lysozyme from LbL coated agarose hydrogels in the presence of phosphate buffer saline (PBS) solution. However, PBS, used to characterize sustained release, does not approximate the properties of natural environment (i.e., blood serum), since it is a protein-free solution. The presence of other proteins may impose a competitive environment for LbL degradation and affect the protein release from the multilayers. To assess this, we performed the lysozyme release in DMEM (containing high D-glucose at 4500 mg/l and anrino acids such as L-glutamine) supplemented with 10% FBS (F BS contains various proteins, including albumin at a concentration ~2.6 g/dL ) and 2% penicillin/streptomycin (antibiotics). Figure 2.8 shows the optical densities of the bacteria in the cell culture medium (without lysozyme), and bacterial solutions containing active lysozyme released from LbL assembled multilayer (as shown in Figure 2.1a; BPEI initiating) on agarose incubated in fibroblast medium at 37°C for the indicated number of days. In the absence of active lysozyme, the bacteria remained intact, as shown by the first bar in Figure 2.8. In contrast, the LbL released lysozyme decreased the amount of live, intact bacteria in the solution (Fig. 2.8, Day 1 — 4) indicating the presence of active lysozyme. This demonstrates that LbL coated agarose can provide controlled protein release in a competitive (culture media) environment, i.e., in the presence of other proteins, amino acids and growth factors. 45 Figure 2.8 Optical densities of the bacteria in cell culture medium (without lysozyme), and bacterial solutions containing active lysozyme released from LbL coated agarose incubated in fibroblast medium for 1-4 days. Cell culture medium was replaced each day. 0.60 a 0.50 i 0.40 i 0.30 t 0.20 ‘l 0.10 t P O O I Bacterial Day 1 Day 2 Day 3 Day 4 solution in media Optical Density (450 nm) 2.3.2.5 Lysozyme Release under Static Conditions, and Direct impregnation (Soaking) 0f Lysozyme in Agarose Hydrogels All the cumulative lysozyme releases as explained above in Figures 2.4 to 2.7 were obtained under dynamic release conditions i.e. the release supernatant was collected and replaced with fresh buffer at the reported time intervals (see Materials and Methods section). On the other hand, Figures 2.9(a-e) shows the lysozyme release corresponding to the Figures 2.4-2.7, with the exception that the releases were obtained under static release conditions i.e. the release supernatant was not collected until the end time point. Figure 2.9e also shows the static release profiles of lysozyme for the case where agarose was impregnated (soaked) with lysozyme, either at pH 3.0 or 7.0, without any LbL assembly formation. Soaking was performed for a period of about 24 hrs (with only one final wash of pH 2.0 for pH 3.0 soaking, and pH 7.0 wash for pH 7.0 soaking), which was 46 greater than the total time in which the agarose was immersed in the protein solution during LbL assembly. Figure 2.9e shows the non-increasing static release profile for the soaked samples, suggesting that lysozyme release from impregnated agarose was likely governed by the equilibrium between the released and unreleased protein. 0n the other hand, Figure 2.9f shows the increasing dynamic cumulative release profiles of lysozyme released from soaked samples. However, the corresponding non-increasing static profile of soaked sample (Figure 2.9e) suggests that the continuous increase in cumulative dynamic release is most likely due to the disturbance of equilibrium as soon as the supernatant was changed. The static releases from the various multilayer assemblies were similar to the aynamic cumulative protein releases from LbLs i.e. all the total static releases from LbL coated agarose increased with time (Figure 2.9(a-e), respectively). This suggests that the release fi‘om the multilayer coated agarose was primarily governed by the degradation kinetics of the multilayers, and not affected by the equilibrium. Therefore, LbL assembly formation, with either three or two components, was certainly required for the sustained release of protein from the agarose. 47 Figure 2.9 (a-e) Static lysozyme release, triggered by physiological pH, corresponding to the Figures 2.4-2.7. (f) Dynamic lysozyme release, triggered by physiological pH, showing release from soaked lysozyme (i.e. direct impregnation of lysozyme) sample. (a) 2500 - +BPE|[5,5]3 zooo - 1500 ~ 1000 ~ 500* Static Release (pg/ml) (b). 1600 _ -x— 4°/ “°° * + 3%: 1200 — + 2% 1000 4 + 1% 600 .‘ 40° - ’1’ 200 - I o W— t 1’ fi fi r V r "___. W. r r__,.__‘ Static Release (uglml) 48 Figure 2.9 continued (C) —x— Lyso[5,5]3PM 3% + LPEI[5,5]3PAA- 3% + BPEI[5,5]3PAA- 3% -—I— LPEI[5,5]3PAA- 1% + BPEI[5,5]3PAA- 1% §§§§“§ s [[111] Statlc Release (pg/ml) § d o 8 l (d) 1400 ‘ +Lyso[1o,1o,5,5]PAA 1200 a +Lyso[15,15]PAA 1000 # +Lyso[5,5]3PAA 800 - 600 ~ 400 ~ 200 - Static Release (pg/ml) 49 Figure 2.9 continued (e) 2°°° ' +[Lyso/PAA115 1800 ‘ + [Lyso/PEG)“ 1500 ~ —)l(— Lyso[5,5]3 1400 ~ 93(- Lysozyme Soaking pH 3 1200 1 —0— Lysozm Soaking pH 7 -O— BPEI[5,5]3 Stetlc Release (pg/ml) § ow}. ’F/ Day, Day? Days Day“ Days 3 1000 _ —9— LPEI[5,5]3 - 3% 900 ~ + Ly50[5.5l3 - 3% 300 .. +BPEI[5,5]3 -3% 700 J -I—— Lyso Soak pH 7 600 — + Lyso Soak pH 3 500 - 400 - 300 a 200 - 100 1 ' 0 I T T [ Cumulative Release (pg/ml) Day 1 ”are 081/3 Day 4 Day 5 50 2.3.2.6 Range of Protein Loading and Release from LbL coated Agarose Hydrogels Figure 2.5c shows the total surface area per unit volume available from the intrinsic pores of the agarose, which is 12.26 mz/cc for 3 wt% agarose as measured by BET. Also, corresponding to the LbL assembly shown in Figure 2.1a (BPEI initiating) and the protein release shown in Figure 2.4a, the decrease in concentration of the stock solution of lysozyme from which the LbL assembly was made on bulk agarose was ~300ug/ml. Therefore, from the total amount of lysozyme loaded onto the agarose after the LbL process and the total surface area of the agarose including the intrinsic pores, the lysozyme loading per unit area of agarose was calculated to be 47.5 ng/cmz. This corresponded to 3 lysozyme loading of 16.2% with respect to dry weight of a 3 wt% agarose hydrogel. Further, the range of lysozyme released from the bulk agarose was ~0.07% on day 1 to ~0.8% (cumulative) at 4 weeks with respect to the dry weight of the agarose, which corresponds to the amount of lysozyme released from the LbL coated agarose shown in Figure 2.4a. Therefore, given the dimensions of the templated scaffold (1.5mm length, 1.5mm width, 2mm height, 200nm channel diameter and 67pm wall thickness (thickness of the wall between two channels)), the amount of lysozyme deposited/dry weight of the scaffold, and the release rate/dry weight of the LbL coated bulk agarose; we calculated a total release of ~60 ng at day l to ~700ng (cumulative) at 4 weeks from a templated agarose scaffold. This calculated amount of total protein released from an agarose scaffold is within the range of 20 — lOOng/ml of BDNF required for neurite outgrth and axonal regeneration'os. However, as described in Figure 2.4b, the 51 lysozyme released was found to be biologically active (as calculated on the basis of activity of freshly prepared lysozyme) during the earlier time points, with decreasing activity at later time points. Further, the range of protein amount released is governed by the wt% of the agarose and the various LbL configurations, as shown in Figures 2.5 and 2.6. Therefore, to obtain the amount of desired active protein release, the gel strength (% agarose), the size of the agarose scaffold, and the LbL configurations can be optimized further to tune the release rate from these scaffolds to higher or lower rates, as needed, for nerve regeneration. These calculations show that this system can provide sufficient protein release, as needed, for performance as a scaffold for nerve system regeneration. 2.3.2.7 LbL Formation at Higher pH and Lysozyme Release Since very low pH conditions might not be suitable for BDNF, therefore we filrther evaluated the lysozyme release with few LbLs formed at a pH 3 or higher. Remember, in all the previous LbL coating experiments over agarose (Figure 2.4-2.9); the pH of PAA, PEG, and wash solutions was 2.0, and pH of lysozyme was at 3.0 (in PBS). Therefore, overall the effective pH could be less than 3.0. We fabricated a two component LbL of lysozyme and PEG (Figure 2.1c), with lysozyme at pH of 4.75 in acetate buffer, and PEG at pH of 2.0 in DI water during LbL fabrication. All the wash solutions (pH adjusted DI water) were at pH 2.0. Figure 2.10a and 2.1% shows the afvnamic (cumulative) and static releases of lysozyme in pH 7.2 PBS from such an LbL coated agarose. The release profiles are similar to the case of lysozyme soaked samples i.e. static release has non-increasing profile, whereas dynamic release has a continuously increasing profile. This again suggests that this release was driven by the 52 equilibrium conditions (similar to lysozyme soaking), and this could be perhaps due to that LbL did not form at these pH conditions. Another interesting observation regarding LbL formation at relatively higher pH (higher than previous configurations of Figures 2.4-2.9) was observed when an LbL of [(Lyso/PEG)5(PAA/PEG)5]3PAA was prepared with all the solutions, including lysozyme, at pH of 3.0 during LbL fabrication. The static releases in this case were absolutely zero (BCA assay provided negative concentration values). However, in the ajmamic releases, lysozyme started to get released at Day 2 onwards (Day 1 was zero). In other words, LbL coated agarose started to release protein only after an exchange of supernatant. This is shown in Figure 2.10c. Non-increasing static release profile again suggests that LbL might not have formed at such pH conditions. We could not suggest a reason for zero release observed at Day 1. 53 Figure 2.10 (a,b) Dynamic and Static lysozyme releases, triggered by physiological pH, with lysozyme pH at 4.75 in acetate buffer during LbL fabrication on agarose. (c) Dynamic and Static lysozyme releases, triggered by physiological pH, with pH of all solutions at 3.0 during LbL fabrication on agarose. A 9 v 1600 l Lyso at pH 4.75 in LbL Fab 1400 4 1200 r 1000 a 800 r 600 r 400 r 200 r + Dynamic Release Cumulative Release (pg/ml) 3 500 - Lyso at pH 4.75 in LbL Fab uh O O 1 NH r‘/——{ + Static Release 200 ~ 100 ~ Static Release (pg/ml) o l 54 Figure 2.10 continued (c v 900 - All at pH 3 in LbL Fab + Dynamic Release -—-— Static Release Release (pg/ml) 2.3.3 Low Molecular Weight H—bonded Multilayer Disintegration on a Planar Substrate The degradation rate of H-bonded multilayers is one of the many factors that control the protein release from the LbL coated agarose gels. Therefore, as a first step in assessing the degradation of PAA/PEG multilayers, we evaluated the degradation behavior of these H-bonded films fabricated on a planar substrate. The degradation kinetics of high molecular weight PEO and PAA multilayer systems on a planar substrate have been previously investigated in detail, and were reported to degrade in about 30 min upon exposure to a pH of 3.5 or higher”. However, the stability or rate of film degradation will also depend on the choice of constituent l mers and their ro erties98, such as their molecular wei ht. The effect of P0 y P P g molecular weight on the growth profile of PAA/PEO multilayers is known”, however to 55 our knowledge, the effect of molecular weight on the degradation kinetics of these H- bonded multilayers has not been studied. Here, the multilayer films on agarose gel were formed using low molecular weight polymers, 15kDa PAA (sodium salt) and 10kDa PEG, which differed from previous studies. Therefore, we analyzed these films for their degradation behavior on a planar substrate using SEM. The thickness of 25 bilayers of lSkDa-PAA/ 10kDa-PEG films, as determined by SEM (Figure 2.11, t=0), was approximately 600nm which is close to previous report with the 25 bilayers of 250kDa PAA and 15kDa PEG films“. However, we observed that the 15kDa FAA and 10kDa PEG films degraded slowly over a period of days (5 — 10 days) (Figure 2.11) rather than minutes. It appeared that the low molecular weight PAA/PEG films did not completely degrade even after 10 days. These results suggest that the disintegration rate of the H- bonded PAA/PEG depends also on the molecular weights of the constituent polymers. 56 Figure 2.11 SEM images of H-bonded films composed of 25 bilayers of 10kDa PEG and 15kDa PAA formed on a planar substrate and exposed to deionized water (DI) (pH 5.6- 6.3) for the time durations indicated. Top panel: SEM images of films after fabrication. Middle panel: SEM images of films after immersion in DI water for five days. Bottom panel: SEM images of films after immersion in DI water for ten days. Same spot on the » films before and after degradation were imaged for comparative analysis. Columns 1, 2 and 3 show three different spots on the film. 2.3.4 Cytophobicity of H-bonded PAA/PEG Multilayers Previous in vivo investigations revealed that templated agarose scaffolds implanted at the site of a spinal cord injury formed a reactive cell layer of adherent leptomeningeal fibroblasts at the interface of the distal end of the scaffold and the host matrixm' 109. This reactive cell layer formed several days after scaffold implantation, and appeared to limit the ability of host axons that have already regenerated into a scaffold to exit the distal end 57 of a scaffold and reinnervate the spinal cord beyond the lesion”’ 109. Therefore, a strategy that provides sustained release of growth factors for enhanced axonal growth, while simultaneously preventing in vivo fibroblast adhesion onto the outer surface of agarose, is highly desirable. The low molecular weight H-bonded PAA/PEG films coated onto TCPS substrates were cytophobic towards fibroblast attachment in vitro for at least two weeks. This was possibly due to the slow degradation kinetics of these films, causing the surface to erode gradually and thus changing the surface topography over a period of days. It has been demonstrated previously that varying the surface topography (the physical features on the “0. As evident from the surface) can regulate adhesion and proliferation of the fibroblasts SEM micrographs (Figure 2.11), the initial surface morphology of PAA/PEG fihns is fairly rough (Figure 2.11, t=0), and becomes rougher as the films degrade over time. Figure 2.30 (lower panel middle image) of PAA/PEG films coated onto the channel wall of a templated agarose scaffold also clearly shows a rough morphology. The changing surface topography of the gradually eroding PAA/PEG films could lead to a rougher surface over time that may not be favorable to fibroblast adhesion. Further, an eroding/degrading substrate cannot easily support cell adhesion. Moreover, PEG is known to resist protein and cell attachmentm’ 112. Figure 2.12a and 2.12b shows the cytophobic behavior of PAA/PEG multilayers coated onto a TCPS substrate with 30.5 and 5.5 bilayers, respectively, for almost two weeks. 58 The cytophobic behavior of the 30.5 bilayers is somewhat different from the 5.5 bilayers, i.e., the 30.5 bilayers prevented cell adhesion from the start, whereas the 5.5 bilayers did not prevent cell adhesion initially but prevented adhesion over time. We provide the following possible reasons for this. LbL films exhibit a non-linear growth leading to partial substrate coverage during the initial growth of up to several bilayers (typically 3-5 bilayers)l 13 . This non-linear initial growth up to 5 bilayers has been shown previously for PEG and the high molecular weight PAA assembly“, and could result in partial film coverage on a surface coated with 5 bilayers of PAA/PEG multilayers. After these initial layers, the growth becomes linear, giving a more uniform surface coverage“ “3. The 30 bilayers of PAA/PEG coated surface is completely covered with a degrading surface that is rough (as shown in Figure 2.11), which would not easily support cell adhesion. On the other hand, the 5 bilayers of PAA/PEG coated films has regions on the surface covered by the degrading/eroding films which would not easily support cell adhesion, whereas the uncoated regions of the film may more easily support cell spreading and adhesion from the start. Therefore, as the fibroblasts spread and proliferate on the uncoated regions of the 5 bilayer films, forming regions of cell spreading and attachment, these cells will continue to spread until they reach the coated regions of the films, and as the latter regions erode the group of attached cells will disengage as a whole from the surface. The degrading PAA/PEG films (both the 5.5 and 30.5 BLs) are not cytotoxic to the cells (Figure 2.12c, as discussed below) as determined by lactate dehydrogenase (LDH) assay, which is a measure of cytotoxicity. 59 The cytophobic behavior does not indicate cytotoxicity of these films. A quantitative lactate dehydrogenase (LDH) cytotoxicity assay was performed on the fibroblasts seeded onto these films. The LDH released into the supernatant after 4 days of culture was collected and measured. As shown in Figure 2.12c, no excess LDH was released into the culture medium from the cells seeded onto these films as compared to the control TCPS substrate after 4 days. This indicates that the PAA/PEG films, which degrade over time to release PAA and PEG polymers into the culture medium, were not toxic to the cells and the unattached, floating cells on these PAA/PEG films were not due to cytotoxicity. Therefore these films, in addition to providing sustained release of protein from agarose, may help also in preventing the reactive cell layer from forming on the implants in vivo. 60 Figure 2.12 (a) Phase contrast microscopy images demonstrating the cytophobicity of the 30.5 bilayers of PAA/PEG multilayers over time (days). Top panel: NIH-3T3 fibroblasts cells on TCPS plates coated with 30.5 bilayers of PAA/PEG. Bottom panel: Fibroblasts on bare TCPS plates (control). (b) Phase contrast microscopy images demonstrating the cytophobicity of the 5.5 bilayers of PAA/PEG multilayers over time (days). (0) Cytotoxicity levels of fibroblast cells after 4 days of culturing on (PAA/PEG)5PAA multilayers built onto TCPS (denoted here as 5.5BLs), (PAA/PEG)30PAA multilayers built onto TCPS (denoted here as 30.5 BLs), and bare TCPS plates. The absorbance values corresponds to the amount of lactate dehydrogenase (LDH) released into the culture supernatant. A higher absorbance value indicates higher LDH release and thus higher toxicity. Serum in the cell culture medium contains a small amount of LDH, which is shown as “background media”. (a) (b) 61 Figure 2.12 continued (C) 75 0.50 - 3 l I Media Supematantl 2 0.40 ~ all I 0.30 r J '5 : 020 . ‘0" O 1 .i. a " - i s 0-10 - o g _vr’_~_f. ». _7_ Edi - g 0.00 ‘7 Y ”F T T— T— 1 m \‘b \‘b ’{b \‘b .n b b o b s §¢G ’§0 ’¢0 '¢0 0?: 9 9 (0° «(.3 60V ‘36’ &*g 60 5°. 0 2.4 CONCLUSIONS We fabricated LbL assembled multilayer films that incorporated proteins into the multilayer structure over agarose, rather than pre-loading the protein directly into the agarose gel. This allowed the protein to be loaded subsequent to the fabrication, and avoided exposing the protein to harsh organic solvents during fabrication of the templated agarose scaffolds. The LbL multilayers consisting of either a three component assembly of PAA, PEG and protein, or a two component assembly of PEG and protein were fabricated at low pH (pH 5 3.0) conditions. These multilayers formed thin films over the surface of the agarose and provided prolonged and sustained protein release at physiological pH conditions for up to a month in certain cases. The lysozyme released was found to be active during the earlier time points, with decreasing activity at later time 62 points. The amount of protein released depended on the concentration of the agarose, the H-bonding and hydrophobic characteristics of the LbL initiating polymers, and the LbL constituents. This suggests that the protein release is a complex phenomenon, governed by both the multilayer degradation kinetics and the agarose porosity. The relative rate of protein released with the three component assemblies was slower than the two component assemblies. The arrangements discussed can be used in clinical trials for axonal regeneration, with either a two component H-bonded biocompatible assembly consisting of PEG and protein, or a three component assembly replacing PAA with another biocompatible polymer such as poly(glutamic acid) or poly(aspartic acid) for slower protein release. The choice of the configuration would depend on the desired rate of protein release. Currently, these combinations are being tested for sustained release of BDNF fi'om templated agarose scaffolds, both in vitro and in vivo, to enhance axonal growth. 63 CHAPTER 3 TIME CONTROLLED RELEASE OF ARABINO- F URANOSYLCYTOSINE (Ara-C) FROM AGAROSE HYDROGELS USING LAYER-BY-LAYER ASSEMBLY 3.1 INTRODUCTION Patterned or templated scaffolds with arrays of uniaxial, uniform diameter channels similar to normal spinal cord have been shown to support linear axonal growth into spinal cord lesion sites in the central nervous system (CNS)2. Agarose hydrogel can be designed to match the mechanical properties of the spinal cord, is biocompatible and bioinert, and more importantly, is stable for the extended period required for regenerating organized axons, and thus is a good choice for nerve regenerationz. Templated agarose scaffolds for nerve guidance have been shown to exhibit excellent integration with host tissuez, nevertheless, a reactive cell layer (RCL) of adherent leptomeningeal fibroblasts forms at the interface of the distal end of the scaffold and the host matrix at the site of injury 14’ 27. RCL forms several days after scaffold implantation, and limits the ability of the regenerated host axons to exit the distal end of the scaffold and reinnervate beyond the lesion site”. Furthermore, the leptomeningeal fibroblasts also form a RCL within the channels of the templated scaffolds. Fibroblasts adhere to the inner wall of the channels and reduce the channel space available for the regenerating axons”. Figure 3.1 shows the RCL formation in these two different cases. 64 Figure 3.1 Diagrams showing the growth of leptomeningeal fibroblasts along the inner periphery of a single channel of templated agarose scaffold (top image), and at the distal end of channel (bottom image). wChannel IMF ——9 Regenerating axon Fibroblasts — reactive cell Iay<7 We propose that the RCL formation could be attenuated, in part, by modulating the substrate cell adhesive property, to render the surface resistive to RCL formation. Creating a cytophobic surface has been achieved previously”, whereby degradablel4 or ”4 multilayers are used to render a surface cytophobic for extended non-degradable periods lasting over weeks. Another possible method to prevent the RCL formation is to design a biodegradable system at the site of RCL formation. However, to further the impact, one could couple the biodegradability with a delivery system that slowly releases, in a controlled manner, a potent drug to inhibit fibroblast cell division without affecting the regenerating axon. The concentration of the drug delivered at the site of injury should be at a controlled rate and as low as possible so that the regenerating axon is not affected while the fibroblasts growth is inhibited. 65 l-B—D-Arabino—fiiranosylcytosine (AraC or Ara-C) is a chemotherapeutic agents used clinically for the treatment of acute leukemia115 . Ara-C is an antimitotic agent often used at low concentrations (10 uM or less) to induce cell death of rapidly proliferating non- neuronal cells, such as astrocytes, in culture. Ara-C, transported into the cell by a membrane transporter‘wng, inhibits DNA synthesis119 as well as the activity of an 120 enzyme, B-DNA polymerase, involved in DNA repair . In addition to dividing cells, the neurotoxic effect of Ara-C can cause apoptosis of postrnitotic neurons ””23 . Suggested mechanisms by which Ara-C causes neurotoxicity is through the generation of reactive oxygen species that lead to oxidative DNA strand breakage122 as well as blocking DNA repair124 . High doses of AraC administered by injection are necessary in chemotherapy, for examples, the AraC administered by injection provides delivery only for up to a few hours due its short half life and thus is not detected in the system at later time points125 or the high doses are necessary for the penetration of AraC into cerebrospinal fluid through blood-brain-banier126. However, the high doses of AraC used in these treatments is cytotoxic and can cause severe secondary side effects, including disorders of the CNS and peripheral nervous systems (PNS)125‘127 . A slow release formulation providing prolonged exposure of AraC to cerebrospinal fluid as compared to a standard injection delivery has been shown to give better response and quality of life in patients being treated for leptomeningeal meningitism. Indeed, several methods have been developed 125.129- over the past decade to provide controlled release of AraC from polymeric matrices 131, however none have used templated scaffolds. 66 Controlled delivery of a low level (preferably less than 10 uM) of AraC at the site of injury could be a potential solution to controlling RCL formation. In this study, our objective was to provide controlled delivery of Ara-C from agarose hydrogel, since the implanted scaffolds experience extensive RCL formation at the site of injury during axonal regeneration. However, the current methods of incorporating drugs into hydrogel structures during the fabrication processls'w' 125' ‘30' ‘3‘ are not compatible with the templated agarose scaffold fabrication processz. The latter exposes drugs to harsh organic solvents, such as tetrahydrofuran, that are used to selectively etch patterning constituents during the templated scaffold fabrication processz, which can be detrimental to the stability of the drug. Layer-by-layer (LbL) assembled multilayers, introduced by Decher”, offer promise in the field of controlled drug delivery, due to their tunable film properties, flexibility in choice of assembly components and ease of processing“ 44. LbL multilayers can be tuned to incorporate varying amounts of drugs or proteins as well as provide sustained release under specific conditions of pH, salt or temperature4345 . Degradable multilayer assemblies, based on sequential embedding of drugs during the fabrication, can incorporate any drug, independent of the molecular weight of the drug90’94. Fabrication of hydrogen bond (H-bond) degradable LbL multilayer films was initially reported by Rubnerp5 and Zhang96. Sukhishvili and Granick demonstrated the pH controlled assembly and degradation of poly(carboxylic acid)-based H-bonded LbL films97’ 98 , which was followed by numerous studies involving these multilayers for different applications, e. g. to generate self-standing floating films, solid polymer electrolyte films, or for patterned 67 delivery of nucleic acids to cells38' 4" 99. The LbL methodology using non-degradable multilayers to provide controlled drug or protein release from synthetic hydrogels, had 20 . . . '2‘, Wthh is not amenable With loaded the drug during the hydrogel fabrication process the fabrication process used for the templated agarose scaffolds. Sustained release of drugs or proteins incorporated within degradable H-bonded films has been recently demonstrated90'”. Here, we employed multilayer LbL assembly of degradable PAA, PEG and Ara-C over agarose, subsequent to the fabrication of the agarose. Carboxylic acid (- COOH).based weak polyelectrolytes form H-bond interactions at low pH (pH < 3.5 in the case of poly(acrylic acid (PAA)) and deprotonate to carboxylate ions (-COO') at high pH, which degrade the H-bonded multilayer assembly”. We demonstrate controlled release of Ara-C fi'om agarose at physiological pH at concentrations amendable to inhibiting fibroblast growth which should minimally affect neuron viability. 3.2 MATERIALS AND METHODS 3.2.1 Materials Poly(acrylic acid, sodium salt) solution (PAA, MW 15,000; catalog no. 416037), poly(ethylene glycol) (PEG, MW 10,000; catalog no. P6667) were purchased from Sigma-Aldrich (USA). l-B—D-Arabino-fiiranosylcytosine (Ara-C) (catalog no. 251010) was purchased from Calbiochem (USA). Ultrapure agarose (Sigma-Aldrich, USA) was dissolved at a required concentration in 18.2 MQ-cm deionized (DI) water (MilliQ, Millipore) at near 100°C. For all the experiments, unless otherwise specified, 2ml of hot agarose solution was poured into 6-well tissue culture polystyrene (TCPS) plates (Costar, Corning, NY). For ultraviolet/visible (UV-vis) absorbance experiments, the hot agarose 68 solution was filled into a 96-well polystyrene plate (Evergreen Scientific, USA). Agarose was allowed to gel at room temperature. 3.2.2 LbL Formation over Agarose PAA, PEG and Ara-C solutions used to fabricate the multilayer assemblies were prepared in DI water to a final concentration of 1 mg/ml and the pH was adjusted to 2.0, unless otherwise specified. DI water adjusted to pH 2.0 was used as the wash solution during the assembly. Multilayer assembly of [(PAA/PEG)5(Ara-C/PEG)5]3PAA (as described in Results and Discussion) was formed over agarose gel (agarose discs) prepared in 6-well plates. The substrates were dipped in PAA, PEG or Ara-C solutions (depending on assembly step, as described in Results and Discussion) for 30 mins followed by 10 mins in a wash solution with agitation to create a multilayer. A Carl Zeiss slide stainer was used to form the multilayers. PAA was kept as the terminating layer in each case. Films, when abbreviated as n.5, denote the PAA/PEG multilayer where n is the number of PAA and PEG bilayers and the “.5” indicates an additional, single terminating layer of PAA. After multilayer formation onto the agarose discs in 6-well plates, the LbL coated agarose plate was left in DI water at pH 2.0 overnight (to assess for the ability of LbL coated agarose to provide controlled release after storage in pH 2.0 DI water). At the start of the experiment, the agarose discs were transferred to a new 6-well plate to avoid the effect of Ara-C (if any) that may be released from the sides of the 6-well plate during cell cultming or release measurement experiments. AraC soaked (impregnated) agarose signifies that the agarose filled 6-well plate is immersed in a solution of AraC (lmg/ml, pH 2.0) in 1X PBS for ~ 24 hrs, washed with 1X PBS (pH 2.0) for 20 min with agitation, 69 and left in 1X PBS (pH 2.0) overnight (similar to LbL coated agarose plate which was also left overnight in DI water at pH 2.0). To determine the incorporation of AraC during the multilayer build-up process, UV-vis peaks of the 96-well plate were measured at 23°C in the range of 200-400 nm and a spectral resolution of lnm with a SPECTRAmax Plus 384 (Molecular Devices). The absorbance/scattering spectrum of bare agarose (non- coated agarose) was subtracted from each LbL coated agarose spectrum. The cumulative amount of AraC loaded within the multilayers was estimated based on the Beer-Lambert law, assuming the agarose hydrogel is a solution. 3.2.3 Cell Culture and Imaging NIH-3T3 fibroblasts were purchased from American Type Culture Collection (Rockville, MD). All cell cultures were maintained in DMEM (catalog 11995, Invitrogen) with 10% FBS and 100 U/ml penicillin plus 100 rig/ml streptomycin (P/S) at 37°C and 10% C02. In the Ara-C—fibroblasts studies, the fibroblasts were seeded at a density of ~ 20,000 cells/ml onto glass cover-slip and placed into a 6—well plate with 2 ml of media (described in section 2.3.1). 3.2.3.1 Cell Culture and Ara-C Release from Agarose Hydrogels To study the effect of Ara-C released from LbL coated agarose in 6-well plates, fibroblasts were cultured onto glass cover-slips (Corning) placed in a 6-well plate. The LbL coated agarose 6-well plates were incubated in cell culture medium at 37°C, without cells initially, and the medium was replaced with fresh culture medium at 30 mins, 16 and 24 hrs. This was performed to flush out the low pH water from the hydrogel, to 70 minimize the effect of low pH on cell growth. After 24 hrs, the cells on the cover-slips were placed faced up onto the agarose discs in the 6-well plates. The cell culture medium was replaced every 24 hrs and the cells were monitored for 3 days, starting from when the cover-slips were placed onto the agarose filled 6-well plates. After this initial 3 days of culture, the cover-slips on the agarose filled 6-well plates were replaced with new cover-slips cultured with fresh cells (as described above), and the cells were monitored again for 3 days. Phase contrast images were collected with Leica DM IL inverted microscope (Bannockbum, IL) equipped with SPOT RT color camera (Diagnostics Instruments, MI) using 10X dry objective. 3.2.4 Ara-C Release Measurements For the Ara-C release experiments, the LbL coated agarose in the 6-well plates were incubated in 2 ml of 1X-PBS (pH 7.4) at 37°C. The buffer was replaced with 2 ml of fresh PBS after each sampling interval, and the collected buffer was stored at -20°C until further analysis. The buffer was replaced with fresh PBS at the following intervals -— 30mins, 16 and 24 hrs after the start of the release experiments (similar to the cell culture experiment described above in section 3.2.3.1), and every subsequent 24 hrs. The concentrations of the Ara-C released from the LbL coated agarose were measured by reverse phase high pressure liquid chromatography (HPLC). HPLC analysis of Ara-C was performed using Waters 2695 Separations Module, Waters 2966 Photodiode Array Detector, 250 x 4.60 mm reverse-phase stainless steel column packed with 4pm particles (Phenomenex, Synergi 4u Hydro-RP 80A). A gradient of water (A) and HPLC grade acetonitrile (B), both containing 0.05% (v/v) trifluoroacetic acid (TFA) (CF3COOH), was 71 used (0-10 min — 5% B, 15 min — 50% B, 20 min — 60% B). Samples (10p.l injection volume from 25 pl in stock vial) were eluted at room temperature with a mobile phase consisting of 0.05% TFA in acetonitrile, at a flow rate of 1 ml/min. The pressure limits were set to 0 — 4000 psi. A standard curve was obtained using pure Ara-C dissolved in 1X PBS to determine the Ara-C concentrations released from the agarose. Ara-C exhibits a UV peak at 272 nm due to the presence of its aromatic ring structure, while PAA and PEG do not exhibit such a UV-vis peak (data not shown). The amount of Ara-C released from Ara-C soaked (impregnated) agarose (i.e., without LbL multilayers of PAA or PEG) were determined using UV-vis spectrophotometer at 272nm. However, since the Ara-C released could be conjugated with the PAA or PEG released from the LbL, the amount of free Ara-C in the LbL released samples were determined using RP-HPLC with a UV-vis detector132 . This evaluation method was necessary for LbL released Ara-C since the PAA or PEG conjugated Ara-C would have different (and unknown apriorz) extinction coefficients from their unconjugated forms. This thereby prevented a direct UV/vis 132. The free Ara-C was eluted at tR ~ 9 mins at 272 nm in spectrophotometer evaluation RP-HPLC. The amount of free Ara-C in the samples was calculated based on the area under the peak corresponding to the retention time of pure Ara-C obtained at 272nm. Cumulative amounts reported were determined by adding the release at each sampling interval. All data are shown as mean i SD from at least 3 independent experiments. 3.3 RESULTS AND DISCUSSION H-bonded degradable multilayers have been demonstrated as a potential method to provide controlled release of proteins from agarose scaffold”. Such multilayers resist 72 fibroblast attachment to the substrate for over a period of two weeks and thereby could be used to control the formation of a fibroblast RCL”. In addition to these fiinctions, if these multilayers can also be timed to provide controlled release of an anti-mitotic drug to arrest the growth of the RCL, then forming multilayers on agarose could be a versatile approach to addressing the challenges of repairing nerve injury. Ara-C contains hydroxyl groups, and a primary amine and carbonyl group, which could potentially form H-bonds with the polymers during LbL assembly. Figure 3.2a illustrates the chemical structure of Ara-C. We incorporated Ara-C as one of the multilayer component, along with PAA and PEG, to build degradable multilayers over agarose. This is one approach to achieving controlled release of Ara-C from the agarose hydrogels, in a fashion similar to the protein release study described previously”. Ara-C is a small molecule, whereas protein is a polypeptide consisting of amino acids. The difference in the physical size and molecular weight of a polymer or polypeptide versus a small molecule could have a significant impact on the multilayer formation and release profiles. Nevertheless, we hypothesized that Ara-C can be incorporated into the multilayer structure based on the primary amine and hydroxyl groups on Ara-C that could form hydrogen bonds with PEG to provide controlled release of Ara-C. Agarose hydrogels have a broad range of pore diameters, of less than lnm up to greater than 500nm74'79. Due to the relatively large average pore size of the agarose as compared to the size of the drug and the absence of strong interactions between the drug and agarose, the only means of entrapping the drug is through entanglements or physical interactions, which would not be able to provide controlled release of the drug. 73 Figure 3.2 (a) Chemical structure of 1-B-D-arabino-fi1ranosylcytosine (Ara-C). (b) Multilayer assembly of PAA, PEG and Ara-C over agarose. Curved line represents the agarose. HO OH /\/\/\/\/\ To test the possibility of controlled release of Ara-C from agarose hydrogels, we assembled multilayers of PAA, PEG and Ara-C ([(PAA/PEG)5(Ara-C/PEG)5]3PAA) on agarose, as shown in Figure 3.2b. Agarose is a physically cross-linked double-helix 3-D gel network of polymer chains interconnected by H-bonding and hydrophobic interactions” 75. PAA with protonated carboxylic groups has hydrophobic domains at low pH conditions133 , and thus can form hydrophobic interactions and H-bonds with agarose under an acidic environment. Thus, we fabricated multilayers, according to Figure 3.2b, on agarose hydrogel at a low pH of 2.0. 3.3.1 AraC Incorporation into Agarose during LbL Multilayer Assembly To assess the incorporation of AraC during the buildup of the LbL multilayers, we measured the UV-vis absorbance spectra of agarose during the intermediate steps of the sequential layer-by-layer dipping process. Figure 3.3a shows the UV-vis spectrum of 74 agarose loaded into [(PAA/PEG)5(Ara—C/PEG)5]n multilayers. Ara-C exhibits a UV peak at 272 nm due to the presence of its aromatic ring structure, while PAA and PEG do not exhibit such a UV-vis peak (data not shown). However, in the building of the multilayers of AraC with PAA and PEG onto the agarose structure, we observed the UV-vis peak of the AraC red-shifted to about 300nm. The rise in the absorbance values with higher number of multilayers indicated that AraC is being loaded within the agarose structure as the number of deposition cycles increased. Further, Figure 3.3b shows an exponential increase in absorbance values at 272 nm as the number of AraC bilayers was increased. This suggests an exponential growth of [(PAA/PEG)5(Ara-C/PEG)5]n films on the agarose, similar to PLL and polypeptide, polysaccharides or nucleic acid-based multilayer films13 4'13 7 . The exponential growth, rather than a linear growth, was beneficial for the increased loading of AraC within the agarose. Thus, the UV-vis characterization suggests that AraC was continually being loaded as the number of deposition cycles was increased. A loading of ~ 17 rig/cc of 3 wt% agarose was estimated for the multilayers of [(PAA/PEG)5(AraC/PEG)5]3 with three additional basal layers of AraC, as shown in Figure 3.3b. 75 Figure 3.3 (a) UV—vis spectrum of agarose loaded with multilayers of (PAA/PEG)5(AraC/PEG)2(PAA/PEG)5(AraC/PEG)[(PAA/PEG)5(AraC/PEG)5].,. n represents the number of repetitions of sequence [(PAA/PEG)5(AraC/PEG)5]. In the plot, P represents (PAA/PEG) and A represents (AraC/PEG) multilayers and the 5 represent the number of bilayers. P5A5P5Al is the base multilayer for each case in the UV-vis measurements. (b) Plot of AraC absorbance at 272 nm as a function of increasing number of AraC layers. (a) 0.8 1 —~- P5A2P5A1(P5A5)4 0.7 + P5A2P5A1(P5A5)3 + P5A2P5A1(P5A5)2 —- P5A2P5A1 Absorbance (a.u.) O .5 I i I Y 1 l l i e e e e e e o ’iré'vé‘i‘ W®W°Pe° 6" '9' 03" sfoéo «3‘ 599.9,? Wavelength (nm) (b) 0.60 - g 0.55 0.50 0.45 ~ 0.40 -. 0.35 0.30 . . P5A2P5A1 P5A2P5A1(P5A5)2 P5A2P5A1(P5A5)3 P5A2P5A1(P5M)4 Absorbance at 272 76 3.3.2 Qualitative Evaluation of the Effect of Agarose Released Ara-C on Fibroblast Growth LbL coated agarose discs, without cells, were initially incubated in culture medium for up to 24 hrs, with the medium replaced at 30 min, 16 hrs, and 24 hrs. This first 24 hrs released some of the Ara-C from the LbL coated agarose, prior to culturing the discs with the cells. Subsequently, cover-slips containing cells were placed on top of the LbL coated agarose discs. Figure 3.43 shows the phase contrast images of fibroblasts cultured on a cover-slip in contact with LbL coated agarose discs. After one day of culture with LbL coated agarose discs (i.e. Day 1, which is 48 hrs since exposing the LbL coated agarose to physiological pH — see Materials and Methods), the effect of Ara-C on the cells are noticeable as compared to the control cells exposed to LbL coated agarose without Ara- C. As shown in Figure 3.4b, the cell number decreased significantly as compared to the controls, suggesting that free Ara-C was released from the LbL coated agarose within Day 1 (48hrs). Figure 3.4c shows that effect of AraC on cells at Day 4 and onwards is not as significant as observed in Figure 3.4a. This suggests that either no free AraC was released at Day 4 (120 hrs) onwards or the amount of free AraC released was less than amount of AraC required to inhibit cell division. The other possibility is that released AraC might be conjugated to PEG or PAA inhibiting its anti-mitotic function. AraC conjugated with PAA or PEG might not be an active form of AraC, as it has been suggested in a previous study that AraC covalently conjugated with PEG renders it inactive until the hydrolysis of PEG-AraC conjugate releases free AraC13 2. 77 Figure 3.4 Fibroblasts cultured on a cover-slip in contact with LbL multilayer ([(PAA/PEG)5(Ara-C/PEG)5]3PAA) coated agarose. Prior to exposure to the cells, the LbL coated agarose discs were incubated in cell culture media (without any cells) for 24 hrs, with flesh culture medium replaced at 30 min, 16 hrs, and 24 hrs. (a) Cells were monitored for 3 days (indicated as Day 1 (48 hrs), Day 2 (72 hrs), and Day 3 (96 hrs)), (b) The number of cells cultured on the covers-slips in contact with LbL multilayer coated and non-coated agarose. Day 0 implies fresh cells, i.e. just before placing the cells in contact with LbL coated agarose. (c) After 3 days, cells on agarose were replaced with fresh cells cultured on new substrate and were again monitored for next 3 days (indicated as Day 4 (120hrs), Day 5 (144 hrs), and Day 6 (168 hrs)). Scale bar represents lOOum. Time in parenthesis represent the age of the LbL coated agarose, i.e. it denotes the time since the start of the multilayer degradation in culture media, whereby the initial 24 hrs of degradation was performed in the absence of cells. (a) Control ' ‘ '1 . Control " “ . ...: Fresh Cells Day 1 Day 2 Day 3 (post 24 hrs) (48 hrs) (72 hrs) (96 hrs) 78 Figure 3.4 continued (b) a 600] +Control E500 +LbL Multilayer : 400 — 3 300 ~ ’5 1 c 200 a 100 ~ 0 0 . Q N q, ‘5 (c) Control Control Control . Control Fresh Cells Day 4 (post 96 hrs) (120 hrs) Day 6 (168 hrs) Figure 3.5 shows the fibroblasts cultured on a cover-slip placed in contact with 3) agarose discs without LbL (control, top panel), or b) agarose discs coated with PAA and PEG but 79 without Ara-C, i.e. (PAA/PEG)15,5 multilayer on agarose (bottom panel). The number of PAA layers on (PAA/PEG)1 5_5 coated agarose were the same as in the ([(PAA/PEG)5(Ara-C/PEG)5]3PAA) coated agarose with Ara-C. As evident from Figure 3.5, PAA and PEG released from the agarose do not alter the fibroblast growth as compared with the control. PEG, but not PAA, is FDA approved and non-toxic. However, as illustrated in Figure 3.5, multilayers of PAA and PEG do not appear to be toxic to the cells. In a previous study, the cytotoxic effect of PAA released from a similar LbL configuration was quantitatively assessed and found to be non-toxic“. These results suggest that the reduced growth rate of the fibroblasts observed in Figure 3.4 was due to the release of the Ara-C fi'om the LbL coated hydrogels, and not due to a cytotoxic effect of PAA or PEG. Figure 3.5 Fibroblasts cultured on a cover-slip in contact with LbL multilayer ((PAA/PEG)15_5) coated agarose, without Ara-C. Cells were monitored for 3 days (indicated as Day 1 (48 hrs), Day 2 (72 hrs), and Day 3 (96 hrs)). Scale bar represents lOOum. Time in parenthesis represent the age of the LbL coated agarose, i.e. it denotes the time since the start of multilayer degradation in culture media, where the initial 24 hrs of degradation was performed in the absence of cells (with the medium without cells replaced at 30 min, 16 hrs, and 24 hrs). Control Fresh Cells Day 1 Day 2 Day 3 (post 24 hrs) (48 hrs) (72 hrs) (96 hrs) 80 3.3.2.1 Dose Response of Pure Ara-C on Fibroblast It is not possible to determine from Figure 3.4a whether the reduced growth of the cells is due to free Ara-C that has continued to be released at subsequent time points, i.e. Day 2 (72 hrs) and Day 3 (96 hrs), or due to the continued effect of the Ara-C released and taken up by the cells on Day 1 (48 hrs). Depending on the concentration, Ara-C once taken up by the cells can inhibit cell growth for a long period (discussed below). Whether the Ara-C released on Day 1 continues its effects depend, in part, on the concentration of the Ara-C released. To shed light on this question, we added varying concentrations of pure Ara-C directly to the fibroblasts for 24 hrs and monitored the cells for 72 hours. This provided a qualitative assessment of the effective concentration of Ara-C and the length of time the Ara-C was effective in the cells. Ara-C was added to the cells for 24 hrs, with the media (without Ara-C) replaced every subsequent 24 hrs and the cells monitored for up to 72 hrs (Figures 3.6a and 3.6b). The concentration of Ara-C added ranged from 0.25 rig/ml to 20p.g/ml. As evident fi'om Figures 3.63 and 3.6b, the effectiveness of the Ara-C decreased with concentration (20ug/ml > 1.40ug/ml > 0.70pg/ml > 0.25ug/ml). Cell growth in the presence of LbL released AraC (Figure 3.4b) appeared to be roughly similar to the cell growth observed in cultures with AraC directly added at concentrations between 1.4 —— 20pg/ml (Figure 3.6b). The same cell seeding density was used in all cases shown in Figure 3.6a. Although the cell growth in the 0.70pg/m1 and 0.25ug/ml Ara-C cultures was slower than the control cells at each time point, the cell growth was faster than in the cells cultured with LbL released Ara-C for these concentrations. Thus, given that this is a qualitative assessment 81 of the Ara-C released from the LbL coated agarose, we can only estimate that the Ara-C concentration released from the LbL coated agarose discs may range from 1.40 to 20ug/ml on Day 1 (48 hrs). There are two possibilities, it could be that either a high concentration of AraC (near to 20ug/ml) was released within Day 1 (48 hrs), or the AraC release at low concentrations was maintained for up to Day 3 (96 hrs). Although this approach cannot be used to provide a quantitative estimate of the Ara-C release from the LbL coated agarose, it does provide some indication of the range of Ara-C released and how long the effect of the Ara-C taken up on Day 1 lasts. To obtain a more accurate estimate of the Ara-C released from the LbL coated agarose disc at each time point, a more quantitative measure of the amount of Ara-C released was performed with RP- HPLC. 82 Figure 3.6 (a) The effect on fibroblast growth of directly adding pure Ara-C. Varying concentrations of Ara-C were added once, for the first 24 hrs and subsequently replaced with non-Ara-C containing media. The fibroblasts were monitored for up to 3 days. The effectiveness of the Ara-C decreased with concentration. Scale bar represents 100nm. (b) The number of cells as a firnction of time. Day 0 implies fresh cells, i.e. just before adding the AraC to cultured cells. The growth rate of the control cells (data not plotted) was similar to those plotted in Figure 3.4b. (a) Control Control . Control _ . j _ i . -0.70 pg/ml. 83 Figure 3.6 continued (b) 180 . +0.70 uglmi NE 150 3 +1.40 uglmi +20u lml g 120 - 9 O .n 9 ~ E 0 5 60 — E 30 ~ 0 l l l 41 Q N W ‘5 A A 4. A 0" 0° 0° 0” 3.3.3 Quantitative Evaluation of Free Ara-C 3.3.3.1 Quantitative Evaluation of Free Ara-C in a Mixture of PAA-PEG-Ara—C The released Ara-C could be conjugated with either PAA or possibly with PEG released from the LbL. Ara-C could conjugate with PAA by forming an amide bond between the primary amine on Ara-C and the protonated carboxylic acid group on PAA at low pH. This would reduce the amount of free Ara-C as the relative concentrations of PAA and PEG increase. To confirm this, we prepared a solution of Ara-C, PAA and PEG, where the amount of Ara-C was kept constant while the amounts of PAA or PEG were increased. The RP-HPLC analysis showed that the amount of free Ara-C decreased as the relative amounts of PAA and PEG increased with a fixed amount of Ara-C (Figure 3.7, where 6.25 pg of total Ara-C was added to each sample). 84 Figure 3.7 RP-HPLC measurements of the amount of free Ara-C in the solution mixtures ‘ of PAA, PEG and Ara-C as a function of increasing PAA and PEG amounts (no LbL). X- axis indicates the amount of both PAA and PEG, individually, in the solution e.g. 100 pg on x-axis indicates that 100 pg of PAA and 100 pg of PEG was added to the solution. Total amount of pure Ara-C added to each solution was 6.25 pg. 38* a E e—‘l 8 E4— ’ 2‘, r . £2- < E0 T i T 1 l n- 0 50 100 150 200 250 PAA, PEG Amounts (pg) 3.3.3.2 Quantitative Evaluation of Agarose Released Ara-C Figures 3.8a and 3.8b show the cumulative and non-cumulative concentrations, respectively, of free Ara-C released from the LbL coated agarose discs at the time points noted. The time points shown in Figure 3.8b correspond to the time points shown in parenthesis in Figure 3.4a, i.e. amount of time the fibroblasts cultured on cover-slips were placed in contact with LbL coated agarose discs. The average concentration of free Ara-C released fi'om LbL at each time point was ~ 1 pg/ml (Figure 3.8a and 3.8b). This amount falls within the range shown in Figure 3.6b. Therefore, the qualitative (as described in section 3.3.2. 1) and quantitative assessment of Ara-C release correlate. 85 Figure 3.80 and 3.8d demonstrate the cumulative and non-cumulative concentrations, respectively, of Ara-C (or free AraC) released from the Ara-C impregnated (soaked) agarose. The concentration of Ara-C released was much higher at each time point, i.e. more of the Ara-C was released than was released from LbL coated agarose. This shows that LbL coated agarose provided better controlled release of Ara-C than the Ara-C impregnated agarose. The concentration of Ara-C released from LbL coated agarose was significantly lower than the levels at which the nerve cells in culture are deleteriously affectedm‘ 122’ 138’ 139. Ara-C induces apoptosis in cultured cerebellar neurons in a concentration dependent manner, with an ECso value of ~60pMm. Ara-C can enhance neuronal survival at low concentrations (10 pM = 2.43 pg/ml) by reducing the cell division of non-neuronal cells present in neuronal culture”8 . Also, it has been shown that an initial pulse delivery of Ara-C supplied twice at the concentrations of 10 pM did not deleteriously affect the Schwann cells (a chief supporting factor in nerve regeneration) while successfully eliminating a large number of fibroblasts in a human peripheral nerve culturem. Therefore, in view of these previous studies, the amount of agarose released fiom LbL coated agarose falls within a permissible limit, such that the released Ara-C should not affect the axonal growth negatively at the site of injury, but should be able to inhibit the growth of leptomeningeal fibroblasts around the scaffold. 86 Figure 3.8 Non-cumulative and cumulative free Ara-C concentrations released from: (a,b) LbL coated agarose, and (c,d) non-coated Ara-C soaked agarose, both exposed to 1X PBS at physiological pH. (a) i 3 1 With LbL Multilayers B 3 0 3 2 i 2 .. ° 1 a 1 1 < ; g l u. 0 i — #4 .0 e e 69‘ 1'06 $56 ‘5 ’\ ’1' (b) 3 6 With LbL Multilayers N 5 5~ 0: U s 5: 4 a E 3 — E 3 .3. 2 ‘ s 1 A E 3 ° 0 s” t‘" «9 «'9 a?“ 53$ ’0'“ a?" 87 Figure 3.8 continued m e y ..IH. I 9 w «e M Var L b L m o m. w + 9&9 . ex . ll. 0 \ .e _l a i Q5 2 8 4 0 1 2.59: omega”. ONE ooh“. (C) Without LbL Multilayers 96 6 5 4 3 2 1 0 :53 3.3.3". 024. 62... 3:33:50 ((1) 88 Further, we observed that the amount of AraC released from LbL coated agarose was reduced if the concentration of bulk AraC solution at the beginning of LbL formation was reduced fi'om lmg/ml to 500pg/ml, as evident from cells exposed to LbL released AraC when the bulk concentration of AraC was 500pg/ml (Figure 3.9). In all the other cases discussed in this study, the bulk concentration of AraC at the beginning of LbL formation was 1mg/ml. This reduction in released AraC could be due to reduced AraC loading and thus less release of free AraC from the multilayer, as it is known that the amount of polymer adsorbed within the multilayer structure reduces with decreasing polymer concentration at the time of LbL formationmo. Figure 3.9 Fibroblasts cultured on a cover-slip in contact with LbL multilayer ([(PAA/PEG)5(Ara-C/PEG)5]3PAA) coated agarose, when the bulk concentration of AraC at the beginning of LbL formation was 500 pg/ml. Prior to exposure to the cells, the LbL coated agarose discs were incubated in cell culture media (without any cells) for 24 hrs, with fresh culture medium replaced at 30 min, 16 hrs, and 24 hrs. Cells were monitored for 3 days (indicated as Day 1 (48 hrs), Day 2 (72 hrs), and Day 3 (96 hrs)). Scale bar represents 100pm. Time in parenthesis denotes the time since the start of the multilayer degradation in culture media, whereby the initial 24 hrs of degradation was performed in the absence of cells. Control . Control Control Control Fresh Cells Day 1 Day 2 Day 3 (post 24 hrs) (48 hrs) (72 hrs) (96 hrs) 89 3.4 CONCLUSIONS Here, we show the controlled release of Ara-C from agarose to control reactive cell layer formation of fibroblasts in and around implanted scaffolds during axonal regeneration in central and peripheral nervous system. LbL multilayers of PAA/PEG/Ara-C were formed over agarose to delay the release of Ara-C from agarose. The inhibitory affect of released Ara-C on the growth of fibroblasts was demonstrated. The controlled release of free Ara- C from LbL coated agarose was observed for up to 96 hrs. Further, the amounts of Ara-C released per day were significantly low so that they would not affect the axonal growth. Since the prolonged Ara-C exposure at high concentrations is detrimental to neuronal culture, therefore the release of effective Ara-C only up to a certain period of time and at low concentrations was beneficial for axonal regeneration in vivo applications, To control the reactive cell layer in vivo, the microspheres or some other microstructures of agarose can be coated with LbL, washed for a few number of cycles at physiological pH to neutralize the pH and remove initial burst of Ara-C, and injected after a certain period of time post-implantation of scaffold. 90 CHAPTER 4 MULTILAYER MEDIATED FORWARD AND PATTERNED SIRNA TRANSFECTION USING LIN EAR-PEI AT EXTENDED N/P RATIOS 4.1 INTRODUCTION RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing process triggered through small interfering RNAs (siRNAs)3l' 32 which serves as a powerful therapeutic tool”’ 34 in gene therapy. An important aspect of gene therapy for regenerative medicine and organized tissue formation is to manipulate the location of transfected cells, requiring the generation of gene expression patterns in spatially controlled environments62' 63. Patterned delivery of DNA has been demonstrated with cells seeded onto modified surfaces, where vector-DNA complexes were immobilized onto chemically modified surfaces, including self-assembled monolayers (SAMs), using different patterning techniques63‘ 141‘ 142. Delivery of patterned siRNA from a substrate to adherent cells for high-throughput functional genetic analysis has been demonstrated with reverse transfection based RNAi microarrays143 ' ”4. Reverse transfection plates the cells at the time of transfectionm, whereas forward transfection plates the cells to allow them to first attach and grow, prior to transfection. Reverse transfection-based approaches for gene delivery or RNAi rnicroarrays requires that the cells must be able to adhere to the surface containing the expression vector, or the substrates must be chemically modified to immobilize the non-adherent cell linesms. Gene delivery from a substrate depends, in part, on the vector-nucleic acid complex that is bound to the surface“. Various 142 parameters such as surface charge, hydrophobicity/hydrophilicity , rigidity of the cell 146 adhesion substrates all contribute to the molecular interactions between the vector and the polymer on the surface. Any chemical modification of the substrates that may 91 enhance cell adhesion could adversely affect the release of the vector-nucleic acid complexes from the surface and thus interfere with cellular internalization of the polymer-nucleic complexes“, and efficient gene delivery. The present study describes a method for forward transfection of siRNA, yielding micron-sized patterns of transfected mammalian cells. With this method, the cells are cultured separately from a degradable LbL assembled multilayer arrayed with the siRNA, thereby separating the two issues, the complex release fiom, and the cell adhesion on the substrate. The layer-by-layer (LbL) assembly method introduced by Decher and co-workers39’ 4° for multilayer thin film formation is an attractive approach for controlled release of biomolecules from surfacesm. LbL thin films provide flexibility in terms of their choice of substrate and constituent components, surface patterning techniques, fabrication conditions, and tunable structural properties”. Other advantages include their ease of preparation and cost-effectiveness. Different patterning techniques can be employed to conjugate biomolecules, such as nucleic acids, to multilayer structures. Soft-lithographic micro-contact printing (pCP)64’ 65 is one such technique, which has emerged as a platform of choice for biochips and drug delivery applications“. Various “inks”, including, proteins, DNA, RNA, and polyelectrolytes have been used in pCP to pattern surfaces without the need for dust-free environments and harsh chemical treatments“. A previous method of in vitro localized transfection of cultured cells from multilayer thin films did not involve patterned delivery of DNA from these filmsm. Nonetheless, LbL thin film application of reverse transfection of DNA to form cell microarrays have been 92 previously demonstrated”, but the method is not easily applied to siRNA due to the enhanced susceptibility of siRNAs to degradation as compared to DNAs33' 51. Approaches to embed the polymer and nucleic acid alternatively to form LbL films149 or embed the polymer-nucleic acid complexes within the multilayers150 have not involved patterned delivery. Thus, spatially controlled delivery of siRNA based on thin film chemistries has yet to be realized. Here, we describe the application of a LbL assembled degradable multilayer film for patterned delivery of siRNA using a forward transfection approach. The transfection process involved the following steps (Figure. 4.1). (1) pH controlled, biocompatible and degradable multilayers were fabricated using LbL assembly under acidic conditions“. (2) Nanoparticles of vector-siRNA complexes prepared at physiological pH conditions were used as “ink” in pCP to form patterns on multilayer substrates. (3) Multilayer substrate containing patterned nanoparticles laid on top of cells at physiological pH conditions degraded the multilayer and formed patterns of transfected cells. This method is a variation of the forward transfection technique; herein denoted as the multilayer mediated forward transfection (MFT) method. Quantification of MP T efficiencies with linear polyethylenimine (LPEI)-siRNA nanoparticles found nitrogen/phosphate (N/P) ratios 2 30 gave significant transfection (2 60%). MFT of patterned siRNA provides an efficient and simple approach to spatially controlled siRNA delivery for tissue engineering applications. This method also provides a proof-of concept study towards the eventual development of a forward transfection-based cell microarray. 93 Figure 4.1 Diagram illustrating the multilayer mediated forward transfection (MFT) of cationic vector complexed siRNA for patterned delivery. Stamped (l) “Br-NA Vector nanoparticles Buffer Buffer Nano- particle ink Nanoparticle ink Q (h) ,“ze Kan PDMS stamp (a) . gcrflPligln-S 3"; 3‘ 1ft at physiological pH cat at P t“ . . e“.‘.',-,*. »‘.‘.\\1 "br‘: n- ' ‘a.- _._ .Kq.\“ .' .I-fl , . . fl“: ‘2‘:“v'e'. I 3].", ‘-!“.‘-‘ >\.L_I;\\" ‘ . "‘fie‘. , “_ei, ...,4“ \‘~ ”mg“: / ““2 \\1.,' 4 ~“ n“ .\ "i'.,"”. , ) J 1 J, i nth' ~ 1' i.» . wt%": .w‘w' .- “273}? 5 ‘ . v « -.-. v - ~. \ t“: r. ' ‘ . I » r 5‘." .1 ~ ' .t'x‘.'}?i.*'iy‘.1t9' ‘ 'f \_.j’ ‘L'_-‘ r. ‘ w‘) 1‘ '. 0., x, _ ‘1)k‘g‘gi 5, . . . L. {..t a _ - ~ I,‘ l “"‘ 7 r - . 0 ~ .- PDMS Stamp +<—‘ Air dry PDMS stamp ‘ x i. Hf” / . \x for 45 min Stamped 6,53314‘ ' (Cell culture medium: ’ ‘- nanoparticles ‘ physiological pH) PDMS ‘ . * Transfected cells (c) ‘ ‘ 53‘?” Q; (0 gaff-M . , ' ' I'l , - \\ —' g4 . i . \R ' ' ,/{ O 0 .1} '1‘; “if?!” ' (PANPEG)n.5 film ‘fxxl, // 1' ' K/ln age»? (Cell culture medium: fabricated at pH 2 ‘ “*7 ’ physiological pH) Efficient MFT is contingent on the formulation of the vector-siRNA nanoparticles. Normal forward transfection (NFT) was evaluated for a suitable range of nanoparticle formulations for MFT, using 25kDa LPEI as the non-viral gene delivery vector. Among the polymeric vectors, PEI is the gold standard for gene delivery, but its high transfection 46. 47 efficiency is often associated with high cytotoxicity . In addition, despite the superficial similarities of siRNAs and DNAs, their distinct characteristics, such as 33' 51‘ 59, results in significant differences in their optimal molecular weight and topography delivery formulation with transfection reagents”. Therefore, delivery vehicles and transfection conditions must be designed and optimized to cater to their individual 94 46-57 requirements for efficient delivery. The use of PEI as a polymeric DNA transfection reagent has been studied extensively, with PEI-DNA complexes analyzed for a broad 43'50' 54‘“: 58, up to a N/P ratio of 135”. However, to 0111' knowledge, range of MP ratios reported studies with siRNA transfection has been limited to narrow ranges of PEI- siRNA compositions (mostly limited to N/P ratio of 10)51'53’ 56‘ 57, until recently15 1. A recent study characterized ketal modified and unmodified branched PEI (BPEI)-siRNA complexes up to a N/P ratio of 10015], but a similar N/P ratio analysis have not been performed with LPEI. 22kDa LPEI was reported unable to provide in vitro siRNA transfection, while 25kDa BPEI successfully mediated siRNA transfection (with 200nM siRNA) at N/P ratios up to 852. However, BPEI is more cytotoxic than LPEI46’ 52. Thus, there is a need for a less toxic form of PEI that can provide siRNA silencing (preferably, better silencing), at similar or lower siRNA concentrations than used previously with BPEI. Therefore, in this study we evaluated a broad range of LPEI-siRNA N/P ratios (ranging from 5 to 90) using 25kDa LPEI as the transfection reagent. For comparison, 25kDa BPEI was also evaluated as a transfection reagent. Our results indicated LPEI to be a better siRNA transfection reagent than BPEI using siRNA concentration of 200nM. We characterized the LPEI-siRNA nanoparticles for this range of N/P ratios. We found that (1) complete incorporation of the siRNA was achieved at N/P ratio near 90, which produced transfection efficiency greater than 90% and nanoparticles ~50nm in size, (2) partial incorporation of the siRNA was observed at N/P ratios between 30 to 75, which produced transfection efficiencies greater than 80%. The nanoparticle size was ~150nm 95 for N/P ratios between 30 to 60 and less than 100nm for N/P ratio of 75, and (3) further reduction in the incorporation of siRNA was observed at N/P ratios of 5 and 15, no transfection was observed at N/P ratio of 5 and ~40 % transfection was observed at N/P ratio of 15. The nanoparticles size was greater than 200nm for N/P ratio of 5 and ~150nm for N/P ratio of 15. Finally, N/P ratio of 30 for LPEI-siRNA nanoparticles was optimal for achieving high NFT (~85%) transfection efficiency with minimum cytotoxicity. 4.2 MATERIALS AND METHODS 4.2.1 Materials Poly(acrylic acid, sodium salt) solution (PAA, Mw 15,000; catalog no. 416037), Poly(ethylene glycol) (PEG, Mw 10,000; catalog no. P6667), and branched polyethylenimine (BPEI, Mw 25,000; catalog no. 408727) were purchased from Sigma- Aldrich Chemical (Milwaukee, WI). The linear polyethylenimine (LPEI, Mw 25,000; catalog no. 23966) was obtained from Polysciences, Inc (Warrington, PA). Poly(dimethylsiloxane) (PDMS) from the Sylgard 184 silicone elastomer kit (Dow Corning, Midland, MI) was used to prepare stamps. Bamstead Nanopure Diamond (Bamstead International, Dubuque, IA) purification system with a resistance of > 18.2 MQ cm was used as a source for deionized (DI) water. Solution pH was adjusted using HCl or NaOH. Layer-by-layer (LbL) assembled multilayer films were made on quartz slides (Technical Glass Products, OH) that were cut to fit 12-well tissue culture polystyrene (TCPS) plates (Costar, Corning, NY) Lipofectamine 2000 (LF2k) transfection reagent (1mg/ml), OptiMEM reduced serum medium (catalog no. 31985), Dulbecco’s Modified Eagle Medium (DMEM), penicillin, streptomycin, were purchased 96 from Invitrogen (Carlsbad, CA). Fetal bovine serum (F BS) was purchased from Biomedia Corp (Foster City, CA). HeLa cells were purchased from American Type Culture Collection (Rockville, MD). Custom synthesized siRNA with sense sequence 5’- GGUGAAGGUAGAUCAAAGAdeT-3’ and anti-sense sequence 5’- UCUUUGAUCUACCUUCACCdeT-3’, targeting human double-stranded RNA- dependent protein kinase (PKR) mRN A was purchased from Dharrnacon (Chicago, IL), and diluted to a concentration of 80pM. Fluorescein and Alexa Fluor-555 conjugated double-stranded RNA (dsRNA) oligomers (Block-iT Fluorescent and Block-iT Alexa Fluor Red Fluorescent Oligos) (Invitrogen, Carlsbad, CA) were used to demonstrate patterned delivery in MFT. 4.2.2 Cell Culture HeLa cells were maintained in DMEM with 10% FBS and lOOU/ml penicillin plus lOOpg/ml streptomycin (P/S) at 37°C and 10%COZ. For transfection and cytotoxicity studies, HeLa cells were cultured and grown to complete confluency in 12-well plates in 1.2 ml of P/S free DMEM supplemented with 10% F BS. 4.2.3 Degradable Layer-by-Layer (LbL) Multilayer Fabrication Hydrogen (H)—bonded (PAA/PEG)n_5 multilayers were fabricated at a deposition pH of 2.0. PAA and PEG polymer solutions used to fabricate multilayer assemblies were prepared in DI water to final concentrations of 1 mg/ml. The pH of both solutions was adjusted to 2.0 and filtered with a 0.22 pm cellulose acetate filter (Corning, NY). DI water adjusted to pH 2.0 was used as the wash solution. PAA consists of C00” terminal 97 groups, which due to their acidification forms H-bond with PEG molecules at low pH conditions“. These H-bonded multilayers degrades upon immersing into physiological pH conditions“. Different numbers of bilayers of PAA and PEG were prepared with PAA as the terminating layer in each case. Quartz slides were cleaned in piranha solution (7:3; concentrated sulfuric acid: 30% hydrogen peroxide), dried under N2 gas and further cleaned using a plasma cleaner (Harrick Scientific Corporation, NY) for 10 min at 0.15 Torr and 50sccm flow of 02. A Carl Zeiss slide stainer was used to deposit multilayers on quartz substrates. Quartz substrates were immersed in lOOmM LPEI solution (pH ~7.4) for 30 min to provide an initial positive charge and then rinsed in DI water for 3 min with agitation. Subsequently, the substrates were dipped into PAA solution for 15 min followed by 3 min in wash solution with agitation. The substrates were then dipped into PEG solution for 15 min followed by 3 min in wash solution with agitation to create one bilayer. The dipping cycle was repeated to form multilayer films. Multilayer films are abbreviated as (PAA/PEG)“, where n is the number of PAA and PEG bilayers and the “.5” indicates an additional, single terminating layer of PAA. 4.2.4 PDMS Preparation Patterned PDMS stamps were created by curing degassed prepolymer and initiator (10:1) mixture against a microfabricated silicon master in an oven overnight at 60°C, as described elsewhere“. The masters consisted of features: parallel lines from 200-250 pm and squares from 200-700pm. Non-pattemed PDMS stamps were prepared against a plane silicon wafer as the master. PDMS stamps were cut to the size of multilayer 98 substrate in order to obtain uniform transfer of nanoparticles with minimum loss during the stamping process. 4.2.5 PEI-siRNA N anoparticle Formation lOOmM LPEI and lmM BPEI stock solutions (molarities with respect to repeat units of the polymer) were prepared in DI water and adjusted to pH 7.2. Volumes of LPEI or BPEI mixed with siRN A were calculated for the different nitrogen/phosphate (N/P) ratios (the ratio of protonable amine groups on PEI to phosphates on siRN A). OptiMEM was used as a buffer to prepare nanoparticles in all cases, except for the zeta(§)-potential analysis. N/P ratios of 5, 15, 30, 45, 60, 75 and 90 were used for LPEI-siRNA and N/P ratios of 5, 10 and 15 were used for BPEI-siRNA formulations. Solutions of PEI and siRNA were prepared separately in OptiMEM buffer (at physiological pH) at the calculated concentrations (at room temperature), and mixed within 5 minutes after their preparation. For agarose gel electrophoresis assay, UV/vis, C-potential, DLS, NFT and cytotoxicity studies; PEI and siRNA solutions were prepared in separate volumes of lOOpl, giving 200p] of mixed volume. For MF T, C—potential analysis of the nanoparticles released from the multilayers, SEM and AF M; PEI and siRN A solutions were prepared in separate volumes of 12.5p1 and mixed to give 25 pl of nanoparticles. PEI-siRNA mixture was kept at room temperature for 30 minutes prior to use in transfection or characterization studies, unless specified otherwise. 4.2.5.1 N/P Ratio Calculation 99 (N/P) =(n1v X MEI X VPEI) /(np X nsiRNA> (41) where, = N/P ratio Mp5] = Molarity of PEI stock solution (based on the repeat units) VPE] = Volume of PEI stock solution used at a particular N/P ratio nN = Number of nitrogen atoms per repeat unit of PEI (nN = 1 for 25kDa LPEI and 11 for 25kDa BPEI) np = Number of phosphates per siRNA molecule (np = 40 for the selected siRNA) nsiRNA = Number of moles of siRNA 4.2.6 N anoparticle Stamping (Microcontact Printing) onto Multilayers After multilayer and nanoparticle formation, the next step of MFT was stamping of the nanoparticles onto the multilayer using PDMS as the stamping elastomer. PDMS stamps were plasma treated for 2 minutes and drop-coated with nanoparticles. The plasma treatment facilitated spreading of the nanoparticle ink, enabling temporary electrostatic interactions between the nanoparticles and the SiO" groups on the PDMS. To minimize loss of the nanoparticles, the stamps were air dried for 45 minutes rather than dried with N2 assistance. As the nanoparticle ink dried, the PDMS recovered partial hydrophobicity causing the dried nanoparticles on the surface to have weaker interactive forces with the PDMS. Nanoparticles were transferred to (PAA/PEG)n,5 multilayers through pCP. PAA (pKa ~ 5) when incorporated into a multilayer assembly remains partially ionized even at the pH of 2.0 152. During stamping, the weak binding of the nanoparticles to the PDMS 100 facilitated their transfer to the partially negative PAA terminated multilayer. Non- patterned stamps were used for quantifying the transfection efficiency. 4.2.7 Normal Forward Transfection (NFT) and Multilayer mediated Forward Transfection (MFT) HeLa cells were transfected with different nanoparticle formulations for 48 hr and the cell-culture medium was not changed post-transfection. NF T: LPEI-siRNA nanoparticle solution (200pl) was added to the cultured cells in 1 ml of fresh cell culture medium. For NFT, the final concentration of siRNA was 200nM, unless specified otherwise. For comparison, LF2k (2pg) and BPEI were also used as transfection reagents, with 40pmoles (533nM) and 240pmol (EZOOnM) final concentration of siRNA, respectively. MF T: Multilayer quartz substrates containing the LPEI-siRNA nanoparticles were sterilized under UV light for at least 15 minutes. Prior to transfection the cell culture medium was removed, and the quartz substrate was placed top-down onto the cultured cells. 1.2 ml of fresh culture medium was added. For MFT, 240pmol of siRNA (5 200nM final concentration in NFT) was used, unless specified otherwise. For comparison, LF2k (2pg) with 40pmoles of siRN A (E33nM final concentration in NFT) was also evaluated. 4.2.8 Characterization 4.2.8.1 Agarose Gel Electrophoresis Assay Relative amount of free siRNA in the LPEI-siRNA nanoparticle preparation at each N/P ratio was evaluated by a gel retardation assay. NanOparticles were prepared as described 101 above at a constant siRNA concentration of 200nM at each N/P ratio. A 15p1 aliquot of the samples with 3 pl of loading buffer (Bio-Rad, CA) was loaded on a 0.8% agarose gel prepared in 1X Tris-boric acid-EDTA (TBE) buffer (Bio-Rad, CA). Electrophoresis of the LPEI-siRNA nanoparticles was run in 1X TBE buffer at 110V for 30 min. siRNA bands were visualized using SYBR gold nucleic acid gel stain (Invitrogen) and a UV transilluminator. 4.2.8.2 Ultraviolet Visible (UV /vis) Absorbance LPEI-siRNA nanoparticle solutions prepared from 750nM final concentration of siRN A at the different N/P ratios were diluted in 1.2m] of OptiMEM buffer (at physiological pH). Higher siRNA concentration was used to obtain measurable absorbance values which did not alter the calculation of the amount of siRNA incorporated into the nanoparticles. UV/vis peaks were measured in a 10mm path length quartz cuvette at 25°C and with a wavelength interval of lnm using SPECTRAmax Plus 384 (Molecular Devices, CA). 4.2.8.3 Scanning Electron Microscopy (SEND and Atomic Force Microscopy (AFM) Constant siRNA amount of 240pmol (E 200nM final concentration in NFT) was used with varying concentrations of PEI. SEM images of air dried nanoparticles were collected with field emission JEOL 6300F electron microscope. AF M images were collected in the tapping mode using 300kHz silicon probes (Vista probes) with a Nanoscope IV multimode scope from Digital Instruments (Santa Barbara, CA). 102 4.2.8.4 Dynamic Light Scattering (DLS) LPEI-siRNA nanoparticle solutions prepared from 750nM final concentration of siRNA at the different N/P ratios were diluted to 3.0ml of total volume in OptiMEM buffer (at physiological pH). Hydrodynamic particle size of LPEI-siRNA nanoparticles was determined by DLS at 25°C with a 90Plus/BI-MAS multi-angle particle size analyzer, Brookhaven Instruments Corp, NY. The wavelength of the 15mW solid laser used was 660nm, and the scattering angle used was 900. Dust filter value of 30 was used. Refractive index for particles in each sample was taken as 1.50 + 0i. Refractive index and viscosity of the aqueous suspension used was 1.33 and 0.89cP, respectively. Measurements were performed within 30 min of mixing the LPEI and siRNA for nanoparticle preparation. Each measurement was performed for 10 runs per sample, each run of 2 minutes. Intensity-weighted size distribution in the multirnodal size distribution (MSD) analysis mode (based on Non-Negatively constrained Least Squares (NNLS) algorithm in MAS OPTION software from Brookhaven) showed bimodal distribution of particles, with a primary population in a size range less than 500nm and a second population in a range greater than 5pm. PEI-nucleic acid complexes tend to form aggregates over time in the buffer solutions 55’ 153. These aggregates were detected in the second population of particles. Since PEI-siRNA complexes greater than 150nm, which is the size limit for non-specific cellular uptake through clathrin-coated pitsm, have been reported unable to mediate gene silencingsz, only the primary population size of the particles are reported. Mean and standard deviation were plotted by the number-weighted size distribution in MSD analysis mode. Particle sizes were corroborated by scanning electron and atomic force microscopy. 103 4.2.8.5 Zeta (Q—potential LPEI-siRNA nanoparticles were prepared in nuclease free DI water, as explained above. siRNA concentration was kept constant at or equivalent to 750nM. Higher amount of siRNA was used to obtain detectable count ratessz. C-potential values of “direct complexes” (i.e., nanoparticle complexes immediately after their formation and diluted to final volume of 1.5 ml of nuclease free DI water) and “multilayer released complexes” (i.e., nanoparticle complexes released from multilayers in a volume of 1.5 ml nuclease free DI water) were measured in polystyrene cuvettes at 25°C using ZetaPALS, zeta potential analyzer (Smoluchowski model), Brookhaven Instruments Corp, NY. 4.2.8.6 Fluorescence Analysis of Patterned Delivery Confocal laser scanning microscopy (CLSM) images were obtained using Olympus Fluoview 1000 laser scanning confocal microscope. Conventional fluorescence images were collected using Leica DM IL inverted microscope (Bannockbum, IL) equipped with SPOT RT color camera (Diagnostics Instruments, MI). 4.2.8.7 Real-Time Quantitative Reverse Transcriptase- Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from cells with RNeasy mini kit (Qiagen, Valencia, CA) and depleted of contaminating DNA with RNase-fi'ee DNase (Qiagen). Equal amounts of total RNA (1 pg) were reverse-transcribed using an iScript cDNA synthesis kit (Bio-RAD). The first-strand cDNA was used as a template. The primers used for qRT-PCR analyses of human PKR (5'-CCTGTCCTCTGGTTCTTTTGCT-3' and 5'- 104 GATGATTCAGAAGCGAGTGTGC-3')‘55 , and human GAPDH (5'- AACTTTGGTATCGTGGAAGGA-3' and 5'-CAGTAGAGGCAGGGATGATGT-3') were synthesized by Operon Biotechnologies, Inc. (Huntsville, AL). RT-PCR was performed in 25-pl reactions using 1/ 10 of the cDNA produced by reverse transcription, with 0.2 pM of each primer, 1 X SYBR green supermix from Bio-RAD, and at an annealing temperature of 60 °C for 40 cycles. Each sample was assayed in three independent RT reactions and triplicate reactions were performed and normalized to GAPDH expression levels. The cycle threshold (CT) values corresponding to the PCR cycle number at which fluorescence emission in real time reaches a threshold above the base-line emission were determined using MyIQTM Real-Time PCR Detection System (Bio-RAD). 4.2.8.8 Cytotoxicity Tests HeLa cells were cultured with different nanoparticle formulations (with 200nM of siRNA) for 48 hr and the supernatant was collected and stored at -80°C until analysis. Cells were washed with phosphate buffered saline (PBS) and kept in 1% triton-X-IOO in PBS for 24 hr at 37°C and 10%COz. Cell lysate was collected, vortexed and centrifuged at 500rcf for 10 min. Cytotoxicity was measured using cytotoxicity detection kit (Roche Applied Science, Indianapolis, IN) as the fraction of lactate dehydrogenase (LDH) released into the medium, normalized to the total LDH (released + lyzed). 4.2.9 Statistical Analysis 105 All experiments were performed at least three times, and representative results are shown. All data, unless specified, are shown as the mean i SD. for indicated number of experiments. Student t-test was used to evaluate statistical significances between different treatment groups. Statistical significance was set at p<0.01, unless specified otherwise. 4.3 RESULTS AND DISCUSSION A broad range of N/P ratios (ranging from 5 to 90) of the LPEI-siRNA nanoparticles was characterized for their degree of siRNA incorporation into the nanoparticles. Concomitantly, we find a blue shift in the UV/vis absorbance of the polymer-nucleic acid complexes with increasing siRNA incorporation into the nanoparticles, as a fimction of N/P ratio. These nanoparticles were further characterized for their zeta ((1)-potential, sizes, normal forward transfection (NFT) efficiencies and cytotoxicities. LPEI as the delivery vector provided high transfection efficiencies with 200nM siRNA as compared with ~33nM, a standard concentration used with Lipofectamine 2000 (LF2k)”6, and therefore 200nM was used to evaluate the LPEI formulations. Subsequently, the LbL assembled multilayer mediated forward transfection (MFT) of siRNA was demonstrated, and the transfection efficiencies quantified and compared with LPEI and LF2k as the transfection reagents. 4.3.1 Physical Property Evaluation of LPEI-siRNA Nanoparticles at Different N/P Ratios 4.3.1.1 Agarose Gel Electrophoresis 106 To determine the degree of incorporation of siRNA into the nanoparticles at varying N/P ratios, we performed agarose gel electrophoresis of LPEI-siRNA nanoparticles ranging fi'om N/P ratio of 5 to 90, at a constant siRNA concentration of 200 nM (Figure 4.2). At 200 nM siRNA and no LPEI (N/P = 0), the siRNA is completely unbound (i.e., all free siRNA) (lane 1); at 200 nM siRNA and 31 pg/mL LPEI (N/P = 90), the siRNA appears to be completely bound, i.e., no free siRNA appears in the gel (lane 8). Therefore the gel suggests that all the siRNA incorporated into the nanoparticles at N/P ratio greater than 75 and near 90. Figure 4.2 Agarose gel electrophoresis of LPEI-siRNA nanoparticles at various N/P ratios with 200nM siRNA concentration. Numbers indicate the N/P ratios. (0 N/P ratio indicates the naked or free siRNA). 0 5153045607590 F a ._ _ :‘ .. {,3MK' MK 4.3.1.2 Ultraviolet-Visible (UV/vis) Spectroscopy We observed a maximum peak for LPEI in DI water at 240nm for all the concentrations used in this study, without siRNA or any additional components (Figure 4.3a). Note, this 107 is different from previous spectrophotometric methods that detected for PEI, where the PEI was either complexed with copperm, or a protein based assay was used“. We observed a LPEI peak at 244nm when suspended in OptiMEM buffer (a buffer used for transfection studies), without siRNA or any additional components (Figure 4.3b). The difference in the LPEI peak in water vs. in buffer could be due to the presence of salts in the buffer solution, and the resulting change in effective dielectric function159 of the LPEI-buffer solution from that of the LPEI-water solution. We measured the UV/vis absorbance of the LPEI-siRNA nanoparticles as a function of increasing N/P ratios. The peak absorbance wavelength of the LPEI-siRNA nanoparticles underwent a linear blue shift away from 260nm (characteristic peak of nucleic acid, Figure. 4.3c inset) as the LPEI concentration increased. The peak gradually shifted towards lower wavelengths with increasing N/P ratios; shifting to 256nm at N/P ratios of 5, and 244nm at N/P ratio of 75 and higher (Figures 4.30 and 4.3d). The plasmon band of the nanoparticles at N/P ratio of 75 (and higher ratios) was the same as the plasmon band for LPEI without siRNA in OptiMEM buffer solution. Indeed this gradual UV shift has been observed with bimetallic “core-shell” and alloy nanoparticles. Mallik et aim0 showed that progressive covering or encapsulation of gold particles by silver layers resulted in a UV/vis blue shift. As the silver covered the gold, the plasmon band was silver dominated. Similarly, core-shell type silver—gold alloy nanoparticles showed a red shift with a single intermediate absorbance peak as concentration of the gold in the nanoparticles increasedm. Their results indicated that the peak shift depended on the ratio of the two metals. Based upon these literature results and our gels results, we 108 suggest that the gradual shift in the UV peak correlate with the increasing incorporation of the siRNA into the nanoparticles, resulting in less free or naked siRNA with increasing amount of LPEI at the higher N/P ratios (at a constant siRNA concentration), reaching complete incorporation of the siRNA at N/P ratio greater than 75. Figure 4.3 Ultraviolet-visible (UV/vis) absorption spectra of LPEI suspended at varying concentrations in: (a) DI water, or (b) OptiMEM buffer, without siRN A, showed maximum peaks at 240nm and 244nm, respectively. (“LPEI concentration (E N/P ratio)” corresponds to the concentration of LPEI for a given N/P ratio calculated for siRN A final concentration of 750nM). Same peak positions were observed for all the other LPEI concentrations used in the range for nanoparticle fabrication. (c) UV/vis absorption spectra of LPEI-siRNA nanoparticles show a blue shift with increasing N/P ratios. (Inset: Maximum UV/vis peaks of siRNA and LPEI in OptiMEM are 260nm and 244nm, respectively). ((1) Plot of the wavelengths corresponding to the absorbance peak of LPEI- siRNA nanoparticles as a function of the N/P ratio. Abs. Abs. La) .JU’E' W = 240: Water) (we: Amax . 244; OptiMEM Butter) 1 W2 t’ l 5—5e.1pgzmr (a NIP 45) ' (b):’“‘ t' t —5a.1,.9m (a up 45) 2. t' ‘t i- 474-2991."! 1' 1119135)} 0 8 t g - -1742pglmt(l NIP 135) 0 8 it I x ' : . o 1: ‘x’ “ 4’ i ()43 041 . . r . 4i éuhv in" j 4 200 240 280 320 360 (k) 200 240 280 320 360 0.) Abs. kmax nm 1 2 (c) NIP 13,: LPEI(244nm) ( ) (d) ‘ a; “.‘(SIRN A 2 5 0:; 0.8 __ ' (260nm) 04f W30 N”) 45 245i 200 240 280 320 360 (7») 0 5 15 30 45 60 75/90/ (N/P) Ratio LPEI Only 109 4.3.1.3 Particle Size Analysis Scanning electron microscopy (SEM) images were taken for N/P ratios from 5 to 90 (Figure 4.4a). Several of the sizes were further confirmed with atomic force microscopy (AFM) (Figure 4.4b). Overall, the size of the nanoparticles decreased with increasing N/P ratios, with significant changes in size for N/P ratios in the range of 5 to 15 and 60 to 75. The size of the nanoparticles was greater than 200nm at N/P ratio of 5, decreased to ~150nm at N/P ratios between 15 and 60, decreased further to less than 100nm at N/P ratio of 75, and ~50nm at N/P ratio of 90. The hydrodynamic sizes of these nanoparticles were measured using dynamic light scattering (DLS) (Figure 4.4c), and found to be similar to the sizes obtained with SEM and AF M. 110 Figure 4.4 Variation in the size of LPEI-siRNA nanoparticles as a function of N/P ratio. (a) Scanning electron microscopy (SEM) images of the nanoparticles and their sizes as a function of N/P ratio. Scale bar represents 500 nm, and the error bars indicate the standard deviations of at least ten measurements performed on three different scanned areas per sample. (b) Top images - Atomic Force microscopy (AFM) images of LPEI- siRNA nanoparticles at N/P ratios of 30 and 90 (using 240pmol of siRNA). Middle images — Particle size distribution at these N/P ratios. Bottom images — Section analysis of three representative particles of different sizes within a given image. (c) Histograms of DLS determined nanoparticle sizes for N/P ratios of 5, 45, 60 and 75 show a decrease in the average particle size with increasing N/P ratio. (a) 5 15 30 45 60 75 9O NIP Ratio 111 Figure 4.4 continued (b) 150.0nm 730nm 0.0nm 20° 111930 A180 . Avg=127.40:20.02 nm 5160 5140 3120 ”100 '3 so ~ “g 50 0. 4o - m l 2031110457910 Particle Number on image nm 1 100- l . ,1 JK , . 0 _- J «‘«Kmumvflae5-kw "E's-Lit - 100 - Section Analysis (mesa) 0 10 pm 20 QOnm 0 10.0 20.0 30.00”“ 30 €25 Avg: 22p 39 t 3. 72 nm E :20 .5 W15 % E10 0- 5 0 811296741351210 Particle Number on Imaae 30 i y I). Y 5 i i it. «...! . ...-’»M."~"‘Z‘”W"°-._-st——--~~—- 0 ._-,_;J~__,'4' .. I. 1", “.... ‘ fl Section Analysis (NIPQO) - 30 . . 0 10 “m 20 30 112 Figure 4.4 continued (e) NIP Ratio 1:» 217-13.2.9.9..rmm ..NIE FE‘19,1§;1§%:§$-?2;5 N." (S-d-L moi ‘ 100» ' 1 m* m. b so» 5 so» 3 70» 8 701 g a). g 60f Z 50, Z 50» 40. 4o} 30. . 30L 202- i 202- ~ 10? 1 10;» L 1 ”‘76:,“ 200 360100010000 io““"”“""‘1‘éo zoo ”@“ibbb‘”“‘""“"“”“‘1‘dooo Diameter (nm) Diameter (nm) 11 NIP Ratio 60: 155.6 a: 7.9 nm (s.d.) 11o [Ni/P Ratior75: 175.7 ; 8.§ nm (so) 1Wr ‘ 1 100' so? 90- Got 80;? g m. J g m: . gee» g 602- z 50 4 2 5o. 40L 40' . 30:. ‘ 30 2°? . ‘ 2° . 1053 10- . ‘i‘o ‘ ‘ ‘ ‘iéo 200M5651ooo1oooo 90““‘” 106M266M663‘i60éiw""mwi‘déoo Diameter (nm) Diameter (nm) 4.3.1.4 Zeta (Q—potential Analysis C-potential was measured to assess the relative charge of the nanoparticles at the different N/P ratios. As more siRNA was incorporated into the nanoparticles, the C-potential shified to more positive values (Figure 4.5). A negative C-potential value was obtained at N/P ratio of 5 (~ —10mV) with its magnitude less than that of pure naked siRNA (~ —30 113 mV). This suggests that most of the siRNA remain in free form at low N/P ratio of 5, which is corroborated by the agarose gel (Figure 4.2). N/P ratio of 15 shifted the 8;- potential from negative to positive, suggesting more siRNA incorporated into the nanoparticles than at N/P ratio of 5. Further increase in N/P ratios to between 30 and 75 increased the C-potential to 20-25mV, suggesting enhanced siRNA incorporation. At N/P ratio of 90 the C—potential reached ~30mV, similar to the C—potential of pure LPEI at an equivalent LPEI concentration to N/P ratio of 90. The C-potential measurements corresponded with the UV/vis and agarose gel electrophoresis results indicating complete incorporation of the siRNA at N/P ratio near 90. C—potential was also measured immediately after the stamped nanoparticles were released from the multilayers at 370C. C—potential values were negative for N/P ratios 5 60, and positive for N/P ratios of 75 and 90. In addition to the nanoparticles, PAA and PEG were released upon multilayer degradation, presenting the possibility of further interaction of PAA or PEG with the released nanoparticles. The presence of PAA itself in the solution or PAA coating of the nanoparticles, or PEG followed by PAA coating of the nanoparticles could result in negative C-potential at the lower N/P ratios (5 to 60). Interestingly, enhanced gene silencing was observed with MFT, as compared to NFT, for N/P ratio of 5 (discussed below)), which may be indicative of further PAA or PEG interaction with the released nanoparticles (discussed further in section 4.3.5). Such an increase in MFT efficiency was absent at N/P ratios of 15 to 90, suggestive of the presence of PAA or PEG in solution with minimal interaction with the nanoparticles. These N/P ratios (5 to 60) gave negative C-potentials. The positive C—potential values at 114 the higher N/P ratios of 75 and 90, albeit slightly lower than that obtained with the “direct complexes”, suggested minimal coating, if any, of these nanoparticles or that the higher LPEI concentration minimized the effect of PAA and PEG in solution. Figure 4.5 Zeta-potential values of LPEI-siRNA nanoparticles as a function of N/P ratio, immediately after they were formed (Direct Complexes) and released upon multilayer degradation (Multilayer Released Complexes) in deionized water, measured at 250C. ”LPEI (N/P90) only” corresponds to the amount of LPEI for N/P ratio of 90 without siRNA. Error bars indicate the standard deviation of three measurements of ten runs per sample. 50 $40 éaod 20~ 10. 6- Direct Complexes + Multilayer Released Complexesj 750nM NIP 5 NIP 15 NIP 30 NIP 45 NIP 60 NIP 75 NIP 90 LPEI siRNA (NIP90) Only Only 4.3.2 Multilayer mediated Forward Transfection (MFT) for Patterned siRNA Delivery, and Effect of the Number of Bilayers For MFT, the multilayer substrate containing the nanoparticles was positioned over a confluent monolayer of cultured mammalian cells. The physiological pH of the culture medium resulted in the disintegration of the multilayer and release of the nanoparticles 115 from the multilayer and delivery to the cells. Figures 4.6 and 4.7 shows the patterned delivery to cells via MFT. Placing the substrate on top of cells was not detrimental to health of the cell. This was evident from the cell images in Figures 4.6, 4.7, 4.8, and from the high yields of total RNA extracted from the cells after transfection (see qRT-PCR characterization in Materials and Methods Section for RNA extraction process; RNA yield data not shown). A similar procedure of placing the substrate over the cultured cells has also been previously demonstrated by Lynn and co-workers, where the authors show the non-pattemed localized DNA delivery from a LbL assembly”. 116 Figure 4.6 Fluorescent images demonstrating patterned siRNA delivery to HeLa cells with multilayer mediated forward transfection (MFT) using (PAA/PEG)6.5 multilayer assembly, fluorescent dsRNA oligomers (lOOpmol) and Lipofectamine 2000 (LF2k, Sug). Nanoparticles and HeLa cell patterns transfected with: (a) Alexa F luor SSS-labeled oligomers, (b) F luorescein and Alexa F luor SSS-labeled oligomers (overlaid images). Top panel- CLSM images of LF2k-fluorescent oligomer nanoparticles arrayed onto multilayer. Middle and bottom panels- HeLa cell patterns transfected with fluorescent oligomers and their corresponding phase contrast images acquired using CLSM (middle panel) and conventional fluorescence microscopy (bottom panel). Scale bar represents 500 um. (a) The degradation kinetics of PAA/PEG multilayers reported by Ono and Decher“, showed that less than 7 bilayers of PAA/PEG did not release the upper films as self-standing, floating films, however bilayers greater than 7 released the upper films within 30 minutes. Here, we find the release of the nanoparticles for patterned delivery and transfection efficiencies were independent of the number of bilayers. We evaluated 117 patterned delivery and quantified MFT efficiencies (quantification discussed in section 4.3.3) for 6.5 bilayers (Figures 4.6 and 4.9b) and 30.5 bilayers of multilayers (Figures 4% and 4.7) and found they were similar. Since stamping of the nanoparticles onto (PAA/PEG)n.5 multilayers formed only an additional monolayer of particles (instead of a complex film), this could explain the thickness independent release of the nanoparticles fi'om the multilayers. Figure 4.7 Conventional fluorescence microscopy images demonstrating square patterned siRNA delivery to HeLa cells with MFT using (PAA/PEG)30,5 multilayer assembly, fluorescent dsRNA oligomers (40pmol) and Lipofectamine 2000 (LF2k, Zug). Top Image- Fluorescence image of LF2k-fluorescein labeled oligomer nanoparticles arrayed onto multilayer. Middle and Bottom images- HeLa cell patterns transfected with fluorescein labeled oligomer; and their corresponding phase contrast images. Scale bar represents 100 um. 118 To confirm that the transfection is due to the release of the complexes upon degradation of the film and subsequent cellular uptake of the complexes, we evaluated a plasma- treated bare quartz substrate (negatively charged) which should be similar to a non— degrading multilayer; both are non—degrading and bind the complexes to its surface. We stamped the complexes onto the plasma-treated quartz substrate, and observed no transfection after 48 hrs, suggesting that the complexes were not released and did not penetrate into the cell merely through cell/complex interaction (Figure 4.8). 119 Figure 4.8 LPEI-siRNA nanoparticles stamped on a plasma-treated quartz did not release from the substrate to the cell culture medium. These nanoparticles remained intact on the quartz even afier 48 hrs. There was no transfection observed with the cells. (a) Conventional fluorescence microscopy image of the intact patterns of LPEI-fluorescein labeled dsRNA oligomer nanoparticles, imaged on the quartz substrate, were stamped onto plasma-treated quartz and placed onto the cultured cells for 48 hrs. The edges of the intact patterns on the quartz are clearly visible. Scale bar represents 200 um. (b) Conventional fluorescence microscopy image of HeLa cells (corresponding to image a) after contact with the stamped quartz substrate for 48 hrs showed no appreciable transfection. No patterns of transfected cells observed in image b. Guidelines were created manually for areas (A1 and A2), representing where the patterns should have transferred to the cells had the MFT been successful. Very few cells, if any, show fluorescence in image b. (0) Phase contrast images of the untransfected HeLa cells corresponding to image b. (d) qRT-PCR of PKR gene expression levels in HeLa cells, 48 hr post-contact with LPEI-siRNA nanoparticles stamped on a plasma-treated quartz, indicating no transfection. MFT and NFT efficiencies for N/P ratio 45 are shown for comparison. S is a > 0 ..J C .9 O ll 0 L- Q 060 X ill 0 C O 0 m X Q '8 Cells Only Plasma Treated MFT- NIP 45« NFT~ NIP 45 Bare Quartz 30.58Ls with Stamped Complexes 120 4.3.3 Evaluation of Transfection Efficiency: Real-time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) Analysis To evaluate the transfection efficiencies of MFT, non-pattemed PDMS stamps were used to stamp nanoparticles containing custom designed siRNA targeting the double-stranded RNA-dependent protein kinase (PKR) gene. MFT efficiencies with LPEI (at different N/P ratios) and LF2k as transfection reagents were compared with those of NFT using LPEI, BPEI (at different NIP ratios) and LF2k as transfection reagents (Figures 4.93 and 4.9b). NFT efficiency using LF2k with 33nM siRNA gave more than 80% silencing. Gene silencing was not observed for LPEI with 33nM of siRNA (at the highest NIP ratio of 90). In support, a previous study demonstrated that to achieve sufficient silencing 200nM of siRNA was required with BPEI at N/P ratios of 6 and 852. Therefore, we selected a siRN A concentration of 200nM for the gene knockdown evaluations with LPEI. There was no NFT observed with LPEI at N/P ratios of 5, 40% silencing at N/P ratio of 15, and more than 80% silencing at N/P ratios of 30 and higher (Figure 4.9a). BPEI, similar to LPEI, showed no silencing at N/P ratio of 5, however, its transfection efficiency was better than LPEI for higher N/P ratios. BPEI at N/P ratio of 10 gave similar while BPEI at N/P of 15 gave higher level of silencing as compared with LPEI at N/P ratio of 15. Conversely, BPEI was more cytotoxic than LPEI at these N/P ratios (see section 4.3.4). MFT efficiencies were lower than NFT at similar N/P ratios for the LPEI-siRNA nanoparticles. This reduction in efficiency may be due to either the loss of nanoparticles 121 during the stamping process, with some of the nanoparticles sticking to the stamp and not transferring to the multilayer, or potential dissociation of the nanoparticles, i.e., LPEI sticking to the PDMS and exposing the nucleic acids. This reduced efficiency was also evident with the stamping of the LF2k-siRNA nanoparticles (used 40pmol siRNA E 33nM in NFT, a standard concentration used with LF2k156) in the MFT process (Figures 4.9a and 4.9b). To assess whether the preparation time was a factor, the NFT efficiency of LF2k-siRNA complexes (typical NFT) was evaluated after 20 mins (standard Preparation time of LF2k-siRNA complexes156 ) and 2.5 hrs (average process time of MFT), and found to be similar. The large standard deviations associated with MFT may be attributed to the experimental variation introduced through the manual (non- automated) transfer of the nanoparticle particles by the pCP process. MFT of siRNA can be performed with any transfection reagent, as long as the nanoparticles remained stable and functional through-out the stamping process. MFT can be performed on any cell type, in addition to the ones described here. MFT efficiencies were _>_ 60% for LPEI-siRNA nanoparticles at N/P ratios 2 30. When compared to NFT efficiencies, there was an unexpected increase observed in MFT efficiency for nanoparticles at N/P ratio of 5. This may be attributed to interaction of the larger nanoparticles (greater than 200nm) with the PEG and PAA upon multilayer degradation in the culture medium, enhancing transfection at N/P ratio of 5 (discussed firrther in section 4.3.5). The small amount of silencing observed with “LPEI (N/P 45) Only” mock sample in Figures 4.9a and 4% is essentially noise in the qRT-PCR characterization. 122 Figure 4.9 qRT-PCR quantified expression levels of PKR gene knockdown in HeLa cells 48 hr post-transfection of siRNA delivery using 25kDa LPEI or 25kDa BPEI at different N/P ratios, or Lipofectamine 2000 (LF 2k, Zug) with: (a) normal forward transfection (NFT) and (b) multilayer mediated forward transfection (MFT). Amount of siRNA was 240pmol (5 200nM final concentration in NFT) and transfection reagent was LPEI, unless specified otherwise. Numbers in parenthesis denote the amount of LPEI corresponding to the noted N/P ratio of 45, but without siRNA. BLs denote the number of bilayers. Error bars indicate the standard deviations of RT-PCR reactions on three independent samples. #, #p<0.01 as compared with “cells only” (i.e. cells without any transfection reagent or siRNA). Wp<0.05 as compared with “cells only”. *p<0.05 compared with “N/P 5 in Figure 4.9(a)”. (a) ‘ "1a)N‘om'ar'samaid‘migrant...('ur‘néniagaaes 123 Figure 4.9 continued (b) g ‘30 ' (b) nun1139a"a';aia.aiaagea'Transfectioninflame..." ..r 1.00 ###-* I 3' g 1 mm #41 r 1 ‘ .3 0.80 4 l' m 2 0.00 .1 l g- I l I u: 0.40 I I I ; g l o 0.20 : o . g 0.001 n. 6 <\ o ‘0 °’ " " ” ” q g c c v v v v V V V V 9 3 of? '3’ '3’ ‘3’ '3’ t? ‘3’ ’3' ‘3‘” e‘ 4’ 4? 93° 5'? "9 ”9 1‘? «§‘ 6? ‘3? 0 § ‘5 § ‘8 ‘5 \5 \5 § 12‘ \8 a." 6” it, e " Q? 45) 6° 3“ gr v a- s § § § a} 3Q ~2- <5" 5" Q j' 4» ___—- v # 4.3.4 Cytotoxicity Evaluation: Transfection Reagent and N/P Ratio Optimization Cytotoxicity measurements along with transfection efficiencies were used to determine the optimum N/P ratio and transfection reagent for NFT and MFT. A good transfection reagent must provide high transfection efficiency with minimal cytotoxicity. LDH release was used to assess the cytotoxicities of the LPEI-siRNA, BPEI-siRNA and LF2k- 33nMsiRNA complexes during NFT (Figure 4.10). The cytotoxicity level for BPEI-siRNA N/P ratio of 10 was much higher than for LPEI- siRNA nanoparticles at N/P ratios from 5 to 30. Increasing the BPEI-siRNA N/P ratio to 15 increased cytotoxicity (Figure 4.10) as well as transfection efficiency (Figure 4.9a), 124 137. i! till ‘5) g.»- “a 1.x a: 1‘0 but the transfection efficiency was still lower than for LPEI-siRNA nanoparticles at N/P of 30. Within a narrow range of N/P ratio (less than 10) evaluated previouslysz, BPEI was reported to provide higher transfection efficiency than LPEI. Our results are similar to previous findings at the low N/P ratio range; however, at a high N/P ratio of 30, LPEI provided higher transfection efficiency and lower cytotoxicity than BPEI at the low N/P ratio of 10 (Figures 4.9 and 4.10). Therefore, LPEI at N/P ratio of 30 was deemed a better choice over BPEI, based on transfection efficiencies and cytotoxicities. However, LPEI if added to cells at very high concentrations, even in the absence of siRNA, is toxic to the cells. Therefore, as the N/P ratios increases greater than 45, i.e., as more LPEI is added, the observed toxicity increases, as expected. The cytotoxicity of LPEI-siRNA nanoparticles at N/P ratio of 30 was found to be comparable to that obtained with the LF2k-33nMsiRNA complexes (Figure 4.10). However, LPEI at N/P ratio of 30 was a better choice than LF2k for MFT, since lower siRNA amounts cannot provide high MFT (Figure 4.9b), and to increase the siRNA amounts in the LF2k-siRNA complexes would require higher amounts of LF2k, which would finther enhance the toxicity (Figure 4.10). 125 Figure 4.10 Cytotoxicity levels in HeLa cells 48 hr post-NFT of siRNA delivery using 25kDa LPEI or 25kDa BPEI at different N/P ratios, or Lipofectamine 2000 (LF2k, Zug). Concentration of siRNA was 200nM and transfection reagent was LPEI, unless specified otherwise. Numbers in parenthesis denote the amount of LPEI or BPEI corresponding to the amount of PEI at the noted N/P ratio but without siRNA. Error Bars indicate the standard deviations of %LDH release of three independent samples. *p<0.01 compared with “N/P 45” and “BPEI-N/P 10”. LDH Released (%) 4.3.5 Relationships between Degree of siRNA Incorporation, §~potential, Size and Transfection Efficiencies of LPEI-siRNA nanoparticles Based on the UV/vis and agarose gel electrophoresis results, the plasmon peak shifted to the blue region, as the concentration of LPEI increased, and reached that of pure LPEI when all the siRNA incorporated into nanoparticles and no free siRNA remained. The average nanoparticle size decreased as the N/P ratio increased. Nanoparticle size greater than 200nm with negative C-potential may be inferred to have less siRNA incorporated given the large amount of unbound siRNA in lane 1 of the agarose gel (Figure 4.2) and the maximum UV/vis peak was at 256nm (Figure 4.3c). This was associated with no gene silencing (N/P ratio 5). Nanoparticle size ~150nm with positive C—potential between 20- 126 25 mV may be inferred to have more siRNA incorporated since lanes 2-7 of the gel show less unbound siRNA and the position of the UV/vis peak shifted to intermediate wavelengths. Complete siRNA incorporation into nanoparticles may be inferred at nanoparticle sizes 550nm with C-potential Z30mV (similar to the C—potential of pure LPEI at an equivalent concentration) since lane 8 of the gel shows no free siRNA and the UV/vis peak shifted to 244 nm, which is the maximum peak for LPEI. A possible explanation for the decreasing size of the nanoparticles could be that at increasing amounts of LPEI and a fixed amount of siRNA (i.e., increasing N/P ratios), there may be more electrostatic repulsion between the LPEI molecules at a given level of electrostatic attraction between LPEI and siRN A, thus contributing to smaller sized particles. Correspondingly, minimal silencing was observed at N/P ratio of 15 and greater than 80% gene silencing was observed for N/P ratios ranging from 30 to 75 . Transfection efficiency was increased fiirther at N/P ratio 90. Therefore, the smaller size nanoparticles, with 25kDa LPEI, provided more efficient NFT efficiencies. Particle size of ~150 was observed to be the cut-off size for efficient cellular uptake, which agreed with previously reported size limit of 150nm for non-specific cellular uptake through clathrin-coated pits154 . N/P ratio of 15 was an exception, despite their particle size of 150 nm, they gave lower transfection as compared with the higher N/P ratios from 30 to 60 (Figure 4.9a). However, the smaller blue shift and low C—potential at N/P ratio of 15 could explain, in part, the reduced transfection efficiency. 127 A possibility exists that some of the small nanoparticles observed at the high N/P ratios are aggregates of LPEI alone in solution. However, since we observed high level of silencing at these N/P ratios, this coupled with the fact that nanoparticles larger than 150nm are reported unable to provide siRNA silencing”, we believe the small size LPEI nanoparticles contain siRNA molecules and are not likely aggregation of LPEI. The lower C—potential values for the nanoparticles released from the multilayers suggest that the released PAA or PEG from the film may also interact with LPEI-siRNA nanoparticles. Increase in transfection efficiencies at N/P ratio of 5 is observed for MFT as compared to NFT. Amino and carboxylic acid pendant PEG chain coating on PEI/DNA complexes have been shown to increase their transfection efficiencies, even at 162 153, 163 3 negative C-potentials . Also, PEG is known to reduce nanoparticle aggregation modulating the complex properties (i.e., surface charge, size, and complex-cell interactions) leading to improved transfection efficiencies in some casesm’ 164. These previous studies support the possibility that PAA or PEG could alter the properties of larger nanoparticles (greater than 200nm) in solution to enhance the MFT efficiency at N/P ratio of 5. 4.3.6 Hypothesis for number of 25kDa LPEI molecules per siRNA molecule ”LPEI(siRNA) = [(MLPE/ X VLPE1(N / P)comp) / nsiRNA]X [FWLPEI / MW LPEI] (4.2) where, "mg/(siRNA) = Number of LPEI molecules encapsulating a single siRNA molecule 128 VLPEmv / p)comp = Volume of LPEI stock solution used at N/P ratio of complete encapsulation MW LPEI = Molecular weight of LPEI (25 kDa) F WLPEI = Monomer molecular weight of LPEI (43 g/mol) Substituting VLPEIW / Hemp from equation (4.1) into equation (4.2) and taking my =1 for LPEI, we get; nLPE1(siRNA) = (N / P)comp X (np) X (FWLPEI / MW LPEI) (4.3) where, mmp = N/P ratio which provides complete encapsulation As explained in section 4.3.1.1 and 4.3.1.2, complete encapsulation of siRNA by 25kDa LPEI occurred at N/P ratio near 90. If we assume N/P ratio of 90 provides complete encapsulation, then from equation (4.3), anE[(SiRNA) is estimated to be ~ 6 with 25kDa LPEI. Although evidence of the number of LPEI molecules that may surround a siRNA molecule does not exist, a model has been put forth on the number of atelocollagen (a highly purified type I collagen) molecules that could surround a single siRNA 165 165 molecule . Svintradze and Mrevlishvili proposed a molecular model for the interaction/binding of a collagen triple helix and siRNA through hydrogen bonding. They propose that 5-8 atelocollagen molecules may arrange around a siRNA molecule to create a fiber complex. Collagen triple helix in aqueous solution orients as a stiff rod-like helix 129 structure, which Svintradze and Mrevlishvili’s model suggests arranges around the siRNA with both their longitudinal axes aligned in the same direction. The hydrogen bonds between atelocollagen and the phosphates on the siRNA is proposed to form either directly (atelocollagen-siRNA(PO4)) or through water molecules (atelocollagen-H20- siRNA(PO4)). There is evidence in the literature that multiple LPEI molecules orient in a planar zigzag florrnd66,l67 (discussed below), raising the possibility that LPEI is similar to atelocollagen in that they both are planar molecules, one planar zigzag and the other rod-like, respectively. Note the overall dimensions of LPEI are slightly smaller than atelocollagen (Table 4.1). The LPEI may bind with the phosphate groups on the siRNA in a similar fashion as atelocollagen with siRNA. The binding may be through either electrostatic interactions between the nitrogen on LPEI and the phosphate on siRN A or through direct hydrogen bonding between the nitrogen on LPEI and the hydroxyl group on the siRNA (or through a water bridge, similar to atelocollagen). In light of this, it is possible that there could be multiple 25kDa LPEI molecules that surround a siRNA molecule. Table 4.1 Average dimensions of an atelocollagen, a 25kDa LPEI and a siRNA molecule. Atelocollagen LPEI (Planar siRNA (double- (Triple helix, stiff, zig-zag) stranded, A- rod-like)” form)165’ ‘63 Length ~300nm ~214nm ~5.6nm Diameter ~ 1.5 nm < 0.66 nm ~ 2.6 nm Multiple LPEI chains have been shown to stack together in aqueous form. Yozo Chatani 1166.167 et a confirmed from X-ray structure analysis that hygroscopic LPEI is present as 130 crystalline dihydrate in aqueous state. The lattice parameters of the crystalline LPEI dihydrate monoclinic unit cell are a = 13.26 A, b = 4.61 A, c (fiber axis) = 7.36 A and fl = 101.00 (where a, b, c represent the lattice dimensions and ,6 is the angle between a and c). Each unit cell contains 4 LPEI monomeric (-CH2CH2NH-) units (with each structural repeat unit having 2 monomeric units) and 8 water molecules166 . The polymer chains orient in a planar zigzag with alternating stacks of polymer chains and water molecules arranged in parallel along the be plane. These chains are held together by multiple hydrogen bonds between the NH groups and water molecules. Taking into account these parameters, one can estimate the length and diameter of a single LPEI chain containing 581 monomeric units (see Table 4.1). In the presence of siRNA it is not known exactly how the LPEI chains interact with siRNA. But given what is known about LPEI in the aqueous state and the proposed interaction of atelocollagen with siRNA, it is possible that the LPElzsiRNA complexes could be present at a 6:1 average ratio. This average ratio could take on a number of configurations; the two extreme cases would be i) 6 individual LPEI polymer chains surrounding a siRNA, similar to the atelocollagen and siRNA scenario, or ii) a single unit of 6 alternating stacks of LPEI/water attached to the siRNA molecule, which is entropically unlikely. It is more likely that the real scenario is in between these two extremes, for example, multiple, i.e. , 3 LPEI chains with 2 alternating stacks of LPEI/water units, surrounding a siRNA molecule, or multiple LPEI chains surrounding several siRNA molecules or some variation thereof. To fully explore the interaction of 131 LPEI and siRNA and the resulting structure would require further investigation that is beyond the scope of this study. 4.4 CONCLUSIONS The MFT method provided a forward transfection approach that used degradable multilayers for patterned siRNA delivery to cultured cells. Forward transfection provides an advantage over reverse transfection in terms of tuning the substrate chemistry, such as the multilayers for polymer-siRNA immobilization, separately from the substrate modification that may be required for cell adhesion. Lower siRNA amounts (~40 pmol E 33nM siRNA in NFT) did not provide sufficient MFT efficiencies, whereas higher siRNA amounts (~24O pmol E 200nM siRNA in NFT) provided 2 60% transfection efficiencies of LPEI-siRNA nanoparticles at N/P ratios greater than 30. Therefore, based on the cytotoxicities and transfection efficiencies, LPEI at N/P ratio of 30 was a better choice than LF 2k for MF T, even though they both gave comparable NFTs. We selected and characterized N/P ratios ranging from 5 to 90 for their UV/vis spectra, relative amounts of siRNA incorporated into the nanoparticles, C—potentials and nanoparticle sizes. UV/vis shift to 244nm, C potential of 30mV, and complete siRNA incorporation was achieved at N/P ratio near 90. NFT efficiencies increased with decreasing nanoparticle size and increasing N/P ratio. 25kDa LPEI was found to be a highly efficient transfection reagent at N/P ratio of 30, providing greater than 80% transfection efficiencies at siRNA concentration of 200nM, better than BPEI. Finally, MFT provides a method for forward transfection of siRNA, yielding micron-sized patterns of transfected 132 mammalian cells, and may be a potential approach for developing cell microarrays based on forward transfection. 133 CHAPTER 5 FABRICATION OF LINEAR-PEI NANOPARTICLES USING HIGH SHEAR RATE MIXER 5.1 INTRODUCTION Polymeric nanoparticles have emerged as the efficient drug delivery carriers that can be fabricated and loaded with therapeutic molecules using numerous strategies, and can carry these molecules to inside the cells. Their efficient delivery to cells is contingent on their physical and chemical properties, such as size and shape”’ 60’ 61 , and investigating these properties has been the focus area of many research studies these days. Size of the LPEI-siRNA nanoparticles is known to play an important role in efficient delivery of the therapeutic molecule, siRNA, to the mammalian cells”. In a previous study, LPEI-siRNA nanoparticles were fabricated at different compositional concentration ranges, and evaluated for the relationships between their varying sizes and transfection efficiencies. Nanoparticle size less than 150nm was found to be required for the efficient uptake Of LPEI-siRNA complexes by cells”. However, the problem associated with such nanoparticles is their aggregation in solution over a period of time lead to large and polydisperse particles, which is compromising for the enhanced cellular uptake and also for other nano-size based applications. Therefore, it is necessary to obtain uniform sized and non-aggregating nanoparticles for their high efficiency in nucleic acid transfection. In addition to size, another important parameter of the nanoparticles is their shape, which can play an important role in efficient cellular uptake, but have not been evaluated in- depth till so far. The effect of the different shapes of micro and nanoparticles60 on uptake by the phagocytic cells or macrophages (cells which can engulf large, > 0.5pm, 134 particulate targets) has been reported recently by Champion and Mitragotrim. These authors reported that there is an inter-play between size and shape of particles for their uptake by macrophages, where shape of the particles plays a critical role in initiating the phagocytosis and size impacts the completion of phagocytosis“. However, the effect of particle shapes on the delivery of DNA or the therapeutic siRNA carrying nanoparticles has not been reported yet. One of the constrictions in particle shape investigations of nanoparticles for different applications is the lack of commonly applicable fabrication techniques for creating particles of different shapes and uniform sizes. Inter-polymer complexes (IPCs) results from the interaction of two or more polymers in a solutionlw'177 and is one of the many techniques employed to fabricate polymeric nanoparticles. The main driving force associated with the IPCs formation is the gain in entropy of the system, with forces such as electrostatic interaction (forming inter- polyelectrolyte complexes (IPECs))169'm, H-bondingm’174 and hydrophobic interaction contributing to the complexation of interacting polymers175 . The concept of inter-polymer mixing has been under investigations since many years, much before the commencement of LbL assembly technique in 90’s”’ 95‘ 96’ 173' 174. The properties associated with IPCs are directly related to the properties of multilayersm; such as, the equilibrium between the IPCs formation and multilayer stability/erosion in a solution, both are governed by the type of polyelectrolyte chains, the ratio of their lengths and charge, ionic strength, and pH 176 of solutions . The size and polydispersity of the IPCs dispersions formed are known to be influenced by polymer structure (e. g. length), polymer mass, the ratio between charges of polyelectrolytes, polymer mixing ratio, pH, concentration, and temperature 170' ”1' 173' 135 m. A new aspect on the preparation and properties of IPECs forming nanoparticles of different sizes has been illustrated by Dragan, Mihai and Schwarz, where these authors showed that rate of addition of polyelectrolytes (rate of titration or mixing) can have significant impact on the dimensions of the resulting nanoparticlesm. As discussed above, particle shape can play an important role in efficient cellular uptake. Among the polymeric vectors, PEI is the gold standard for gene delive 8' 46’ 47. However, the current protocols for PEI mediated siRNA or DNA delivery38' “6'57 are based on conjugating these nucleic acids to PEI or modified PEI, which is the post complexation nanoparticle formation and do not yield the particles with novel shapes. Therefore, as an alternate strategy for a more efficient gene delivery, LPEI nanoparticles with novel shapes and/or uniform sizes can be prepared before siRNA or DNA complexation to PEI. These particles can further be used to make inter LPEI-siRNA complexes and the novel shape and/or uniform sizes of these complex particles can possibly help in enhanced siRNA delivery to cells. Due to increased surface area of the pre-forrned nanoparticles, the use of pre-formed PEI nanoparticles may be advantageous than using PEI polymer solution, as this may result in increased siRNA loading to nanoparticles leading to an efficient siRNA delivery. 5.2 MATERIALS AND METHODS 5.2.1 Materials The linear polyethylenimine (LPEI) (Mw 25,000, catalog no. 23966; and Mw 2500, catalog no. 24313) was obtained from Polysciences, Inc (Warrington, PA). Pluronic F68 136 (triblock copolymer of polyethylene glycol and polypropylene glycol) was purchased from Sigma-Aldrich (USA). Bamstead Nanopure Diamond (Bamstead International, Dubuque, IA) purification system with a resistance of > 18.2 M!) cm was used as a source for deionized (DI) water. 5.2.2 LPEI-IPC Micro and Nano-Particles Formation Unless specified otherwise, all the particles were fabricated using 25kDa LPEI. Chloroform (CHC13) or ethanol were used as the organic solvent in which LPEI was dissolved at the different concentrations (as specified in Results and Discussion), and water or water-glycerol mixture was used as the non-solvent in which Pluronic F 68 (henceforth referred as F68) was dissolved at a concentration of 5mg/ml at the room temperature. LPEI in chloroform was dissolved at temperatures > 60°C. Chloroform and water were pre- saturated with each other before solution preparations. To fabricate LPEI-IPC (i.e. LPEI-F68) micro and nano-particles, separate solutions of LPEI and F68 were prepared prior to homogenization at high shear rates, and the two solutions were then mixed to emulsify under high shear mixing conditions. Glycerol was used to increase the viscosity of solvent mixture in some cases. To dissolve F68 in high volume % glycerol, F68 was mixed with glycerol at the mixing speed of 30m/s for 2 min (where the final vessel temperature was 60°C and the solution obtained was milky in appearance). Henceforth, the emulsion system of LPEI in chloroform and F68 in water or glycerol will be referred as LPEI(CHC13)-F68(H20) or LPEI(CHC13)-F68(y%Glycerol) in this study, where y denotes the volume % of glycerol in non-solvent water. 137 The two solutions of LPEI-CHClg or LPEI-ethanol(20ml) and F68-H20 or F68(y%Glycerol)(60ml) were homogenized in mixer vessel at the different circumferential speeds of 10m/s, 20 m/s, 30 m/s, 40m/s or 50 m/s, with the coolant continuously circulating outside the mixing vessel at 4°C or 20°C temperature to control the final vessel temperature. The total solution volume in mixer vessel was 80ml. Mixer vessel was kept either open to atmospheric pressure, or vacuum was applied through a side vent. In the conditions, where vacuum was applied, the total solution volume in mixer vessel was 60 ml (20 ml of polymer in solvent, and 40ml of surfactant in non- solvent), unless specified otherwise. Vacuum was removed after 20—30 see after stopping the mixer. High shear rates resulted in abrupt increase in solution temperatures. 5.2.3 Scanning and Transmission Electron Microscopy (SEM and TEM) SEM images of air dried particles were collected with field emission JEOL 6300F scanning electron microscope. TEM images of nanoparticles were collected with JEOL 100CX transmission electron microscope. Nanoparticles were drop coated on the Formvar coated TEM copper grids and were not stained with any heavy metal compound. Staining was not done purposely, so that the conclusive images showing the hollow nature particles (if any) can be judged based on the differences in contrast due to two polymers (LPEI and F68) and organic residual deposit of CHCl3, only. 138 Unless specified otherwise, if there was any phase separation, then the particles for SEM and TEM were collected from the water phase after solution stabilization. All the emulsions made at any percentage of glycerol were dialyzed extensively against water using a dialysis membrane of molecular weight cut-off value 12000 and 4.8nm pore diameter, before SEM or TEM sample preparation. 5.3 RESULTS AND DISCUSSION 5.3.1 High Shear Rate Mixer We used high shear rate mixing coupled with some other parameters such as the application of vacuum, change of organic phase solvent, variation in solvent viscosity. and different physical parameters such as different turbine geometries (Figure 5. lb) to fabricate LPEI-IPC (i.e. LPEI-F68) particles of varying shapes and sizes. To our knowledge, the use high shear rate mixing coupled with parameters, such as application of vacuum, is a first demonstration for the fabrication of micro- and nano-particle of unique shapes and uniform sizes. The present work involved the use of a high shear rate imparting thin-film spin system, “T.K.FILMICS® Model 80-50”, from PRIMIX Corporation, Japan. A micrograph showing the mixer chamber of this high shear rate mixer is shown in Figure 5.1a. It consists of two concentric cylinders; where inner cylinder (turbine) is the perforated cylinder (of various perforation designs) of diameter 5.20m (Figure 5.1b), and outer cylinder is a solid cylinder with a diameter of 5.8cm. The turbine (inner cylinder) can 139 rotate at high circumferential speeds, viz. 10 m/s, 20m/s, 30m/s. 40m/s, and 50m/s, imparting high shear rates (and thus high shear forces) on the materials being mixed. Figure 5.1 Micrographs showing: (a) mixing vessel of the high shear rate mixer and (b) different turbines designs available (T.K.FILMICS®, PRIMIX Corporation, Japan). (a) Atm Pressure, or Vacuum Overflow Outlet Turbine Circulating Temperature Coolant Probe Inlet for Temperature Control (b) The mixer described above has an open outlet (Figure 5.1a), which can be used for multiple purposes, such as injecting a polymer solution at a desired rate while other components mixing in the chamber, or modulating the pressure of the chamber during components mixing. In this study, this outlet was set to be either at atmospheric pressure, 140 or vacuum was applied during high shear rate mixing. Application of vacuum during emulsification at extremely high shear rate mixing led to particles of interesting shapes. The mixing speed during particle formation, when coupled with other suitable parameters, can have an impact on the physical properties of resulting particles. For example, it was recently demonstrated that a single emulsion process of poly(d,l-lactic) acid (PLA) polymer with Pluronic F68 as a surfactant generated bowl shaped micro and name-particles at high shear rate mixing under specific conditions of temperature, time 178 and high viscosity . An emulsion process involves the following steps: the polymer is dissolved in a solvent and this solvent is added to a non-solvent like water containing surfactant (stabilizer); which is then altogether homogenized to form an emulsion containing polymer-solvent droplets and subsequently solvent can be evaporated or diffused outm’ 180. However, the emulsion process requires a very selective set of conditions for polymers and solvents, as two of the prime conditions are that the solvent and non-solvent should be immiscible and polymer should not be soluble in non-solvent, even at the conditions such as high temperature achieved during homogenization. This can sometimes impose limitations on the combinational choice of polymers and solvents that can be used with this process for particle fabrication. On the other hand, the method of IPCs formation is amenable to the choice of solvents, where all the polymers can be mixed and homogenized in same solvent. 141 5.3.2 Fabrication of LPEI-[PC Micro and Nano-particles under High Shear Rate Mixing To fabricate LPEI-[PC particles, we followed the approach of a single emulsion process. The particle formation was based on the physical interactions, namely H-bondingm, between hydrophilic cationic LPEI and surfactant F 68 mixed together at high shear rates. The structures of LPEI and F68 are shown in Figure 5.2. The melting point of LPEI is 73- 79°c (Polysciences Inc.), and the boiling point of CHC13 is 61 .2°c. Figure 5.2 Structural formulae of linear polyethylenimine (LPEI) and Pluronic F68. (a) H + +' f H N H N n CH3 0 H OH x Y z LPEI (0.5 mg/ml) in CHC13 and F68 (5mg/ml) in water were homogenized at the 'J—f— 12 (1)) different mixing speeds of lOm/s, 20m/s, 30m/s, 40m/s and 50m/s for 15 min each. LPEI in chloroform was injected to F 68-water solution in mixing vessel while running when the vessel temperature was stabilized. High shear rate mixing led to increase in temperature as a function of speed, and yielded particles of different sizes. The 142 temperatures were less than 45°C for speeds less than or equal to 40m/s (with higher temperatures at the higher speeds), and reached greater than 61 2°C (the boiling point of CHCI3) at the speed of SOm/s. Due to the formation of crystalline hydrates, LPEI in the free base form is insoluble in water at temperatures below 600C166' 181. However, LPEI can be dissolved in water at temperatures higher than 60°C. Similarly, LPEI can be dissolved in organic solvent chloroform (CHC13), but only at high temperatures. Thus, due to low temperatures (<600C) at speeds S 40m/s, it can be assumed that LPEI did not dissolve in water phase during mixing and remained dissolved in CHC13 phase. In such a condition, we expected the formation of LPEI containing CHC13 droplets to get dispersed in water i.e. the formation of oil in water (o/w) emulsion. However, the particle suspension was not observed to be stable after homogenization, and there was a chloroform-water phase separation for all the samples mixed at speeds less than 40m/s (Figure 5.3). This phase separation could be due to much higher density of CHC13 than water, resulting in LPEI-CHC13 droplets to settle down and form an interface with water. Interestingly, at the mixing speed of 40m/s, there was no phase separation observed even in the presence of chloroform and the particle suspension was stable for long periods, although the highest temperature achieved was still less than 45°C (less than boiling point of chloroform). Due to increased temperatures, more of the CHCl3 was evaporated at higher speeds and was completely boiled-off from the solution at the speed of 50m/s. Solution overflowed through the vent of mixer at the mixing speed of 50m/s, when the temperature reached above the boiling point of chloroform. Table 5.1 shows the summary of temperatures and phase separation as a function of mixing speeds. 143 Table 5.1 Parameters and conditions obtained during and after high shear rate mixing when LPEI(CHC13) was injected to vessel after F68(H20) reached at maximum temperature in mixer. All the temperatures shown are with-in the error limits of :tZOC. Total run time was 15 min, and coolant temperature was maintained at 4°C. Mixing Speed Injection Temp Final Temp CHCl3 - H20 Phase and Time separation 10 m/s 60 C; 50 sec 60 C Heavy 20 m/s 110 C; 50 sec 12° C Heavy 30 m/s 210 C; 70 sec 230 C Moderate 40 m/s 4F)C; 80 sec 430 C No Phase separation 50 m/s 620 C; 60 sec 660 C No Phase separation (No CHC13 left) Figure 5.3 Micrographs showing particle suspension obtained after homogenization of LPEI (in CHC13) and Pluronic F68 (in H20) solutions at the mixing speeds of lOm/s, 20m/s, 30m/s, 40m/s and 50m/s (fi'om left to right, respectively) without any subsequent solvent diffusion or evaporation. LPEI(CHC13) was injected to vessel after F68(H20) reached at maximum temperature in mixer. Phase separation was observed for the speeds of 10 m/s, 20m/s, and 30 m/s. 144 Figure 5.4 shows the SEM images of particles obtained after emulsifying the two solutions at various mixing speeds, and after evaporation of the solvent phase (chloroform) from droplets (recovered from water phase after phase separation) dried on a substrate. The average particle size was found to increase with increase in mixing speed from 10m/s to 40m/s. This increase in particle size is well correlated to the more evaporation of chloroform at higher mixing speeds. As more of the chloroform was evaporated, given that the temperature was below the melting point of LPEI so that LPEI could not melt, the particle size was found to be large. This could possibly be due to more expansion of polymer creating more voids within the droplets for the evaporation of solvent phase from droplets. The particle sizes were non-uniform at all the mixing speeds and a clear trend of more aggregation (coalescence) of particles was observed with increasing mixing speeds. Particles obtained at the speed of 50m/s, where the vessel temperature was above the boiling point of CHC13, showed maximum level of coalescence. This high coalescence of LPEI at high temperatures (high speeds), particularly at the speed of 50m/s, could be due to the absence of solvent (chloroform), where LPEI particles could possibly form inter hydrogen bonds among themselves and caused coalescence. Overall in the LPEI(CHC13)- F68(HzO) emulsification process at the high shear rates (Table 1, Figures 5.3 and 5.4), particle size was less and phase separation was more at the lower mixing speeds where less chloroform was evaporated due to low mixing temperatures. Particle aggregation was more at the higher mixing speeds. 145 Figure 5.4 SEM images of the particles obtained after homogenization of LPEI (in CHCl3) and Pluronic F 68 (in H20) solutions at the mixing speeds of 10m/s, 20m/s, 40m/s and 50m/s, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. LPEI(CHC13) was injected to vessel after F68(HzO) reached at maximum temperature in mixer. The meshed structure observed in images is the aluminum oxide filter membrane (sample substrate) through which the particles were filtered and dried. 146 5.3.2.1 Effect of High Viscosity and High Shear Rate Mixing on Particle Shape Glycerol can be used as a solvent to increase the system viscosity, where higher volume percent of glycerol give more viscous solution. Viscosity of glycerol decreases with increase in temperature182 . The decrease in viscosity of 100% glycerol is between 3 to 1.5 folds over the increment of 10°C starting from 0°C to 100°C, with more significant decrease (3 to 2 folds decrease) at temperature increments in lower temperature range (0- 50°C) and less significant decrease (2 to 1.5 folds decrease) at temperature increments in higher temperature range (so-100°C)‘32. The hypothesis behind increasing the system viscosity in LPEI(CHC13)- F68(Glycerol/HzO) emulsion system was to apply high shear force on softened or melted LPEI by the virtue of viscosity, so that LPEI can obtain some novel shape on re- solidification. This can be done at the temperatures higher than the melting point of LPEI (73-790C) i.e. at high mixing speeds. However, the limitation of increasing the temperature to such high values is that chloroform (solvent for LPEI) would boil-off (boiling point of chloroform 61 .20C) and the system viscosity would decrease due to high temperature. To keep the system viscosity as high as possible at high temperatures, we dissolved F68 in 100% glycerol instead of adding any water to form LPEI-IPC (i.e. LPEI- F68) particles. LPEI (1 mg/ml) in CHC13 and F 68 (5mg/m1) in water were homogenized at the different mixing speeds of lOm/s, 30m/s, and 50m/s for the different times, as mentioned in Table 5 .2. LPEI(CHC13)-20ml and F68(100%glycerol)-60ml solutions were added in mixing 147 vessel and mixed at high shear rates. High shear rate mixing led to increase in temperature as a fimction of speed and time, and yielded particles of different sizes. Similar to as discussed above in section 5.3.2, the particle suspension was not observed to be stable after homogenization, and chloroform-water phase separation was observed for all the samples at low mixing speeds (< 30m/s) (Figure 5.5). At the mixing speed of 30m/s and above, there was no phase separation observed where most or all of the chloroform was evaporated due to high temperature. Table 5.2 shows the summary of temperatures and phase separation as a function of mixing speeds. Table 5.2 Parameters and conditions obtained during and after high shear rate mixing of LPEI(CHC13)-F68(l00%Glycerol). All the temperatures shown are with-in the error limits of :l:2°C. Coolant temperature was maintained at 4°C. Mixing Speed Final Temp Run Time CHC13 - Glycerol Phase separation 10 m/s 8” c 15 min Heavy 30 111/8 600 C 10 min No Phase separation 50 m/s 750 C 45 sec No Phase separation (No CHC13 left) 148 Figure 5.5 Micrographs showing particle suspension obtained after homogenization of LPEI (CHC13) and Pluronic F68 (100%Glycerol) solutions at the mixing speeds of lOm/s, 30m/s, and 50m/s (from left to right, respectively) without any subsequent solvent diffusion, evaporation or dialysis. Phase separation was observed for the speed of 10 m/s. Figure 5.6 shows the SEM images of particles obtained after emulsifying LPEI(CHC13) and F68(100%Glycerol) at various mixing speeds, and subsequent dialysis of the emulsion mixture in water. The average particle size was found to increase with increase in mixing speed from 10m/s to 30m/s. This increase in particle size is well correlated to the more evaporation of chloroform at higher mixing speeds, as also discussed above in section 5.3.2. As more of the chloroform was evaporated, given that the temperature was below the melting point of LPEI so that LPEI could not melt, the particle size was large. This could possibly be due to more expansion of polymer within the droplets creating more voids for the solvent phase to come out from inside the droplets. At 30rn/s, where most of the chloroform was evaporated, a slight effect of the shear force due to high viscosity is observed as illustrated by the somewhat non-spherical LPEI-IPC particles. 149 Particles of shapes other than perfectly spherical i.e. of rectangular, triangular, trapezoidal shapes were obtained. Contrary to the hypothesis discussed above for the effect of viscosity on particle shape, aggregation (coalescence) of particles was observed at the mixing speeds of 50m/s (where the vessel temperature was above the boiling point of CHC13 and melting point of LPEI) instead of obtaining particles of different shapes by the virtue of viscosity on melted LPEI. This high coalescence of LPEI at the speed of 50m/s could be due to the absence of solvent (chloroform), where LPEI particles could possibly form inter hydrogen bonds among themselves and caused coalescence. Had the melted LPEI been dissolved in a solvent, it can be expected for the formed particles not to stick to each other as they would have more affinity (bonding) to their solvent than with each other. In such a case, one can expect for the melted LPEI dispersed in solvent to take a novel shape when mixed at a high shear rate in a viscous solution. Overall in the LPEI(CHCl3)-F68(100%glycerol) emulsification process at the high shear rates (Table 5.2, Figures 5.5 and 5.6), particle size was less and phase separation was more at the lower mixing speeds where less chloroform was evaporated due to low system temperatures. Particle aggregation was observed at 50 m/s where system approached the temperature of melting point of LPEI. In addition to its effect on the particle shape, another noticeable effect of glycerol was that it helped in reducing particle aggregation/coalescence at the temperatures less than 150 the boiling point of solvent (chloroform). In the absence of glycerol, there was a tendency of LPEI particles to coalesce due to inter hydrogen bonding along with the evaporation of chloroform even at temperatures lower than boiling point of chloroform (Table 5.1). Glycerol might have shielded the hydrogen bonding causing less coalescence of particles. Figure 5.6 SEM images of the particles obtained after homogenization of LPEI (CHCl3) and Pluronic F68 (100%Glycerol) solutions at the mixing speeds of 10m/s, 30m/s and 50m/s after subsequent dialysis of the emulsion mixture in water. The meshed structure observed in images is the aluminum oxide filter membrane (sample substrate) through which the particles were filtered and dried. 151 5.3.2.2 Effect of Polymers, High Viscosity and High Shear Rate Mixing on Particle Shape It has been shown previously that a single o/w emulsion of poly(d,l-lactic) acid (PLA) dissolved in ethyl acetate and F68 in water can give nanoparticles of various sizes and shapes, depending on mixing speed, temperature, time and viscositym. PLA is a biocompatible and biodegradable polymer183 , while LPEI is a promising siRNA transfection carrier”. Therefore, we studied effect of PLA and LPEI interaction in an emulsion process to fabricate the nanoparticles composed of PLA and LPEI. F68 has hydrophobic as well as hydrophilic groups, where hydrophobic groups help in forming the emulsion droplets of polymer-solvent and hydrophilic groups help in stabilizing the droplets in water. Here, we tried to stabilize the PLA-ethyl acetate droplets with LPEI in water, instead of using F68 in water. LPEI is a hydrophilic polymer, and also can form hydrogen bonds using —NH groups on its polymer chain. Therefore, we expected water dissolved LPEI to stabilize the ethyl acetate dissolved PLA based on the N-H---O hydrogen bonding. Figure 5.7 shows SEM image of the particles obtained after emulsification of PLA (5mg/ml) dissolved in ethyl acetate saturated with water (solvent- 20ml) and 2.5 kDa LPEI (4.3mg/ml) dissolved in a solution (non-solvent- 60ml) of 50 %v/v glycerol in water saturated with ethyl acetate, run at the mixing speed of 50m/s for l min. The final vessel temperature reached in this case was about 78°C, which was above the boiling point of ethyl acetate (77.1°C). Similar to as discussed in sections 5.3.2 and 5.3.2.1, since the vessel temperature reached above the boiling point of ethyl acetate, aggregation 152 (coalescence)'of particles was observed causing all the particles to stick with each other due to inter hydrogen bonding between LPEI molecules. Formulated particles might be composed of PLA and LPEI in some ratio, which needs to be confirmed further. Similar to as discussed above in section 5.3.2.1, a slight effect of the shear force due to high viscosity, where most of the solvent was evaporated, was also observed as evident from the somewhat non-spherical particles in Figure 5.7. Particles of shapes other than perfectly spherical were obtained. A phase separation was observed in solution after the emulsion process, perhaps due to non-dissolved particles settling down to the bottom of the solution. Figure 5.7 SEM images of the particles obtained after homogenization of PLA (in ethyl acetate) and 2.5kDa LPEI (in 50%glycerol-water) solutions at the mixing speeds of 50m/s and after subsequent dialysis of the emulsion mixture in water. 5.3.2.3 Effect of Vacuum and High Shear Rate Mixing on Particle Shape Reduction in pressure reduces the boiling temperature of a liquid. Therefore, we hypothesized that sudden application of vacuum during the mixing at high shear rate can 153 cause sudden forced release of solvent from the polymer-solvent droplets, eventually causing a cavity in formulated particles resulting hollow or bowl shaped particles. However, as high shear rate causes rapid temperature increase which can cause solvent evaporation before the application of vacuum, therefore care need to be taken to control the mixing rate to observe the effect of application of vacuum during mixing. LPEI (1 mg/ml) in CHC13 and F 68 (5mg/ml) in water were homogenized at the different mixing speeds of 10m/s, 20m/s, 30m/s, 40m/s and 50m/s for 2 min each. Vacuum was applied after 1 min of run in each case. High shear rate mixing led to increase in temperature as a firnction of speed, and yielded particles of different sizes. The temperatures were less than boiling point of chloroform for all the mixing speeds. As mentioned above, due to the formation of crystalline hydrates, LPEI in the free base form is insoluble in water at temperatures below 600C166' 18‘ but can be dissolved at higher temperatures. Similarly, LPEI can be dissolved in organic solvent chloroform (CHCl3), but only at high temperatures. Due to low temperatures (<600C) at all the speeds, it can be assumed that LPEI did not dissolve in water phase during mixing and remained in CHCl3 phase. Table 5.3 shows the summary of vessel final temperatures as a function of mixing speeds run under vacuum. An interesting trend of phase separation was observed for the mixing speed of 50 m/s, depending on the timing of vacuum applied during the homogenization process. If the vacuum was applied during run after 60 sec of mixing, particles were precipitated and moved down to bottom of solution after days long sitting. And, if the vacuum was 154 applied before run, particles were precipitated and moved to top of solution after days long sitting. Currently, we do not know the reason behind this phenomenon of difference in phase separation. Figure 5.8 shows the SEM images of particles obtained after emulsifying the two solutions at various mixing speeds under vacuum applied after run of 60 sec, and after subsequent evaporation of the solvent phase (chloroform) from droplets (collected from the water phase after phase separation) dried on a substrate. In this case, contrary to the condition where no vacuum was applied (sections 5.3.2 and 5.3.2.1), the particle size was found to decrease with increase in mixing speed up to 40 m/s. This could possibly be due to more removal of chloroform from within the droplets due to sudden application of vacuum, thereby reducing the particle size at higher speeds (and also changing the particle shape, as discussed below). The slow rate of evaporation of chloroform where no vacuum was applied (sections 5.3.2 and 5.3.2.1, and Figures 5.4 and 5.6) in comparison to the sudden evaporation of chloroform after applying vacuum during run, may have caused reverse trend of increasing and decreasing particle sizes with increase in mixing speeds, respectively. No coalescence of particles was observed, except at the mixing speed of 50m/s where the vessel temperature was near to the reduced boiling point of CHCl3. Mixing speed of 50 m/s showed high level of coalescence, as in the case of no vacuum. As described above, this could be due to vacuum induced evaporation of chloroform at 50m/s (vessel temperature 57°C, which is less than normal boiling point of chloroform) so that LPEI particles could get bonded with each other due to inter- hydro gen bonding. 155 Figure 5.8 also shows the presence of a film deposition, where LPEI-F68 particles seems to be embedded within the film. This may be due to the organic matter deposition as the chloroform was evaporated from the substrate during SEM sample preparation. In Figures 5.8 and 5.9, sample droplets were collected after shaking the suspension obtained after emulsion process in mixer, and air dried on a substrate. Figure 5.8 SEM images of the particles obtained after homogenization of LPEI (in CHC13) and Pluronic F 68 (in H20) solutions at the mixing speeds of lOm/s, 20m/s, 40m/s and 50m/s under the vacuum (applied during run after 60 sec of mixing), and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. The meshed structure observed in images is the aluminum oxide filter membrane (sample substrate) through which the particles were filtered and dried. As shown in Figure 5.9, an interesting trend regarding the deposition of organic residue after the evaporation of chloroform upon droplet drying on a substrate, and localization of particles was observed using SEM for the particle suspensions obtained at different 156 mixing speeds. During the sample preparation for SEM for particles obtained at all the mixing speeds, particles were localized only within the regions from where chloroform was evaporated (Figure 5.9). Importantly and interestingly, there was less spreading of chloroform residual matter at the low mixing speeds and more at the higher mixing speeds. Figure 5.9 Low magnification SEM images of the particles obtained after homogenization of LPEI (CHC13) and Pluronic F68 (H20) solutions at the mixing speeds of 10m/s, 20m/s, 40m/s and 50m/s (clockwise from top-left, respectively) under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. 157 Interesting novel shapes of particles were obtained at the mixing speeds of 30 m/s and 40 m/s. Further, particles at the speed of 40 m/s were at the nanoscale and homogeneous in size. Figures 5.10 and 5.11 shows the SEM and TEM images, respectively, of particles obtained at the mixing speed of 30 m/s. As evident from these images, most of the particles were formed with a crater on their surface. As discussed above, this could be due to sudden release of solvent (chloroform) from the LPEI-chloroform droplets in the reduced pressure surroundings with vacuum applied during the high speed mixing. Sizes of the particles at the mixing speed of 30 m/s were in the range of few hundred nanometers to more than a micron. For TEM imaging, samples were not stained with any heavy metal, as mentioned in Materials and Methods section. Samples, as shown in Figure 5.11, were collected after shaking the particle suspension in emulsified solution. In the TEM images (Figure 5.11), background appear dark and the particles appear light, as contrary to a typical T EM image where usually background appears light and sample appear dark due to more less transmission of electrons through sample. However, here ‘as the sample contained chloroform and it formed an organic layer after its evaporation from the surface, therefore the background appears dark and particles appear light. Moreover, multiple particles stacked on top of each other can also be seen in images. Half-cut or crater-shaped particles can be seen in these TEM images, corroborating the SEM results. 158 Figure 5.10 SEM images of the particles obtained after homogenization of LPEI (in CHCl3) and Pluronic F68 (in H20) solutions at the mixing speed of 30m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. 159 Figure 5.11 TEM images of the particles (corresponding to Figure 5.10) obtained after homogenization of LPEI (in CHCl3) and Pluronic F68 (in H20) solutions at the mixing speed of 30m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. Figures 5.12 and 5.13 shows the SEM and TEM images, respectively, of particles obtained at the mixing speed of 40 m/s. Figure 5.12 illustrates the uniformity of nanoparticles formed at the mixing speed of 40m/s, where the particle sizes were in the range of 50-150nm. Figure 5.13 shows the crater formation on the surface of smaller 160 range of particles of less than 100nm in size, formed at the mixing speed of 40m/s. As discussed above, crater formation could be due to sudden release of solvent (chloroform) from the LPEI-chloroform droplets in the reduced pressure surroundings with vacuum applied in-between the high speed mixing. Samples, as shown in Figure 5.13, were collected after shaking the particle suspension obtained after emulsion process in mixer. In the TEM images (Figure 5.13), background appears dark and the particles appear light. As discussed above, this was due to the formation of an organic layer after the evaporation of chloroform from the surface which formed a darker background than particles. The dark appearing circles surrounding the particles could be the F 68 surfactant surrounding the light appearing LPEI particles. Figure 5.12 SEM images of the particles obtained after homogenization of LPEI (in CHC13) and Pluronic F68 (in H20) solutions at the mixing speed of 40rn/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. 1 i 1 l l l i 161 Figure 5.13 TEM images of the particles (corresponding to Figure 5.12) obtained after homogenization of LPEI (in CHCI3) and Pluronic F68 (in H20) solutions at the mixing speed of 40m/s under the vacuum, and after subsequent solvent evaporation fi'om the emulsion dr0plets on a planar substrate. 1110 11111 10011111 . ' ‘ 511 nm “‘1 51) 11111 5011111 ' 511nm Figure 5.14 shows the TEM images of particles obtained at the mixing speed of 50 m/s under vacuum. In this case, particles were collected from water phase after settling down the solution for long period of time. Therefore, as opposed to Figures 5.11 and 5.13, the background here appears light and particles appear dark, as in a typical TEM image. In some cases, blisters or crater formation can be seen in particles due to the sudden evaporation of chloroform on applying vacuum while mixing, similar to that at the speeds of 30m/s and 40m/s. 162 Figure 5.14 TEM images of the particles obtained after homogenization of LPEI (in CHCl3) and Pluronic F68 (in H20) solutions at the mixing speed of 50m/s under the vacuum, and after subsequent solvent evaporation from the emulsion droplets on a planar substrate. 100nn1 ..-__ __HW... Overall in the LPEI(CHC13)-F68(H20) emulsification process under vacuum and at the high shear rates, particle size was less at the high mixing speeds. Due to chloroform evaporation at the sudden application of vacuum during mixing, there was a crater or blister formation on the surface of particles, mostly at the mixing speeds of 30rn/s and 40 163 m/s, and to a less extent at 50 m/s (Table 5.3). Blister or crater formation was more at 30 and 40 m/s and less at 50m/s, because all of the chloroform was not evaporated at former speeds due to lower temperatures, whereas most of the chloroform was evaporated at the speed of 50m/s due to temperature reaching near the pressure reduced boiling point of chloroform. Table 5.3 LPEI(CHC13)-F68(HZO); Run time = 2 min; Vacuum applied after run of 1min in each case. Chiller temperature was maintained at 4°C. All the temperatures were with- in the error limits of iZOC. Mixing Speed Final Temp Particle Shape 10 m/s 60 C Mostly Spherical 20 m/s 1 10 C Mostly Spherical 30 m/s 220 C Mostly Blistered or Crater-shaped 40 m/s 390 C Mostly Blistered or Crater-shaped 50 m/s 570 C Few Blistered or Crater-shaped 5.3.2.4 Effect of High Viscosity, Non evaporating and Miscible Solvent, Vacuum and High Shear Rate Mixing on Particle Shape As mentioned above, the hypothesis behind increasing the system viscosity using glycerol in LPEI(CHC13)-F68(Glycerol/H20) emulsion system was to apply an high shear force on softened or melted LPEI (melting point 73-790C) by the virtue of viscosity, so that LPEI on re-solidification can obtain a novel shape. However, one of the limitations of increasing the shear rate is that it led to increased system temperature which caused chloroform to evaporate during the mixing (boiling point of chloroform 612°C). Absence of a solvent caused formed LPEI particles to coalesce with each other. In the presence of 164 a solvent, formed particles can be expected not to stick with each other as they would not form inter hydrogen bonds with each other. Rather, the LPEI-solvent droplets would combine with each other in the presence of a solvent, and under the high viscous force and high shear mixing rate they can be expected to take an elongated particle shape. In order to overcome the limitation of pre-mature solvent evaporation, we used ethanol as a solvent for LPEI instead of chloroform. Ethanol is miscible with non-solvent water and has a boiling point of 784°C which is in the melting point range of LPEI (73-790C). To keep the system viscosity high, we mixed a solution of 66%v/v glycerol in water (without surfactant) (60ml) with 2.5 kDa LPEI (4.3mg/m1) in ethanol (20ml) at the high shear rate mixing speed of 50m/s for 2 min, and vacuum was pre-applied i.e. before starting the mixer. Figures 5.15 shows the rod and loop shaped LPEI particles formed under these conditions. In another case, we mixed 100%v/v glycerol (40ml) with 2.5 kDa LPEI (4.3mg/ml) in ethanol (20ml) at the high shear rate mixing speed of 50m/s for l min. Figures 5.16 shows the rod shaped LPEI particles formed under these conditions. In this case, vacuum was either pre-applied before starting the mixer, or applied after 30 sec starting the mixer (as indicated in Figure 5.16). Final vessel temperature reached was in between 79-870C (above than melting point of LPEI) for 66%v/v as well as 100%v/v glycerol and LPEI mixing. In the latter case, rod shaped LPEI particles are much bigger in size than as compared in former case. Perhaps, the effect of solvent and non-solvent miscibility, high system viscosity and boiling point of ethanol within the melting range of LPEI, coupled with high shear 165 mixing rate under reduced pressure, resulted in coalescence and elongation of formulated particles. Increase in glycerol percentage increased the size of aforementioned rod-shaped particles. We believe that the main contributing factor resulting in elongation of coalesced particles in this system, as compared with the ones discussed above, is the miscibility of solvent and non-solvent in the melting point range of LPEI. The particle shapes were mostly rods and loops. 166 Figure 5.15 SEM images of the particles obtained after homogenization of LPEI (in ethanol) and 66 v/v% glycerol at the mixing speed of 50m/s under vacuum and after subsequent dialysis of the emulsion mixture in water. Coolant temperature was set at 20°C. 167 Figure 5.16 SEM images of the particles obtained after homogenization of LPEI (in ethanol) and 100 v/v% glycerol at the mixing speed of 50m/s under vacuum applied before run (left column) and under vacuum applied 30 sec after run (right column), and after subsequent dialysis of the emulsion mixture in water. Coolant temperature was set at 20 C. 168 5.3.3 Hypothesis for LPEI particle Aggregation at High Shear Rate Mixing Multiple LPEI chains are known to stack together in their hydrated forrn166‘ ‘67. Chatani et 01166’ 167 confirmed from X-ray structure analysis that hygroscopic LPEI is present as crystalline dihydrate. The lattice parameters of the crystalline LPEI dihydrate monoclinic unit cell are a = 13.26 A, b = 4.61 A, c (fiber axis) = 7.36 A and ,6 = 101.00 (where a, b, 0 represent the lattice dimensions and fl is the angle between a and c). Each unit cell contains 4 LPEI monomeric (-CH2CH2NH-) units (with each structural repeat unit having 2 monomeric units) and 8 water molecules“. The polymer chains orient in a planar zigzag with alternating stacks of polymer chains and water molecules arranged in parallel along the be plane. These chains are held together by multiple hydrogen bonds between the NH groups and water molecules. Given what is known about LPEI in the hydrated state, our hypothesis is that in the absence of a solvent, LPEI can possibly form H-bonds with each other and thus coalesce to form sticky particles altogether as discussed above. The presence of a solvent, such as glycerol or chloroform, might not allow LPEI to form inter hydrogen bonds, as LPEI would have more affinity to solvent in that case. 5.3.4 Hypothesis to Reduce N anoparticle Aggregation The guidelines for IPC formation, such as the effect of polymer concentration38’ 124’ 170,171, ”3'175 or the rate of polymer additionm, can help in fabricating the LPEI-IPC nanoparticles. 169 It is interesting to note that size of the stable inter-polyelectrolyte complex (IPEC) nanoparticles has been shown to be independent of polymer concentrations after charge neutralization of polyanions and polycations mixed in a solution, and forms constant sized nanoparticles of (e. g. ~150nm) at any charge ratio beyond the charge neutralization using certain polyelectrolytes (such as poly(diallyldimethylammonium) chloride (PDAC) and a hydrophobic sulfonated poly(styrene) (SPS) derivative, and others) at a given rate of polymer addition170 . With excess of polycation before the charge neutralization, the size of the stable colloids are less than 150nm and are dependent on the contour length, molar mass and concentration of the polyelectrolytes. The particle size suddenly increases to a constant size (e.g. 150nm) at the condition of charge neutralization and that increased particle size is explained in terms of aggregation forming bigger particles by collision and hydrophobic interactionsm’ 177. With increased amount of added polyanion to a given amount of polycation at the conditions afier charge neutralization, the excess amount of polyanion help in preventing the fiirther collisions and hydrophobic interactions leading to constant particle size. The particle size after charge neutralization was also shown to be dependent on rate of polymer addition. If the rate of addition is decreased after charge neutralization, then this increase the particle size as this implies that the concentration of added polyanion is low in comparison to a condition of high rate of addition at any given time pointm' 177. This hypothesis can be extended to explain the behavior of the sizes and a possible hypothetical way to prevent aggregation of LPEI-siRNA IPECs that we described in Chapter 438. The size of the LPEI-siRNA IPECs was about 50m at the N/P ratio of 90, 170 where near complete incorporation of siRNA into nanoparticles38 and charge neutralization was observed. These nanoparticles have the tendency to aggregate over a period of time. However, according to the explanations as described above, if we add the LPEI in solution after the LPEI-siRNA nanoparticle formation at the N/P ratio of 90, then this excess LPEI should be able to prevent the collisions and aggregations of already formed nanoparticles. Similarly, N/P ratios higher than 90 (where excess LPEI would be added before LPEI-siRN A complexation) may also prevent the aggregation of small sized (~50nm) nanoparticles. This can provide a potentially useful method to avoid the problem of aggregations in a complex particle mixture, which is particularly a wide known problem with PEI-nucleic acid complexes. This hypothesis can serve as a guideline to fabricate LPEI-IPCs using other secondary polymers, instead of siRNA, at high shear rates. 5.4 CONCLUSIONS The objective of this study was to fabricate LPEI based inter-polymer complex (IPC) (LPEI-IPC) (i.e. LPEI mixed with a polymer) nanoparticles of novel shapes and/or uniform sizes using high shear rate mixing conditions. Uniformed size nanoparticles (50- 150nm) were obtained by mixing LPEI(CHC13) and F68(water) at the high shear rate mixing speed of 40 m/s under the reduced pressure surroundings, where vacuum was applied 1 min after starting the mixing. Also, novel shaped micro and nano-particles (blistered or crater-shaped particles) were obtained in the similar conditions at the mixing speeds of 30m/s and 40 m/s. In summary, uniformed size nanoparticles with novel shapes were obtained by mixing LPEI(CHC13) and F68(water) at the high shear rate mixing 171 speed of 40 m/s under the reduced pressure surroundings. In the range of 10-40 m/s mixing speeds, the particle sizes decreased with increase in mixing speed run under atmospheric pressure, and particle sizes increased with increase in mixing speed when vacuum was applied. Further, rod shaped micro-particles were obtained by mixing LPEI (ethanol) and F68 (glycerol in water) at the high shear rate mixing speed of 50 m/s under the reduced pressure surroundings. Increase in glycerol percentage increased the size of such rod-shaped particles. High coalescence of formed particles was observed in all the cases where emulsion system temperature reached above the boiling point of solvent chloroform (612°C). This could be due to LPEI chains adhering to each other in the absence of a solvent, based on the inter hydrogen bonding due to secondary amines on LPEI. Coalescence of particles was also observed even at the temperatures less than the boiling point of solvent, but to the less extent when solvent is completely evaporated. Thus more evaporation of solvent during emulsification led to more coalescence of particles. Under controlled conditions, this process can be used to create an interconnected chain of particles of various sizes. An effect of glycerol (i.e. high viscosity) was observed as to reduce the particle aggregation/coalescence at the temperatures less than the boiling point of chloroform. Effect of high viscosity and reduced pressure coupled with a choice of solvent miscible with non-solvent and having its boiling point in the melting range of LPEI, was observed as these parameters changed the particle shape from spherical or near spherical-shaped particles to rod-shaped particles. 172 CHAPTER 6 CELL ADHESION RESPONSE OF THIN POLYELECTROLYTE MULTILAYERS 6.1 INTRODUCTION A major challenge in the field of tissue engineering is to optimize the surface characteristics to achieve a controlled or desired level of cell adhesion under physiological conditions. The cell adhesive property of the surfaces can be modulated through various physical, chemical and mechanical properties of the surface, individually or in combination, such as the hydrophobicity and hydrophilicity184, surface charge185 , surface roughness or topography” “0, and stiffness'86’188. Layer-by-layer (LbL) assembled polyelectrolyte multilayer (PEM) thin films, introduced by Decher”, provide a versatile approach for altering physical, chemical or mechanical properties of a substrate to address this challenge. Over the past decade, LbL films have shown promise for various clinically relevant biological applications”. For example, cytophobic (cell resistive) LbL thin film coatings on substrates, i.e., implantable hydrogels for nerve repair applicationsz’ 14, have been put forth as a possible method for controlling the growth of leptomeningeal fibroblasts, which hinder the progression of regenerating axons”. LbL fihns have also been applied to create three-dimensional cellular multilayers”, patterned co-cultures185 , microarrays”, biosensors30, functional cell surfaces”, etc. Many of these applications capitalize on the tunability of the cell adhesive behavior on the thin films. Different deposition parameters, such as pH, salt concentration, and number of bilayers (deposition cycles) during LbL fabrication can affect the intrinsic properties of the LbL films, such as surface roughness, stiffness, degree of hydration, and thickness. These key 173 factors/properties define the cytophobic or cytophilic characteristic of the surfacem' 152’ 190. Excessive increase in the thickness of the multilayers may result in adverse cellular 134, 152, 190, 191 adhesion or suboptimal cellular response, i.e., by reducing the available space in the nerve regeneration scaffoldsz’ 14. The multilayer thickness can be altered by 152, 193-195 196, 197 varying the pH15 2' 191’ 192, salt concentration , temperature , or the number of 134, 193 bilyers during film deposition The lack of cell adhesion on PEMs at the nano- to micrometers thickness range has been reported to be due to film swelling, hydration, and mobility of the polymer chains within the filmsm’ 152. An increase in the swelling, intrinsic hydration, and higher chain mobility result in less interpenetrating multilayersm’ 152' 191’ 192' 197400, thereby increasing the thickness (in terms of swelling) and roughness, while reducing the stiffness of the l 200 PEMsm’ 97' . The growth of LbL films, i.e. the thickness, can be either linear or exponential, depending on the number of bilayersm' 201'203. Exponentially growing films swell and exhibit hydrogel-like morphology. Changing the number of bilayers and thereby the thickness of the films, changes the adhesive behavior of the cells on these films134’190'204’205. Elbert et al. showed that the spreading of fibroblasts on multilayers of poly-l-lysine (PLL)/alginate, exhibiting exponential growth, is reduced134. In further support, Richert et al. showed that smooth muscle cells exhibit an overall round cell morphology on exponentially growing, thick films (1- 15pm) of PLL and hyaluronic acid (HA), which otherwise spread out more on thinner filmsl90. In contrast, the factors that govern the 174 adhesive behavior of linearly growing films, such as poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA)'99, or poly(diallyldimethylammonium chloride) (PDAC)/ sulfonated poly(styrene), sodium salt (SPS)”3’ 140’ 193' 206 depend on whether they are weak or strong PEMs. Film swelling and cell adhesive behavior of linearly growing, weak and strong polyelectrolyte PEMs have been attributed to the 15 91 . . 2’ l and the salt concentratron15 2, respectively. fabrication/post-fabrication assembly pH Further, in a recent study, Hillberg et al. showed that linearly growing chitosan/alginate fihns (~100nm) improved cell adhesion after cross-linkingzos. Cross-linking the films increased the film stiffness and decreased the surface roughness. They also showed that, increasing the number of bilayers from five to ten of the cross-linked films improved cell adhesion. For linearly growing multilayers, the increase in the number of bilayers as the driving force for the swelling or hydration of the multilayers at a fixed pH or salt concentration has not been reported. To the best of our knowledge, no study to date have evaluated the cell’s adhesive behavior as a firnction solely of the changing number of bilayers for linearly growing films of less than 200nm. Here, we show that increasing the number of bilayers of PDAC/SPS fihns from 10 to 20, corresponding to a fihn thickness of 37.6 nm (~40 nm) to 95.9 nm (~ 100 nm), respectively, switches the films from a cytophilic to a cytophobic surface (Figure 6.1). We demonstrate this effect with bone marrow mesenchymal stem cells (MSCs) and NIH3T3 fibroblasts. The films exhibit cytophobic behavior at 20 bilayers with fiirther decrease in cell spreading and adhesion as the number of bilayers increased. The thickness increases linearly as the number of bilayers increases (see Results and 175 Discussion), causing a shift in the cell adhesion behavior. A factor previously shown to influence the cell adhesion of linearly growing PEMs consisting of strong 152 polyelectrolytes, i.e. high ionic strength of the deposition salts which causes film swelling and hydration, was kept constant in this study. Therefore, the salt concentration cannot explain the switch in the adhesive behavior observed when the number of bilayers (deposition cycles) increases. Figure 6.1 Diagram showing multilayers composed of linearly growing strong polyelectrolytes i.e. PDAC and SPS, fabricated at a deposition ionic strength of 0.1M NaCl, exhibit increased cytophobicity as the number of bilayers increases, as shown in images (A) to (E). Bands with violet and blue colors represents positively charged PDAC and negatively charged SPS polyelectrolyte chains, respectively; and one set of violet/purple colored band represents ten bilayers of PDAC/SPS. Red, green and blue colors inside the cell structure represent actin filaments, focal adhesion contacts and nucleus of the cell, respectively. Image (F) illustrates a previous study152 with a higher deposition ionic strength, the multilayers exhibit more cytophobicity due to swelling and hydration within the multilayer structure. The thickness band in image (F) represents a more loopy configuration of the polyelectrolytes with enhanced swelling and hydration within the multilayer'52 as compared to those in images (A-E). A Ionic Strength Deposition A v Cytophilic Cytophobicity ‘ Cytophobic Substrate stiffness has been shown to play a significant role in determining whether various cells, such as fibroblasts, muscle cells, endothelial cells, myoblasts and primary hepatocytes, adhere to different substrates, such as gels and LbL thin films. In general, 176 186-191, 207-209 cells spread on substrates with higher elastic modulus , i.e. less deformable. The stiffness of LbL films decreases as their thickness increases. The stiffness of um- and nm-scale PEMs has been measured with nanoindentation or AFMm' 190' ’9" 2‘0'212 analysis of buckling patterns213 , quartz crystal microbalance measurements“, piezo rheometer201 on planar PEMs, and osmotic pressure studies on PEM capsules215 ’ 2 16. PEM films show increasing cytophobicity with decreasing fihn stiffnesslgf” 188‘ 190’ 208. Elasticity measurements have been performed on PEM films less than 200 nmm, however, it is unclear whether mechanical-contact measurements overestimate the Young’s Modulus in 217 films less than 300 nm . Given the limitations of the currently available measuring techniques, it is difficult to quantify differences in the elastic moduli (either Young’s or 2'7. The lack of data and ability to accurately measure the shear modulus) of thin films modulus of PDAC/SPS films at thicknesses less than 100 nm make it challenging to determine whether the decreasing film stiffness, as the film thickness increases, is causing the switch of PDAC/SPS films from a cytophilic to a cytophobic surface. To explain the experimentally observed contraction in the cell area as the number of PDAC/SPS bilayers increases, we implemented a two—dimensional, axisymmetric finite element model of the film subject to traction forces generated by the focal adhesions. We correlated the cell focal adhesion contact area to both the mechanical stiffness of the thin LbL films that the cells are able to sense, and the energy required by a cell to maintain a constant traction force. Using the computational simulation, we were able to explain the 114 observed cell adhesion behavior with respect to increasing film thickness (see reference ”4 for model details). 177 6.2 MATERIALS AND METHODS 6.2.1 Materials Sulfonated poly(styrene), sodium salt (SPS) (Mw ~ 70,000), poly(diallyldimethylammonium chloride) (PDAC) (Mw ~ 100,000 — 200,000) as a 20 wt% solution, sodium chloride (N aCl), and epidermal growth factor were purchased from Sigma-Aldrich (USA). Bamstead Nanopure Diamond (Bamstead International, Dubuque, IA) purification system was used as a source for deionized (DI) water with a resistivity of 18.2 MB cm. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, 0.25% trypsin-EDTA, lX-phosphate buffered saline (PBS), and irnmunostaining components (rabbit anti-paxillin antibody, Alexa Fluor 488 goat anti- rabbit IgG secondary antibody, Texas Red-X phalloidin, DAPI, and ProLong Gold mounting medium) were purchased from Invitrogen (Carlsbad, CA). Insulin and glucagon were purchased from Eli Lilly and Co. (Indianapolis, IN). 6.2.2 Polyelectrolyte Multilayer (PEM) Fabrication PDAC and SPS polyelectrolyte solutions used to fabricate the multilayer assemblies were prepared in DI water to final concentrations of lOmM each with respect to the repeat unit of the polyelectrolytes, with an ionic strength of 0.1M NaCl. The deposition ionic strength of 0.1M NaCl was kept constant in fabricating the multilayer assemblies of varying number of PEM bilayers. Solutions were filtered with a 0.22 pm cellulose acetate filter (Corning, NY) before use. Multilayers were fabricated on tissue culture polystyrene (TCPS) plates (Costar, Corning, NY), glass (Corning Glass Works, Corning, NY) (for confocal and AFM imaging), or gold (for ellipsometric measurements) substrates. Glass 178 slides were cleaned with DI water followed by 100% ethanol and dried under N2 gas. Prior to beginning the multilayer fabrication process, TCPS plates and glass slides were further cleaned using a plasma cleaner (Harrick Scientific Corporation, NY) for 10 min at 0.15 torr and 50 seem flow of 02. Gold slides were cleaned in piranha solution (7:3; concentrated sulfirric acid: 30% hydrogen peroxide) (Caution: piranha solution reacts violently with organic material, handle with extreme care), dried under N2 gas and coated with lipoic acid (Sigma-Aldrich) followed by multilayer deposition. Here, plasma treated TCPS or glass, and lipoic acid coated gold are henceforth referred to as “substrates” for multilayer deposition. A Carl Zeiss slide stainer was used to prepare all multilayers. To form the first bilayer, substrate was immersed for 20 min in a PDAC solution, followed by two sets of 5 min rinses in DI water with agitation, and subsequent placement of the substrate in a SPS solution for 20 min. Substrate was then rinsed twice in DI water for 5 min each. Depositing a layer of polycation/polyanion pair was followed by a 2 min ultrasonic cleaning in DI water to remove weakly bounded polyelectrolytes. This process was repeated to build multiple layers, abbreviated as (PDAC/SPS)“, where n represents the number of PDAC/SPS bilayers (BLs), and equals to 10, 20, 30, 40 or 50 with SPS as the topmost layer in each case. Cell adhesion experiments were also performed on multilayers with PDAC as the topmost layer for fibroblast cell type and similar results were obtained (data not shown). After assembly, the films were allowed to air dry and were stored in a covered container under ambient conditions until use. 6.2.3 Cell Cultures 179 All procedures of cell isolation were approved by the Institutional Animal Care and Use Committee at Michigan State University. Multilayer coated substrates were sterilized under UV light using a germicidal 30W UV-C lamp (Philips, TUV 30W/G30T8) for at least 20 minutes prior to cell seeding. Unless specified otherwise, cells on all the surfaces were cultured in FBS supplemented medium. 6.2.3.1 Bone Marrow MSCs Isolation and Culture Bone marrow mesenchymal stem cells were isolated from 6-8 week old Sprague-Dawley female rats as previously described2 18. In brief, femurs and tibias fiom a 6-8 week old rat were dissected and the two ends were cut open. The marrow was flushed out using a needle and syringe. The cell suspension was filtered through 3 65pm nylon mesh to remove bone debris and blood aggregates. Cells were cultured in DMEM (catalog no. 11885, Invitrogen) supplemented with 10% FBS, 100 ug/ml streptomycin and 100U/ml penicillin, and placed in the incubator with a humidified atmosphere containing 5% CO2 at 37°C. Non-adherent cells were removed on the second day after plating. The medium was replaced every 3 to 4 days until the cells reached 90% confluence. Confluent cells were detached by 0.25% trypsin-EDTA and plated at a density of 5x104 cells per ml with 2 ml added to all surfaces studied. 6.2.3.2 Fibroblasts Culture NIH3T3 fibroblasts were purchased from American Type Culture Collection (USA). Cells were cultured in DMEM (high glucose (4.5 g/l) and sodium bicarbonate (3.7 g/l), catalog no. 11995, Invitrogen) supplemented with 10% F BS, 100 ug/ml streptomycin and 180 100 U/ml penicillin, and placed in the incubator with a humidified atmosphere containing 10% CO2 at 37°C. Cells grown to 80% confluency were detached by 0.25% trypsin- EDTA and plated at a density of 3x105 cells per ml with 2 ml added to all surfaces studied. The cell culture medium was replaced with fresh 2 ml medium 24 hrs post cell seeding. 6.2.3.3 Primary Hepatocytes Isolation and Culture Primary rat hepatocytes were isolated from 2-month-old adult female Sprague-Dawley rats (Charles River Laboratories, Boston, MA), using a two-step eollagenase perfusion technique described by Seglen219 and modified by Dunnm. The liver isolations yielded 150x106 to 300x106 hepatocytes. Using trypan blue exclusion, the viability ranged from 90% to 98%. Cells were cultured in DMEM supplemented with 10% FBS, 14 ng/ml glucagon, 20 ng/ml epidermal growth factor, 7.5 rig/ml hydrocortisone, 100 ug/ml streptomycin, lOOU/ml penicillin solution, and 0.5 U/ml insulin, and placed in the incubator with a humidified atmosphere containing 10% CO2 at 37°C. Freshly isolated hepatocytes were seeded at a concentration of 2.5x105 cells per ml with 2ml added to all the surfaces studied. Cell culture medium was replaced daily with fresh 2m] medium for upto a period of week. 6.2.4 Cell Immunostaining Immunocytochemistry was performed 48 hrs post cell seeding on the surfaces at room temperature. Cells were rinsed with PBS, followed by fixation with 4.0% paraformaldehyde in PBS for 15 min, rinsed 3 times in PBS, then permeabilized with 181 0.1% Triton X-100 in PBS for 15 min and washed 3 times with PBS. After washing, cells were blocked in 1% Bovine Serum Albumin (BSA, US Biological) for 30 minutes. Cells were incubated with rabbit anti-paxillin primary antibody (1:50 dilution in 1% BSA solution) for 1 hour followed by three washes in 1X PBS, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:500 dilution in 1% BSA solution) for 1 hour. Cells were washed further, three times in 1X PBS. During secondary antibody incubation, cells were additionally incubated with Texas Red-X phalloidin (51.11 stock per 20011.1 of 1% BSA solution) to visualize actin filaments (data not shown). Cells were then incubated for 5 minutes in 300nM DAPI (Invitrogen) to visualize the nucleus. After two final washes with 1X PBS, dry glass slides were removed from each well, and ProLong Gold mounting medium (Invitrogen) was applied to the stained the cells. Thin cover-slips (22 mm square, Corning) were adhered to the substrates, taking care to avoid air bubbles. Mounted and stained cover-slips were allowed to cure for 24 hours at room temperature in the dark. 6.2.5 Characterizations Confocal laser scanning microscopy (CLSM) images were obtained with Olympus Fluoview 1000 laser scanning confocal microscope using 40X oil objective. Phase contrast images were collected with Leica DM IL inverted microscope (Bannockbum, IL) equipped with SPOT RT color camera (Diagnostics Instruments, MI) using 10X dry objective. Ellipsometric thickness measurements were obtained with a rotating analyzer ellipsometer (model M-44; J. A. Woollam) using WVASE32 software. Substrate parameters (11 and k) of gold were measured before adsorption of lipoic acid, and 182 thickness of lipoic acid monolayer (0.4 :1: 0.09 nm) was subtracted from subsequent thickness measurements to report the final data. Ellipsometric angle of incidence was 75° for all experiments, and the thickness values were determined using 44 wavelengths between 414.0 and 736.1 nm. AFM images were collected in the tapping mode using 300kHz silicon probes (Vista probes) with a Nanoscope IV multimode scope from Digital Instruments (Santa Barbara, CA). The AF M images were corrected for bow/tilt using a plane fit and the errors reported for root-mean-square (rms) roughness values are the standard deviations of measurements on at least three different 5 pm x 5pm scan areas, on at least two independent samples, in a similar manner as reported previously206. Thickness and AFM measurements were performed on films dried just prior to characterization. 6.3 RESULTS AND DISCUSSION 6.3.1 Thickness and Roughness of Poly(diallydimethylammonium) chloride/(Polystyrene sulfonate, sodium salt) (PDAC/SPS) Multilayers The thickness of the PEM films depend on various factors, in addition to the number of 152, 193-195 deposition cycles, such as ionic strength of the deposition salts , pH (for weak polyelectrolytesffi’ 191‘ 192, molecular weightm’ 22‘ and concentration of the polyelectrolytesmo’ 195. The thickness of the PDAC/SPS multilayers under various deposition conditions have been reported extensively in the literature for a single bilayer up to hundreds of bilayersm’ 140’ 193’195‘ 206, however the thickness of bilayer numbers ranging from 10 to 50 have been not been reported for the particular molecular weight of 183 polyelectrolytes (i.e. PDAC/SPS) used in this study. Therefore, as a first step to modeling the cell adhesion response, we measured the thickness of this PEM system for the bilayers ranging from 10 to 50 with ellipsomety. Figure 6.2 shows the ellipsometric thickness of PDAC/SPS multilayers fabricated at NaCl concentration of 0.1M. This is supported by previous studies of PDAC/SPS multilayers, where thickness measurements 113,140, 193, 206 of multilayer growth at low salt concentrations were observed to be linear. Figure 6.2 Ellipsometric thickness of dried (PDAC/SPS)n multilayer film deposited at an ionic strength of 0.1M NaCl, as a function of the number of bilayers n. The thickness errors are reported (significantly small) as the standard deviation of the measurements from at least three different areas on three different samples. The dashed line is a linear fit to the reported data points. 300 - Thickness (nm) .5 —l N N 8 8 2 8 8 O -1— 1 1 r r T I T I I I I ' I 10 BLs 20 BLs 30 BLs 40 BLs 50 BLs Number of Bilayers Surface topography or roughness can also modulate adhesion and proliferation of cells on the surface” “0. In order to assess the changes in the roughness of PDAC/SPS multilayers across the different number of bilayers, we analyzed the surface topography of 30 and 50 bilayers using AFM (Figures 6.3a and 6.3b). The RMS roughness values were 1.924 i 0.15 nm and 1.617 i 0.13 nm, respectively. This corresponded to values 184 reported previously for 10 bilayers of PDAC/SPSZOG. No significant differences in the roughness values were found for 10 to 50 bilayers. This suggests that the surface roughness is not likely a factor for the varying cell adhesive behavior observed at the different number of bilayers, ranging from 10 to 50. Figure 6.3 AF M topographic images of the morphology of (a) 30 bilayer and (b) 50 bilayer dried PDAC/SPS films deposited at an ionic strength of 0. 1M NaCl. Images are 10pm x lOum, and the z-scales are shown. (8) (b) iZSnm :. 25nm 12.5nm 12.5nm Onm 6.3.2 Adhesion of Mesenchymal Stem Cells (MSCs) and Fibroblasts on PDAC/SPS Multilayers PDAC/SPS multilayers, composed of two strong polyelectrolytes, i.e. PDAC and SPS, follow a linear growth profile (Figure 6.2) and behave like a compact solid, exhibiting high ionic cross-linking density even in the presence of water at low salt concentrations. However these films swell at higher salt concentrations, largely due to a decrease in the 194, 222-224 ionic cross-linking density , and exhibit exponential instead of linear growthm. Mendelsohn et al. showed that PDAC/SPS multilayers exhibit cytophilic behavior at 25 185 bilayers when assembled in water without salt. In contrast, 10 bilayers of PDAC/SPS were cytophobic when assembled in water containing 0.25M NaCl152 . The response of the latter was attributed to the increase in swelling of the 10 bilayers of PDAC/SPS due to the salt (0.25M NaCl) in comparison with the 25 bilayers of PDAC/SPS assembled without salt. High salt concentration changes the conformation of the polyelectrolyte chains of the PDAC/SPS multilayers from a rod-like, extended structure to a loopy structure and makes the films thicker and more cytophobic15 2’ 191. However, in the present study, the salt concentration was kept constant for the multilayer assemblies of varying number of bilayers. The water content of PDAC/SPS films depends on the ionic strength of the depositing solutionzzs, and any swelling of the films is attributed largely to the addition of external salt ions193 '195 ’ 222. Since the concentration of the deposition ions was kept constant in this study, the previous explanations of swelling and hydration cannot explain the observed cell adhesion behavior. Yang et al. showed that a single bilayer coating of polyacrylamide(PAAm)/PAA or PAAm/poly(methaacrylic acid)(PMAA) was enough to turn the surface cytophobic to mammalian fibroblast cellsm. Kidambi et al. showed that 10.5 bilayers (i.e. PDAC as topmost layer, ~ 39 nm) were favorable to fibroblast adhesion, but not primary hepatocytes185 . This suggests that even at extremely low thicknesses, the cells are beginning to sense the effect of the underlying film, such that the film thickness is impacting the adhesion. 186 Figures 6.4a and 6.4b show the phase contrast and focal adhesion/nuclei staining images of bone marrow mesenchymal stem cells (MSCs) and fibroblasts, respectively, on PDAC/SPS multilayers assembled with different number of bilayers. As the thickness of the PDAC/SPS multilayers increased with increasing number of bilayers (while keeping all other parameters constant), fewer cells (both MSCs and fibroblasts) attached onto these multilayers. As illustrated by the phase contrast images, a significant difference was found between the 10 and 20 bilayers for both the MSCs and fibroblasts; and also between control (no multilayer) and 10 bilayers for MSCs. In addition, a significant difference in the cell attachment was found between 20 and 30 bilayers for both cell types. Further, as evident from nuclei staining images, the few fibroblasts that adhered on the 30-50 bilayers tended to clump together. In contrast, a smaller fraction of MSCs remained attached but those that remained exhibited less clumping on the 30-50 bilayers. Despite differences in cell adhesion behavior of the MSCs and fibroblasts, both cell types showed reduced adherence onto PDAC/SPS multilayers as the number of bilayers increased under fixed deposition conditions. Similar cell adhesive response was observed for primary rat hepatocytes, i.e., fewer primary cells adhered with increasing number of bilayers (Figure 6.4c). We performed a finite element study to elucidate why cells 114 adhered less to films of increasing number of bilayers (see reference 114 for model details). 187 Figure 6.4 Confocal laser scanning and phase contrast microscopy images of (a) bone marrow mesenchymal stem cells (MSCs) and (b) NIH3T3 fibroblasts, cultured on (PDAC/SPS)n multilayers. Green, red (only fibroblasts), and blue channels show the focal adhesion sites mapped by rabbit anti-paxicillin primary antibody and Alexa Fluor 488 goat anti-rabbit IgG secondary antibody, actin filaments mapped by Texas Red-X phalloidin, and nuclei mapped by DAPI, respectively. Images were immunolabeled 48 hrs post cell seeding. Phase contrast images were obtained just prior to immunostaining. (0) Phase contrast microscopy images of primary rat hepatocytes cultured on (PDAC/SPS)n multilayer coated TCPS substrates (scale bar = 100 um). n represents the number of multilayer bilayers (BLs), as indicated on the images. Non-coated TCPS or glass served as control surfaces. 188 Figure 6.4 continued (8) 51111111 5011111 50.11111 'l‘t'l’S (‘nml'nl 1(1 Ills 200 11111 51111111 51111111 5011111 189 Figure 6.4 continued 0)) 50 11111 50 um TCPS Control 100 um ' 50 um _ -, ‘SOBLS. . 10011111. I, f. U ‘1 0 I‘ “t 190 Figure 6.4 continued (C) - TC PS Control 100 11111 40 BLs Figure 6.5 shows the focal adhesion/nuclei staining images of MSCs cultured in the absence of serum on 10 and 30 bilayers of PDAC/SPS films. The reduced level of energy consumption during active mechanosensing, resulted in a more rounded cell shape and reduced attachment to the surface, the latter is a well known observation in serum free mediumm' 228. The computational results using the measured focal adhesion area show that the stored energy for serum-deprived MSCs is much less than that of serum-treated cells114 (see reference ”4 for details). 191 Figure 6.5 Confocal laser scanning of bone marrow mesenchymal stem cells (MCSs) cultured on (PDAC/SPS)n multilayers in the presence of serum (top panel) and absence of serum for 48 hrs (bottom panel). 11 represents the number of PDAC/SPS bilayers (BLs). Images are control, 10 BLs and 30 BLs starting from left-to-right. Non-coated TCPS or glass served as control surfaces. Green and blue channels show the focal adhesion sites mapped by rabbit anti-paxicillin primary antibody and Alexa F luor 488 goat anti-rabbit IgG secondary antibody, and nuclei mapped by DAPI, respectively. CLSM images were acquired at 40X magnification. Images were immunolabeled 48 hrs post cell seeding. 5011111 5011111 5011111 50 um 50 um 50 11111 6.4 CONCLUSIONS The film thickness is an important parameter to consider in surface modification of LbL multilayers. Excessive increases in film thickness may result in a suboptimal response of biomedical devices. We show that linearly growing ultrathin polyelectrolyte multilayers (PEMs) films of strong polyelectrolytes, poly(diallyldimethylammonium chloride) (PDAC) and sulfonated poly(styrene), sodium salt (SPS), exhibit a gradual shift from 192 cytophilic to cytophobic behavior, with increasing thickness for films of less than 100nm. Previous explanations based on film hydration, swelling or changes in elastic modulus cannot account for the cytophobicity observed with these thin films as the number of bilayers increases. We implemented a finite element analysis to help elucidate the observed trends in cell spreading114 . The simulation results suggest that cells maintain a constant level of energy consumption (energy homeostasis) during active probing and thus respond to changes in the film stiffness as the film thickness increases by adjusting their morphology and the amount of focal adhesions recruited, and thereby attachment onto a substrate1 '4. 193 CHAPTER 7 POLYELECTROLYTE MULTILAYER STAMPING IN AQUEOUS PHASE AND NON-CONTACT MODE 7.1 INTRODUCTION In the need of tissue or organ transplantation for damaged sections in human body, there has always been a shortage of donor supply. Tissue engineering brings in the concept where organs and tissues to be replaced in human body are engineered in labs to meet their current demand for surgery‘. Organs and tissues generally exhibit three dimensional (3-D) cellular arrangements in-vivo. Thus, there is a requirement to generate more and more tissue sections that exhibit layered cellular structures to gain similarity with in vivo tissues and organs”. Traditional methods to create a 3-D cellular environment were generally based on the approaches of placing cell on or within polymeric matrices from natural materials such as collagen or from synthetic polymers“ 229‘ 23°. Sandwiched cell culture is a type of 3-D culture, where the homotypic or heterotypic cell-cell contact of two layers of cells on top of each other with a sandwiched layer of polymer, can enhance the biological activity of one or the other cell type involvedzs' 29. Recently, layer-by-layer (LbL) assembly of polyelectrolytes over a monolayer of cells to create double or multiple strata of cells has been shown as a method to obtain the sandwiched 3-D cell culture systemzs’ 29. Germain et al. found PDAC/SPS multilayer fihns to be one of the most suitable PEMs when deposited as the capping layer using LbL process over a growing layers of cells (in terms of film porosity and cell viability)”. However, the main disadvantage of using LbL technology, as illustrated in these reports, was that the cells were intermittently deprived of cell culture medium for the long time 194 periods required for polyelectrolyte depositions during the assembly process. Application of such harsh fabrication conditions would not be conducive for the health of cells during the LbL process. Here, we propose to create a sandwiched 3-D cell co—culture by multilayer transfer onto a charged “base” substrate in aqueous phase in a non-contact transfer mode, where the base substrate and the stamp do not contact each other during multilayer transfer. Non-contact multilayer transfer can be useful to create a 3-D cellular co-culture with permeable polymer layers sandwiched between two monolayers of cells, deposited by without the need of removing cell culture medium during polymer deposition over cells. Polyelectrolyte multilayer transfer has been shown previously by Hammond and co- workersm’ 232; however those processes were applicable for multilayer transfer upon contact of stamp to the base substrate and under dry conditions. Contact of stamp to the cultured cells and stamping in dry conditions are not suitable for cell culture. Here, we report for the first time, the multilayer transfer by transferring an assembly of poly(diallyldimethylammonium) chloride (PDAC) and poly(styrene sulfonate) (SPS) onto the charged base substrate without contacting the stamp to the base substrate. PDAC/SPS multilayers are impermeable even to low molecular weight (few hundred Daltons) molecules and this characteristic of these multilayers was used as one of the method to characterize the non-contact, aqueous-phase multilayer (NAM) transfer over a monolayer of cells. NAM transfer was dependent on the number of bilayers of multilayer 195 to be transferred, and on the height of the stamping surface from the base substrate with this process feasibleat least from a transfer height of ~200um. 7.2 MATERIALS AND METHODS 7.2.1 Materials Poly(acrylic acid, sodium salt) solution (PAA, Mw 15,000; catalog no. 416037), Poly(ethylene glycol) (PEG, Mw 10,000; catalog no. P6667), sulfonated poly(styrene), sodium salt (SPS) (Mw ~ 70,000), poly(diallyldimethylammonium chloride) (PDAC) (Mw ~ 100,000 — 200,000) as a 20 wt% solution, branched polyethylenimine (BPEI, Mw 25,000; catalog no. 408727), carboxyfluorescein (6-CF) (green fluorescence dye) and sodium chloride (N aCl) were purchased fi'om Sigma-Aldrich (USA). Poly(allylamine hydrochloride) (PAH) and carboxylated polystyrene latex particles (3 pm diameter) were purchased from Polysciences, Inc (USA). Poly(dimethylsiloxane) (PDMS) from the Sylgard 184 silicone elastomer kit (Dow Corning, Midland, MI) or glass (Corning Glass Works, Corning, NY) were used to prepare stamps. Bamstead Nanopure Diamond (Bamstead International, Dubuque, IA) purification system was used as a source for deionized (DI) water with a resistivity of 18.2 MQ cm. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, 0.25% trypsin- EDTA, 1X-phosphate buffered saline (PBS), and immunostaining components (rabbit anti-paxillin antibody, Alexa F luor 488 goat anti-rabbit IgG secondary antibody, Texas Red-X phalloidin, and DAPI) were purchased from Invitrogen (Carlsbad, CA). 7.2.2 Stamps for NAM Transfer 196 Either PDMS or glass were used as the stamp for NAM transfer of BPEI(PAA/PEG)10,5(PDAC/SPS)30,5, and PDMS was used as the stamp for the NAM transfer of PAH(SPS/PDAC).,,. The design of the stamp was in such a way that it allowed for the formation of a capping structure upon inversion over the base substrate (plane stamp with two posts at the extreme ends), to minimize the stress applied to cells. Edge of the stamps was at a height of at least 200nm above the surface of stamp from which multilayer was transferred. To prepare PDMS stamps, 10:1 ratio of prepolymer to initiator was cured at 60°C overnight, over a stack of covers-slips with a total thickness of ~ 200nm adhered on a planar substrate, which served as a master of PDMS stamps. For patterned stamping, a microfabricated silicon master with photolithographically etched patterns was used. Glass stamps of appropriate height were made by joining a stack of coverslips of total thickness of ~ 200nm on the two opposite sides of a long glass slide. 7.2.3 Polyelectrolyte Multilayer Fabrication for non-contact aqueous-phase multilayer (NAM) transfer BPEI(PAA/PEG)10,5(PDAC/SPS)30,5 multilayers were prepared over plasma treated PDMS (2 min plasma treatment) or glass as the substrate. BEPI, PAA and PEG solutions were prepared at a concentration of 1mg/ml. PDAC and SPS polyelectrolyte solutions were prepared to final concentrations of 3mM each with respect to the repeat unit of the polyelectrolytes. All the polymer solutions were prepared in DI water. PDAC and SPS polyelectrolyte solutions were supplemented with sodium chloride concentration of 197 0.5M. All polymer solutions (PAA, PEG, PDAC, SPS), except BPEI, as well as washing baths (pure DI water) were adjusted to deposition pH of 2.0. PAA, PEG, PDAC, and SPS solutions were then filtered using a 0.22pm cellulose acetate filter (Corning, NY) prior to use. BPEI was the first initial layer for LbL assembly and was deposited on plasma treated PDMS or glass at the pH of 10.5, and washed with unadjusted pH DI water. A Carl Zeiss slide stainer was used for the subsequent LbL deposition. Deposition time for each polymer (PAA, PEG, PDAC, SPS) was set to 15 sec (actual run time 30 sec) and washing time at each step was set to 20 see with agitation (actual run time 1 min). PAH(SPS/PDAC)n multilayers were prepared over untreated PDMS as the substrate. PAH solution was prepared to final concentration of 50mM with respect to the repeat unit. PDAC and SPS polyelectrolyte solutions were prepared to final concentrations of 10mM each with respect to the repeat unit of the polyelectrolytes. All the polymer solutions were prepared in DI water. PDAC and SPS polyelectrolyte solutions were supplemented with sodium chloride concentration of 0.1M and then filtered using a 0.22pm cellulose acetate filter (Corning, NY) prior to use. First layer of PAH was deposited on untreated PDMS at the pH of 10.5 for 10 min, and then washed with DI water. Subsequent assembly of (SPS/PDAC)n multilayer was performed using spin coating process. SPS and PDAC were spin coated and washed alternatively at the speed of 3000 rpm for 8 see each. 7.2.4 NAM Transfer process (PDAC/SPS)n multilayers were prepared on glass or TCPS substrate, and multilayer fabricated on stamp was inverted on top of this multilayer in physiological pH aqueous 198 medium for 24 hrs. Stamps were removed after 24 hrs and multilayer transferred substrate was analyzed. For NAM transfer over cells, a layer of cells was cultured up to a confluency of 70% on (PDAC/SPS)n multilayers, and multilayer coated stamps (after UV exposure for at least 20 min) were inverted over cells, as explained above. 7.2.5 Cell Culture NIH3T3 fibroblasts cells were purchased from American Type Culture Collection (USA). Cells were maintained in DMEM with 10% FBS and 100U/ml penicillin plus lOOpg/ml streptomycin (P/S) at 37°C and 10%CO2. Cells grown to 80% confluency were detached by 0.25% trypsin-EDTA and plated at a density of 2x105 cells per ml with 2 ml added to all surfaces studied. 7.2.5.1 Primary Hepatocytes Isolation and Culture Primary rat hepatocytes were isolated from 2-month-old adult female Sprague-Dawley rats (Charles River Laboratories, Boston, MA), using a two-step eollagenase perfusion technique described by Seglen219 and modified by Dunn220 . The liver isolations yielded 150x106 to 300x106 hepatocytes. Using trypan blue exclusion, the viability ranged from 90% to 98%. Cells were cultured in DMEM supplemented with 10% FBS, 14 ng/ml glucagon, 20 ng/ml epidermal growth factor, 7.5 ug/ml hydrocortisone, 100 ug/ml streptomycin, 100U/m1 penicillin solution, and 0.5 U/ml insulin, and placed in the incubator with a humidified atmosphere containing 10% CO2 at 37°C. Freshly isolated hepatocytes were seeded at a concentration of 2.5x105 cells per ml with 2ml added to stamped multilayers. Cell culture medium was replaced daily with fresh 2m] medium for upto a period of week. 199 7.2.6 Cell Immunostaining Immunocytochemistry was performed 48 hrs post cell seeding on the surfaces at room temperature. Cells were rinsed with PBS, followed by fixation with 4.0% paraformaldehyde in PBS for 15 min, rinsed 3 times in PBS, then permeabilized with 0.1% Triton X-100 in PBS for 15 min and washed 3 times with PBS. After washing, cells were blocked in 1% Bovine Serum Albumin (BSA, US Biological) for 30 minutes. Cells were incubated with rabbit anti-paxillin primary antibody (1:50 dilution in 1% BSA solution) for 1 hour followed by three washes in 1X PBS, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:500 dilution in 1% BSA solution) for 1 hour. Cells were washed further, three times in 1X PBS. During secondary antibody incubation, cells were additionally incubated with Texas Red-X phalloidin (5111 stock per 200111 of 1% BSA solution) to visualize actin filaments. Cells were then incubated for 5 minutes in 300nM DAPI (Invitrogen) to visualize the nucleus. After two final washes with 1X PBS, cells were imaged for fluorescence microscopy. 7.2.7 Optical Microscopy Confocal laser scanning microscopy (CLSM) images were obtained with Olympus Fluoview 1000 laser scanning confocal microscope. Phase contrast images of cells were collected with Leica DM IL inverted microscope (Bannockbum, IL) equipped with SPOT RT color camera (Diagnostics Instruments, MI). Brightfield optical images of films were obtained using Nikon Eclipse ME 600 microscope, and conventional fluorescence microscopy images were collected using Nikon Eclipse E 600 microscope (Nikon, NY). 200 7.3 RESULTS AND DISCUSSION 7.3.1 Non-Contact Aqueous-Phase Multilayer (NAM) Transfer We show the transfer of (PDAC/SPS) multilayers from the stamp substrate onto a charged base substrate, where the multilayer transfer was performed without contacting the stamp to the substrate, termed as non-contact aqueous-phase multilayer (NAM) transfer. NAM transfer can be achieved using stamps of various materials, such as polydimethylsiloxane (PDMS) or glass, as far as the stamping surface can be maintained at the desired height (as discussed later). Stamps were made such that only the edges of stamp rested on base substrate and the multilayer transferring surface of stamp did not touch the base substrate (Figure 7.1a). NAM transfer was demonstrated using two different multilayer assemblies fabricated on stamp surfaces viz. BPEI(PAA/PEG)10,5(PDAC/SPS)30,5 and PAH(SPS/PDAC)4,5. These approaches can be used to encapsulate layers of cells between polyelectrolyte multilayers (PEMs), and do not require aspirating the cell culture medium at the time of layer deposition. Furthermore, the second layer of cells can be efficiently grown over to the deposited PEM (Figure 7.1b). Transfer of multilayer assembly BPEI(PAA/PEG)10,5(PDAC/SPS)30,5 was based on the concept of multilayer release from a hybrid assembly of degradable and non-degradable film, introduced by Ono and Decher“. Non-degradable PEMs built on top of degradable multilayers can be released as a result of decomposition of the underlying degradable fihns, called the sacrificial PEM. A sacrificial film breaks down by virtue of a change in pH, resulting in the breakage of hydrogen bonds, and thereby releasing the self-standing41 201 PEM. Poly(acrylic acid) (PAA) and poly(ethylene glycol) (PEG) are the two suitable biocompatible components to make up the sacrificial PEMs, because they form films at low pH which decompose at high pH. Transfer of multilayer assembly PAH(SPS/PDAC)4,5 was based on the process of multilayer transfer printing (MTP), introduced by Hammond and co-workers23 1’ 232, which enables complete transfer of a PEM from a poly((dimethylsiloxane) (PDMS) stamp over to the substrate PEM. In this technique, a hydrophobic layer of poly(allylamine hydrochloride) (PAH) (at high pH) is deposited over a layer of hydrophobic PDMS, and subsequent layers are built over the first layer of hydrophobic PAH. Upon stamping, the strong electrostatic interactions between the topmost layer of the PEM on the PDMS stamp and the substrate results in the release of the entire PEM structure from the PDMS231’ 232. We extend the MTP approach of PEM stamping to aqueous medium in contrast to stamping under dry conditions with air as the medium and without contacting stamp to substrate. 202 Figure 7.1 (a) Scheme showing non—contact aqueous-phase multilayer (NAM) transfer in aqueous medium. On the stamp, the first two initial layers (green and orange layers) represent degradable H-bonded (PAA/PEG)1o,5 multilayers, and next subsequent four layers (dark blue and dark red) represent (PDAC/SPS)30,5 (PDAC as topmost layer) multilayers. On the substrate before NAM transfer (left image), the two layers (light red and light blue) represent (PDAC/SPS)”, (SPS as topmost layer) multilayers. During the NAM transfer, the (PDAC/SPS)30,5 multilayers gets released from stamp and adhere onto (PDAC/SPS)"; base multilayers on the substrate, as shown in right image. The curved lines in surroundings represent liquid medium at physiological pH. (b) NAM transfer onto a layer of cells cultured on a base polyelectrolyte multilayer (PEM). (a) (b) 7.3.2 NAM Transfer Characterization 7.3.2.1 NAM Transfer Characterization: Optical and Atomic Force Microscopy Figure 7.2 shows the optical micrographs of the stamp surfaces before and after NAM transfer of BPEI(PAA/PEG)10,5(PDAC/SPS)30.5 Onto (PDAC/SPS)1o multilayers, as 203 illustrated in Figure 7.1. Negatively charged 6-carboxyfluorescein (6-CF) dye was used to visualize the PDMS stamps after NAM transfer process. BPEI(PAA/PEG)10,5(PDAC/SPS)30.5 multilayers were prepared on PDMS stamps and these stamps were exposed to 6-CF dye before and after NAM transfer, as shown in Figures 7.2a and 7.2b, respectively. 6-CF dye attached to the topmost positively charged PDAC layer on the stamp before NAM transfer, and to the partially exposed positively charged BPEI after NAM transfer. At high pH, PAA/PEG multilayers degrade slowly resulting in a rougher surface over the period of time14'97’98. The partial degradation of PAA/PEG multilayers during NAM transfer could have resulted in partial exposure of bottom-most layer of BPEI to 6-CF dye. BPEI(PAA/PEG)10,5(PDAC/SPS)30,5 multilayers when exposed to 6-CF dye alter NAM transfer, resulted in a partial staining of the stamp indicating some of the degraded regions (causing to attach 6-CF to BPEI) and other regions with only partially degraded (PAA/PEG) multilayers (with no 6-CF attachment) remaining on the surface. This is reflected in highly rough fihn morphology (irregularities) on stamps exposed to 6-CF dye after NAM transfer. Figures 7.2e and 7.1d show corresponding dark field images of PDMS stamps before and after NAM transfer, respectively, of BPEI(PAA/PEG)10_5(PDAC/SPS)30,5 onto (PDAC/SPS)“; multilayers. Figures 7.2e and 7.2f show the optical micrographs of the surfaces of glass as the stamps before and after NAM transfer, respectively. This illustrates that irregular surface features are not present on the BPEI(PAA/PEG)10,5(PDAC/SPS)30.5 films on PDMS before stamping. Partial eroding characteristic of (PAA/PEG) multilayers is visible on this stamp after NAM transfer, as shown Figure 7.2f. 204 Figure 7.2 (a - (1) Stamps before and alter NAM transfer of BPEI(PAA/PEG).0,5(PDAC/SPS)80.5 on top of (PDAC/SPSM. Top panel: Fluorescent images of 6-CF stained PDMS stamp before and after NAM transfer. Middle panel: Dark field optical images of PDMS stamp before and after NAM transfer. Bottom panel: (e, f) Bright field optical images of glass stamps before and after NAM transfer of BPEI(PAA/PEG)10,5(PDAC/SPS)3o.s on top of(PDAC/SPS)10. Stamp Before NAM Transfer Stamp After NAM Transfer 1111111111 10011111 111011111 F igurc 7.3 shows the optical micrographs of the charged substrates before and after NAM transfer of BPEI(PAA/PEG)10.5(PDAC/SPS)30.5 onto (PDAC/SPS)1o multilayers. As 205 shown in Figure 7.3a, negatively charged (PDAC/SPS)10 coated substrate was exposed to negatively charged 6-CF dye before NAM transfer and thus no staining. However, exposure of negatively charged substrate (i.e. (PDAC/SPS)10) to 6-CF dye after the NAM transfer of BPEI(PAA/PEG)10,5(PDAC/SPS)80,5 possibly resulted in positive PDAC at the top and thus a complete staining of the substrate (Figure 7.3b). The initial positive PDAC or negative SPS charge on the glass substrate was used to provide the opposite charge interactions with the topmost layer on stamp, required for NAM transfer to occur. Similar to 6-CF staining, negatively charged 3pm carboxylated polystyrene latex particles were also used to visualize the NAM transfer. As shown in Figure 7.3c and 7.3d, negatively charged polystyrene beads could not get attached to the (PDAC/SPS)“, coated negatively charged substrate, with SPS as the topmost layer before transfer (Figure 7.3b). However, beads attached to the substrate after NAM transfer which deposited (PDAC/SPS)”; multilayers resulting in PDAC as the topmost surface on substrate. 206 Figure 7.3 Substrates before (left column) and after (right column) NAM transfer of BPEI(PAA/PEG)10_5(PDAC/SPS)30,5 on top of (PDAC/SPS)", as illustrated by: (a, b) Fluorescent images of negatively charged 6-CF dye staining of substrates, and (c, d) Optical images of negatively charged 3pm carboxylated polystyrene latex particle addition to substrates. Substrate Before Substrate After NAM Transfer NAM Transfer 111011111 10(111111 3011111 Further, mdPEG acid molecule, which has carboxylic group at one end and methoxy group at other end of the polymer chain, was used to confirm the NAM transfer process. Figure 7.4a shows an image, where mdPEG acid was patterned onto a positively charged substrate (with PAH coated on a plasma treated negatively glass substrate) and subsequently was exposed to 6-CF dye. This resulted in green patterns indicating 6-CF dye attached to PAH on the substrate, and black patterns indicating mdPEG acid exposing methoxy groups on the surface. Similarly, Figure 7.4b shows an image, where mdPEG acid was patterned onto a substrate after the NAM transfer of 207 BPEI(PAA/PEG)10,5(PDAC/SPS)30.5 on (PDAC/SPS)“; multilayers, and subsequently exposed to 6-CF dye. NAM transfer with BPEI(PAA/PEG)1o_5(PDAC/SPS)30,5 multilayers on a negatively charged substrate possibly resulted in PDAC as the topmost surface on substrate. Further, patterning this positive surface with mdPEG and exposing to 6-CF dye, resulted in green patterns of dye attached to PDAC on the surface, and black patterns of mdPEG acid exposing methoxy groups on the surface. Figure 7.4 Surfaces after mdPEG stamping and 6-CF staining on: (a) PAH coated glass, (b) substrate after NAM transfer of BPEI(PAA/PEG)10,5(PDAC/SPS)30.5 on top of (PDAC/SPS)10. 5011111 101111111 To analyze for the NAM transfer of PAH(SPS/PDAC)n film, a PEM of PAH(SPS/PDAC)4,5 was built over a patterned PDMS stamp, and transferred on top of a PEM substrate of (PDAC/SPS)10,5 in aqueous medium. The charge on the topmost layer of the PEM substrate and the PEM on the stamp were kept opposite in order to form electrostatic bonds. To check the efficiency of PEM stamping under aqueous conditions, PDAC was tagged with Rhodamine-B (Rh-B), and was then dialyzed against 0.1M NaCl solution (all PEMs of PDAC and SPS were fabricated at the deposition salt concentration of 0.1M). PAH(SPS/PDAC)n films with Rh-B tagged PDAC, were built on patterned 208 PDMS stamps, and were stamped over substrate PEM of (PDAC/SPS)m under both, dry and aqueous conditions. Figure 7.5 shows the comparison of the PEM stamping under dry vs. aqueous conditions (the latter was in the presence of cell culture medium at physiological pH). The results indicate that there was a partial transfer of PEMs fiom the voids (non-contacting regions) of stamps under aqueous conditions. Figure 7.5 Polyelectrolyte multilayer transfer of PAH(SPS/Rh-PDAC)4_5 over (PDAC/SPS)1o,5 USing patterned PDMS stamp in (A) dry conditions, (B) aqueous conditions (scale bar = 100um). 100 um 7.3.2.2 NAM Transfer Characterization: NAM transfer over a monolayer of cells PEM stamping under aqueous conditions in non-contact mode (NAM process) was further verified with the cell culture on stamped surface after NAM process, using both multilayers i.e. BPEI(PAA/PEG)10_5(PDAC/SPS)30_5 and PAH(SPS/PDAC)4_5. In the arena of tissue engineering, a special emphasis has been given to improve the in vitro cell culture system of primary hepatocytes, which are the parenchymal liver cellsm’ 230. An improved in vitro cell culture system with an enhanced hepatic function would be highly beneficial to meet the current demands for the liver transplantations to treat a 209 number of patients suffering from liver diseasem’ 230. Primary hepatocytes functions are known to get enhanced when cultured in 3-D and polymeric matrices environmentsm' 233’ 234. In vitro 2-D cell culture studies have shown that nonparenchymal cells such as fibroblasts enhances the metabolic activity of primary hepatocytes 235’ 236. Therefore, in this study, we tried to create an in vitro 3-D cell co-culture environment of primary hepatocytes and nonparenchymal cells such as fibroblasts, that can be expected to result in enhanced metabolic activity of hepatocytes than a 2-D culture environment. However, the non-permeable nature of (PDAC/SPS) films towards large molecules precluded the formation of an successful 3-D co-culture (discussed later). In the first approach, we built sacrificial and non-degradable films onto a PDMS stamp, which was subsequently inverted over top of a layer of cells during the NAM transfer process. BPEI(PAA/PEG)10.5(PDAC/SPS)30.5 multilayers when inverted over a non- confluent layer of cells (grown over another base PEM with SPS as the topmost layer) in the presence of cell medium (of pH ~ 7.2), degraded the sacrificial component of the hybrid PEM, and thus released the non-degradable multilayer. Released non-degradable multilayer adhered with the underlying exposed base PEM (with negatively charged top surface) and cells (which has negatively charge membrane) through electrostatic bonding. This resulted in a layer of cells sandwiched between two homogenous PEMs. The second layer of cells (primary hepatocytes) was subsequently cultured atop of the capped PEM film (i.e. transferred film of (PDAC/SPS)80,5 multilayers). The base PEM and the transferred PEMs need to be restricted in terms of the concentration of the salt, as the 152 high salt concentration swells the films and make them cytophobic . The high number 210 of bilayers (>10 BLs) turns the film cytophobic1 14, and primary hepatocytes do not attach well on the PDAC capped multilayers185 . However, these effects has only been realized on the films fabricated on a rigid substrate and not on the self-standing filmsm’ 185. The responses of the self-standing film adhered on surface could be different from those of films fabricated directly on a rigid substrate. Therefore, in this study, in-spite-of the 80.5 bilayers of (PDAC/SPS) and PDAC possibly being the topmost surface in stamped region after NAM transfer, primary hepatocytes adhered to the transferred PEM (Figure 7.6). Figure 7.6 shows the phase contrast and fluorescence microscopy images where primary hepatocytes were cultured over BPEI(PAA/PEG)10,5(PDAC/SPS)30,5 transferred on top of fibroblasts cultured over another base PEM of (PDAC/SPS)10. Thus, this represents a sandwich 3-D co-culture of fibroblasts and primary hepatocytes, where fibroblasts were sandwiched between the two different sets of PEMs. The phase contrast images shows both of the cell types, where primary hepatocytes can be seen cultured partially on top of the fibroblasts. This suggests that there was an intermediate layer of PEMs which acted as a substrate for primary hepatocytes, and as a capping layer for fibroblasts cultured at the top of base layer and below intermediate layer of PEM. This 3-D co-cultured was then subjected to immunostaining, and it was observed that the molecules i.e. dye DAPI, primary and FITC labeled secondary antibodies for vinculin, and TRITC-conjugated phalloidin stained the primary hepatocytes cells, but none of these molecules could stain the fibroblasts cultured underneath. Therefore, in addition to the above discussed characterizations, the immunostaining characterization further strongly suggest that there was an intermediate layer of PEMs between the two cell types and the dye molecules could not permeate that layer of PEMs. Therefore, NAM transfer resulted in multilayer 211 deposition over a layer of fibroblasts cultured on top of base PEMs. Foumier and co- workers encapsulated a cell line using PDAC/SPS multilayer deposition via the sequential layer-by-layer process over a layer of cells in the presence of buffer solutions, and showed that the up to six bilayers of films allowed a bidirectional diffusion of small molecules preserving the cell metabolism after the deposition of multilayers”. Bruening and co-workers shows that multilayers of PDAC/SPS can be used for nanofiltration applications, as these multilayers can selectively exclude certain ions based on the ion size, film architecture and number of layers in fihnm‘m. Typically 3-5 bilayers of PDAC/SPS are sufficient for the nanofiltration. Our results are consistent with these previous studies as we observed that 80.5 bilayers of PDAC/SPS prevented the diffusion of staining dyes of molecular weights 350.25 kDa (DAPI) and higher. As discussed above, high number of PDAC/SPS bilayer (80.5) instead of a fewer layers were chosen with BPEI(PAA/PEG)10,5 multilayers because NAM transfer was not efficient with less number of (PDAC/SPS) bilayers. Interestingly, higher number of (PDAC/SPS) bilayers was found to be usefirl in characterizing the multilayer transfer in NAM transfer based on PAA/PEG degradation. 212 Figure 7.6 Fluorescent (confocal and conventional microscopy) images and phase contrast images of primary hepatocytes and fibroblast co-cultured in 3-D fashion using NAM multilayer transfer process. Successful staining of top layer of cells i.e. primary hepatocytes, and no staining of bottom layer of cells i.e. fibroblasts suggests that the (PDAC/SPS)so.5 multilayers were transferred during the NAM transfer process. Thick (PDAC/SPS)80.5 films inhibited the diffusion of staining dyes to the bottom layer of fibroblasts. 31111211 SHIN“ 5011111 {Hum Since the dyes molecules could not penetrate the intermediate layer of PEMs, therefore we assumed that this layer would have restricted the transport of nutrients required for the support of primary hepatocytes from fibroblasts. Thus, here we did not further 213 characterize the 3-D co-culture in terms of the metabolic activity enhancement of primary hepatocytes. However, dyes and antibodies based staining of this co-eulture provided an absolute method to characterize the transfer of PEM during NAM transfer process. As mentioned above, it is known that primary hepatocytes grow on the SPS capped films fabricated on a rigid substrate, but do not grow on PDAC capped fihns185 . Therefore, in the second approach, PAH(SPS/PDAC)4_5 (SPS as the top layer) was stamped over (PDAC/SPS)10,5 (PDAC as the top layer) and primary hepatocytes were seeded onto transferred multilayer. Figures 7 .7a and 7.7b shows the non-stamped fihns where PDAC was the topmost layer and primary hepatocytes could not attach on that surface. Figures 7.7c and 7.7d shows that cells attached well after NAM transfer of PAH(SPS/PDAC)4,5 on (PDAC/SPS)10.5 multilayer. Figures 7.7e and 7.7f shows the cells attached on to control TCPS plate without any polymer added. During NAM transfer of PAH(SPS/PDAC)4,5 on (PDAC/SPS)10,5, (SPS/PDAC)“ multilayer was transferred on top of (PDAC/SPS)10,5 providing either PAH or SPS as the topmost surface. It has been shown that PAH gets transferred as the top layer of the stamped PEM after stamping of PAH(SPS/PDAC)n in the dry conditionsm. Also, except for the positively charged strong polyelectrolyte PDAC, primary hepatocytes are able to attach on multilayers terminating with positively charged weak polyelectrolytes such as linear or branched poly(ethylenimine) (LPEI or BPEI). Therefore, it can be assumed that primary hepatocytes can also adhere to the multilayers terminating with weak polyelectrolyte PAH. Thus, it was not clear here whether PAH or SPS was the topmost 214 surface after NAM transfer of PAH(SPS/PDAC)4,5 on (PDAC/SPS)10,5, as primary hepatocytes do attach on SPS terminating films185 and can also possibly attach on PAH terminating films. Further experiments, such as coating negatively or positively charged dye on surface after NAM transfer, need to be done to assess for the transfer of PAH during NAM transfer of PAH(SPS/PDAC)4_5 on (PDAC/SPS)10,5. 215 Figure 7.7 Primary hepatocytes cell attachment on the PEMs as an evidence of (SPS/PDAC)” transfer in NAM transfer process giving SPS as the topmost layer, using PAH(SPS/PDAC)4,5 multilayer on stamp. Figures (a) and (b) shows the primary hepatocytes cultured on the multilayer of (PDAC/SPS)10_5 (PDAC as topmost surface) at Day 1 and Day 3. Figures (c) and ((1) shows the primary hepatocytes cultured after NAM transfer of PAH(SPS/PDAC)4,5 on the multilayer of (PDAC/SPS)10.5 at Day 1 and Day 3. (e) and (f) shows the cells on control TCPS substrate at Day 1 and Day 3. Scale bar represents 100pm. .................... Dayl . . D331; ._ (:1) I’l).\(‘ Top nnnnnnn ..... 216 7.3.3 Partial NAM transfer In order to analyze the effect of aqueous phase while NAM transfer, another case were considered in which the bulk of the aqueous phase (cell culture medium) was removed, but the substrate was not kept totally dried at the time of stamping. Multilayers were observed to be partially transferred to the base glass substrate if aqueous phase dried up during the NAM transfer process. This is referred to as partial NAM transfer in this study. As shown in AFM Figure 7.8, for an 80.5 bi-layers of transferred PDAC-SPS films, the average thickness was found to be about 120 nm (which is less than the expected thiclmess of ~ 400nm for 80.5 bilayersm) after NAM transfer under partially dried conditions. Incomplete transfer of the multilayers could be due to the drying-up of the liquid medium before the time required to complete the transfer (a small volume of medium was used in between the two substrates), resulting in fiactal growth of the polymer. Fractal growth of polymer can occur in the case of microcontact printing of a polymer from PDMS stamp, and is highly dependent on the stamp contact timem. The first layer of the polymer becomes strongly attached to the contacting and Oppositely charged surface. At later times, the process of internal restructuring of the PDMS stamp makes it hydrophobic over time (stamp gets dry or dewetting occurs)), and thereafter the second polymer layer becomes weakly physisorbed onto the first layer of the polymer on contacting surface240 . This process is time dependent. In our case, the self-standing films started to transfer on the bottom charged substrate as the sacrificial films started to decompose. But due to the drying-up of the intermediate liquid medium during this process (because of the small volume used), only a few of the initial layers were 217 chemisorbed onto the base substrate and the subsequent layers were weakly physisorbed producing a fractal growth pattern. The presence of aqueous phase was important for NAM transfer in order to obtain a more complete transfer of the films under aqueous conditions. Figure 7.8 AFM analysis showing fractal transfer of multilayers after NAM transfer under partially dried conditions. 50 50 (nm) nm Section Analysis 125 100. Hi '1 l i 1 I" ' l 1' 1 Hi 1') 2‘5 5'0 25 0 ‘73s r..- ~ ' _ 0 0 5 50 (m) It should be noted that there was a critical thickness of multilayers required for NAM transfer to occur successfirlly. All of the NAM transfers using BPEI(PAA/PEG) multilayers, as explained above, were carried out using 80.5 bilayers of (PDAC/SPS) assembled on top of (PAA/PEG) multilayers on stamp. NAM transfer process was feasible for 80.5 bilayers of (PDAC/SPS), but not feasible for transferring 20.5 bilayers of (PDAC/SPS). This was confumed using optical microscopy and ellipsometry (data not shown). Although, the ellipsometry resulted in poor fits of its parameter values when measured for the films transferred using NcMT, this method allowed for the quick determination of multilayer transfer. 218 7.4 CONCLUSIONS Here, we showed the non-contact aqueous-phase multilayer (NAM) transfer to stamp one set of polyelectrolyte multilayer over another set of multilayer. The multilayer transfer in non-contact mode is important for certain applications, such as fabrication of a 3-D cellular multilayer where the two or more different cell types can be cultured on top of each other with the sandwich layer of PEMs in—between. The non-contact transfer of PEM was possible in aqueous phase, as characterized using optical microscopy and cell culture. We demonstrate the multilayer transfer using two different sets of PEMs viz. BPEI(PAA/PEG)10,5(PDAC/SPS)30,5 and PAH(SPS/PDAC),,. The latter PEM transfer was based on the hydrophobic interactions of PAH with hydrophobic PDMS and relatively weak interactions with (SPS/PDAC) multilayers. The former PEM transfer was based on the degradation of PAA/PEG multilayers at the physiological pH thereby releasing the non-degradable (PDAC/SPS) multilayers. The cell culture trials showed that stamped multilayer of (PDAC/SPS)80,5 was impermeable to molecules above than molecular weight of 250 kDa. Transferable multilayer assembly configuration needs to be optimized so that transferred multilayers can be permeable to nutrients and molecules required for cell growth underneath the transferred film. 219 CHAPTER 8 FABRICATION AND CHARACTERIZATION OF THIN SELF - STANDING COMPOSITE-POLYELECTROLYTE MULTILAYERS 8.1 INTRODUCTION Tissues and organs in vivo are structured in three dimensional (3-D) ordered assemblies maintaining their metabolic functions. Metabolic firnction of cells are maintained and improved in long term by sandwiching the cells between two cell culture substrateszs' 236‘ 241’ 242. Layered cellular multilayers using synthetic polymers have been shown created via layer-by-layer (LbL) assembly deposition of polyelectrolyte multilayers (PEMs) over the cells cultured on a substrate. Germain et al. were first to show the layer-by-layer (LbL) deposition of PEMs over a layer of cells by encapsulating a cell line with the multilayer of poly(diallyldimethylammonium chloride) and sulfonated poly(styrene), sodimn salt (PDAC/SPS) fabricated in the presence of buffer solutions”. They found PDAC/SPS multilayer films to be one of the most suitable PEMs (in terms of fihn porosity and cell viability) when deposited as the capping layer using LbL process over a cultured layer of cells. Rajagopalan et al. created LbL constructs comprised of hepatocyte-polyelectrolyte (PE)-hepatocyte layers, hepatocyte-PE-endothelial cell layers, and hepatocyte-PE-fibroblast cell layers, using chitosan and DNA by forming intermediate polyelectrolyte layers between two cell layers”. Matsusaki et al. showed fabrication of 3-D cellular multilayers using LbL process of fibronectin and gelatin separating two layers of fibroblast or separating a layer of smooth muscle cells and vascular endothelial cells”. However, the main limitation of these methods was that the cells were deprived of cell culture medium during LbL multilayer formationzg’ 29. Cells require nutrients to sustain and perform their metabolic functions, and removing the cell 220 medium and intermediate rinsing during PEM deposition may be harmful to the cell viability. In this study, we fabricated mechanically stable dry self-standing composite polyelectrolyte multilayers (SSC-PEMs). These films were on the thickness scale of sub- micron to nanometer, which is much thinner than the previously reported dry self- standing fihn8243'245. We propose and show that SSC-PEMs can be used for creating a 3- D cell scaffold. The described approach does not require keeping the cultured cells deprived of cell culture medium during 3-D scaffold construction. Multilayer properties can be tuned during LbL formation, separately and prior to the cell culture work, and thus the scaffold preparation is not time consuming and not requiring removal of cell culture medium for long times which is beneficial for the cells' viability. Fabrication of dry self standing multilayer thin films has been shown previously by Hammond and co—workers, where authors fabricated carboxylic acid and PEO based H- bonded films over a hydrophobic substrate and detached them from the substrate to obtain about 8-12 pm thick self standing fihnsm’ 245. However, these H-bonded fihns are not suitable for the cell culture applications, primarily because these films degrade quickly when exposed to physiological pH environments” 98. A degrading cell culture substrate cannot support the cell growth. These films were also much thicker than SSC- PEMs shown in this study. One and Decher showed the fabrication of PAH/SPS self- standing films in liquid phase, by immersing the electrostatistically bonded PAH/SPS films fabricated over sacrificial PAA/PEG films deposited on planar substrate, in water at 221 pH of 5.6-6.3“. However, exposure of an adhered film to a liquid phase for either . . 4 . . . . 4 , 24 substrate dissolution2 6 or sacrrficral film dissolution l 7 can potentially alter the stability and pristine state of the filmsm. SSC-PEMs, prepared in this work, can potentially be useful to fabricate either a three-dimensional (3-D) cellular scaffold or free floating cell culture sheets. Sandwich type culture is important in the cases where the homotypic or heterotypic cell-cell contact can enhance the biological activity of one or the other cell type involved. These scaffolds would hold importance to replace the damaged tissues sections in-vivo. 8.2 MATERIALS AND METHODS 8.2.1 Materials Poly(acrylic acid, sodium salt) solution (PAA, Mw 15,000; catalog no. 416037), Poly(ethylene glycol) (PEG, Mw 10,000; catalog no. P6667), sulfonated poly(styrene), sodium salt (SPS) (Mw ~ 70,000), poly(diallyldimethylammonium chloride) (PDAC) (Mw ~ 100,000 — 200,000) as a 20 wt% solution, and sodium chloride (NaCl) were purchased from Sigma-Aldrich (USA). Poly(dimethylsiloxane) (PDMS) from the Sylgard 184 silicone elastomer kit (Dow Corning, Midland, MI) was used to prepare stamps. Bamstead Nanopure Diamond (Bamstead International, Dubuque, IA) purification system was used as a source for deionized (DI) water with a resistivity of 18.2 MO cm. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, 0.25% trypsin-EDTA, lX-phosphate buffered saline (PBS), and immunostaining components (rabbit anti-paxillin antibody, Alexa Fluor 488 goat anti- 222 rabbit IgG secondary antibody, Texas Red-X phalloidin, and DAPI) were purchased from Invitrogen (Carlsbad, CA). 8.2.2 Self Standing Composite-Polyelectrolyte Multilayer (SSC-PEM) Fabrication Multilayers of PAA and PEG followed by the multilayers of PDAC and SPS were fabricated via layer-by-layer (LbL) process onto a hydrophobic PDMS substrate. Thick PDMS blocks were prepared by curing 10:1 ratio of prepolymer to initiator (100g of prepolymer and 10g of initiator in 100mm Petri dish) in an oven overnight at 60°C. PDAC and SPS polyelectrolyte solutions were prepared in DI water to final concentrations of 3mM each with respect to the repeat unit of the polyelectrolytes. PAA and PEG solutions were prepared in DI water to final concentrations of 1mg/ml. No external salt was used during the deposition of PAA/PEG multilayers, whereas PDAC and SPS polyelectrolyte solutions were supplemented with sodium chloride concentration of 0.5M. Since the degradation onset of PAA/PEG PEMs is known to be above pH value of 3.541’ 97’ 98, therefore a pH value of 2.0 was selected for film formation. All polymer solutions (PAA, PEG, PDAC, SPS), as well as washing baths (pure DI water) were adjusted to deposition pH of 2.0. All polymer solutions were then filtered using a 0.22pm cellulose acetate filter (Corning, NY) prior to use. A Carl Zeiss slide stainer was used for LbL deposition. Deposition time for each polymer (PAA, PEG, PDAC, SPS) was set to 15 sec (actual run time 30 sec) and washing time at each step was set to 20 see with agitation (actual run time 1 min). 223 PAA in protonated form possess the hydrophobic characteristics at acidic pH conditions133 . Therefore, the LbL process was started over an untreated hydrophobic PDMS with PAA at a low pH value of 2.0. Self standing films were also obtained when LbL process was started over hydrophobic PDMS with PEG at a low pH value (data not shown). 20.5 bi-layers of (PAA/PEG) multilayer initiating with PAA (i.e. 20 bilayers of PAA and PEG and an additional terminating layer of PAA) were fabricated onto untreated PDMS substrate, and subsequently various bi-layers of PDAC/SPS were fabricated onto top of (PAA/PEG) multilayers. Depending on the number of bilayers of (PAA/PEG) and (PDAC/SPS) multilayers (discussed in Results and Discussion), the combination of these multilayers was mechanically removed from the PDMS substrate simply with the help of a pair of tweezers and razor to give SSC-PEMs consisted of PAA/PEG and PDAC/SPS multilayers. For the facilitated removal, composite films were dried in vacuum before peeling them off from PDMS. In this study, the top component of SSC-PEM implies (PDAC/SPS) multilayers and bottom component implies (PAA/PEG) multilayers. The term “non-degradable multilayer” implies that multilayers were non- degradable in terms of an effect of change in pH. (PAA/PEG) and (PDAC/SPS) fihns assembled on PDMS are referred as “composite films” before their removal from PDMS, and are referred as “SSC-PEM” after their removal from PDMS. 8.2.3 Characterizations 8.2.3.1 Atomic Force Microscopy (AF M) and Scanning Electron Microscopy (SEM) Thicknesses and morphology of composite films were measured using AFM prior to detaching the films from the PDMS substrates. AFM images were collected in the 224 tapping mode using 300kHz silicon probes (Vista probes) with a Nanoscope IV multimode scope from Digital Instruments (Santa Barbara, CA). A portion of the PDMS was obscured for film growth with a scotch tape, which was removed later at the time of AFM analysis. Regions of the film edge were carefully selected such that there was no apparent detachment or folding of film during AF M analysis. However, in-spite-of the careful selection of regions, portions of the film were observed as partially detached from the PDMS and elevated from PDMS at the edge of coated and non-coated regions where measurement was performed. This resulted in slightly high measured values than the expected values for the SSC-PEM thickness (discussed in Results and Discussion). SEM images of SSC-PEM were collected with field emission JEOL 6300F electron microscope. 8.2.3.2 Optical Microscopy Confocal laser scanning microscopy (CLSM) images were obtained with Olympus Fluoview 1000 laser scanning confocal microscope. Phase contrast images of cells were collected with Leica DM IL inverted microscope (Bannockbum, IL) equipped with SPOT RT color camera (Diagnostics Instruments, MI), and optical images of SSC-PEMs were obtained using Nikon Eclipse ME 600 microscope (Nikon, NY). 8.2.4 Cell Culture NIH3T3 fibroblasts and HeLa cells were purchased from American Type Culture Collection (USA). Both cells were maintained in DMEM with 10% FBS and lOOU/ml penicillin plus lOOug/ml streptomycin (P/S) at 37°C and 10%CO2. Cells grown to 80% 225 confluency were detached by 0.25% trypsin-EDTA and plated at a density of 2x105 cells per ml with 2 ml added to all surfaces studied. 8.2.4.1 Three-dimensional (3-D) Cell Culture using SSC-PEM For a 3-D co-culture, cells were cultured on a (PDAC/SPS)10.5 multilayer and maintained up to a confluency of 70%. Then, SSC-PEMs were plasma etched for 10 min at 0.15 Torr and 50 seem flow of 02 using a plasma cleaner (Harrick Scientific Corporation, NY), and were sterilized under UV light using a germicidal 30W UV-C lamp (Philips, TUV 30W/G30T8) for 15 minutes prior to cell seeding. Just prior to SSC-PEM placement over cells, cell culture medium was removed from the culture dish and cell surface was let to dry for a minute or two. Immediately a large piece of SSC-PEM was placed over the media depleted cultured layer of cells on (PDAC/SPS)10,5 multilayer, and fibroblasts were added as a second layer of cells on top of the adhered SSC-PEM (with (PDAC/SPS)30,5 multilayer component of SSC-PEM as the top surface). 8.2.4.2 Cell Immunostaining Immunocytochemistry was performed 48 hrs post cell seeding on the surfaces at room temperature. Cells were rinsed with PBS, followed by fixation with 4.0% paraformaldehyde in PBS for 15 min, rinsed 3 times in PBS, then permeabilized with 0.1% Triton X-100 in PBS for 15 min and washed 3 times with PBS. After washing, cells were blocked in 1% Bovine Serum Albumin (BSA, US Biological) for 30 minutes. Cells were incubated with rabbit anti-paxillin primary antibody (1:50 dilution in 1% BSA solution) for 1 hour followed by three washes in 1X PBS, and then incubated with Alexa 226 F luor 488 goat anti-rabbit IgG secondary antibody (1:500 dilution in 1% BSA solution) for 1 hour. Cells were washed fiirther, three times in 1X PBS. During secondary antibody incubation, cells were additionally incubated with Texas Red-X phalloidin (Sul stock per 200ul of 1% BSA solution) to visualize actin filaments. Cells were then incubated for 5 minutes in 300nM DAPI (Invitrogen) to visualize the nucleus. After two final washes with 1X PBS, cells were imaged for fluorescence microscopy. 8.3 RESULTS AND DISCUSSION In this study, we fabricated self standing composite-polyelectrolyte multilayers (SSC- PEM) in dry state, obtained by the mechanical detachment of (PAA/PEG) and (PDAC/SPS) composite films from the hydrophobic PDMS substrate. These films are termed SSC-PEMs because they consist of two composite films i.e. H-bonded PAA/PEG multilayers and electrostatistically bonded PDAC/SPS polyelectrolyte multilayers. SSC- PEMs thicknesses were within the range of few hundred nm to about a micron, depending on the number of bilayers. PAA/PEG multilayer component contributed more to thickness in comparison to (PDAC/SPS) multilayers, where latter was only in nanometer scale thickness. H-bonded PAA/PEG multilayers can degrade over a period of time in high pH environments” 4" 97’ 9° , whereas PDAC/SPS polyelectrolyte films are non-degradable and remain stable in different pH environments. Therefore, SSC-PEMs when immersed in a solution at high pH values would eventually yield self-standing ultrathin nanoscale films in aqueous phase. 227 SSC-PEMs were applied to create cell sheet or 3-D cellular scaffold consisting of two or more cell types. It was shown that SSC-PEMs can also sustain the cell growth, with film sheet floating in the cell culture medium. 8.3.1 Self Standing Composite-Polyelectrolyte Multilayer (SSC—PEM) Fabrication SSC-PEMs consisted of electrostatistically bonded non-degradable PDAC/SPS multilayers which were assembled over H-bonded degradable PAA/PEG multilayers deposited at pH of 2.0, on hydrophobic PDMS substrate. Carboxylic acid (-COOH) based weak polyelectrolytes form H-bond interactions at low pH (e.g., pH < 3.5 in the case of PAA) which deprotonates to carboxylate ions (-COO') at high pH and degrades the H- bonded multilayer assembly”. PAA possess the hydrophobic characteristics at the acidic pH conditions133 , and thus can be used to initiate LbL process over the hydrophobic substrates, such as PDMS, under acidic deposition conditions. PAA/PEG multilayers were thick and rough films, which are indicated herein as discontinuous submicron sized pillars of PAA/PEG on PDMS substrate (discussed in section 8.3.2). PAA/PEG pillars supported the thin films of PDAC/SPS constructed on top of them. This is illustrated in Figure 8.1. As shown in Figure 8.2, films were easily detachable from the PDMS substrate in dry conditions using a pair of tweezers and peeling them off yielded the mechanically stable SSC-PEMs. Moreover, as PDMS is a flexible substrate in comparison to PEMs, therefore mechanical compression of PDMS created wrinkling (troughs and craters) in dry film as a result of air entrapped in between 228 the PDMS and dry film. Mechanical compression of PDMS further facilitated the removal of films from PDMS. Figure 8.1 Schematic showing assembly of rough and thick (PAA/PEG) multilayers formed followed by smooth and thin (PDAC/SPS) multilayers on hydrophobic PDMS substrate. Figure 8.2 Macroscopic images of SSC-PEM (PAA/PEG)20,5(PDAC/SPS)30,5: (a) half peeled film from PDMS and PDMS block are shown (b) peeled off fihn and PDMS blocks are shown. (a) (b) SSC-PEMs can give a self-standing thin film of PDAC/SPS alone when these self standing films are immersed in a solution at high pH values. This is due to the degradation of PAA and PEG multilayers into the biocompatible PAA and PEG components“. When PAA deprotonates at a high pH value, it breaks-off the hydrogen bond with PEG molecule and thus the PAA/PEG multilayer degrades. However, the 229 PDAC/SPS multilayer remains intact with change in pH due to the strong constituent polyelectrolytes. 8.3.1.1 Critical thicknesses for SSC-PEM It was found that there is a critical thickness for PAA/PEG multilayers as well as PDAC/SPS multilayers for these films to be detachable from the PDMS. When 10.5 bi- layers of PAA/PEG were formed followed by 80 bi-layers of PDAC/SPS, the multilayers were not removable from the PDMS. It was only when number of PAA/PEG bi-layers was increased to 20.5 or above, followed by 60 bi-layers of PDAC/SPS, the fihns were removable. Also, when number of bi-layers of PDAC/SPS were reduced to 20 and built over 20.5 bi-layers of PAA/PEG, the fihns were not removable. Therefore, the critical number of bi-layers of PAA/PEG films was in between 10.5 to 20.5, and for PDAC/SPS it was greater than 60 bilayers. Increasing the number of PDAC/SPS bilayers facilitated in obtaining the large sections of films. In contrast, increasing the number of PAA/PEG bilayers from 20.5 to any higher number did not facilitate the film removal, rather it resulted in increased thickness of SSC-PEMs. Table 1 shows the various combinations of PAA/PEG and PDAC/SPS multilayer bilayers, which were required for film removal from the PDMS. It is important to note that PAA/PEG films were not removable by themselves, if PDAC/SPS were not built on top of them. Further, thickness of PDMS also played a key role in obtaining the SSC-PEMs. If the PDMS block for LbL deposition was too thin, no film could be peeled off from the PDMS. Therefore, there was a critical thickness for PDMS required to remove the 230 composite films from its surface. Thick PDMS blocks were used (see Materials and Methods), however critical thickness values for PDMS are not investigated in detail in this study. Table 8.1 Critical number of bilayers of (PAA/PEG) and (PDAC/SPS) to obtain SSC- PEM from PDMS substrate. (PAA/PEG) - PDAC/SPS — Removable No. of bi-layers No. of bi—layers 10 20 No 10 80 No 100 0 No 100 20 No 20 80 Yes 20 120 Yes 8.3.1.2 Effect of Molecular Weight of PAA and PEG We observed an important aspect regarding film removal of H-bonded films composed of PAA and PEG (or PEO) from a hydrophobic substrate. A previous study showed detachment of H-bonded PAA/PEO films from hydrophobic substrate yielded self standing fihns, which involved high molecular weights PAA (90,000 g/mol) and PEO (4000000 g/mol)243. However, in this study, we used the low molecular weights PAA (15000 g/mol) and PEG (10000 g/mol). Using low molecular weight polymers, we could not detach the PAA/PEG films even at the high number of bilayers. This suggests that there is an effect of the molecular weight of constituent polymer on the surface energy of these films. In support to this observation, our previous work illustrates that films made of these low molecular weight polymers showed the delayed degradation kinetics and helped in releasing the protein from agarose gels for a long period of time”. Therefore, the impact of molecular weight of polymers involved in a H-bonded assembly needs to be 231 investigated in detail, and this can further open up the applications for these films in various fields. 8.3.2 Thickness and Surface Morphology of SSC-PEM: AF M, SEM, and Optical Microscopy 8.3.2.1 Surface Morphology and Thickness of the Composite Film Thickness of the composite multilayers as well as surface topology and roughness were determined using AF M. FTIR and XPS studies were performed to confirm the presence of polyelectrolytes (with distinguishable functional groups) confined within SSC-PEM. Optical microscopy and SEM were performed to check for the surface morphology of SSC-PEMS. As shown in Figure 8.3a, PAA/PEG films on PDMS formed fihns like discontinuous base pillars of varying sizes, with pillar-like structures protruding from the substrate in varying heights. AFM analysis of (PAA/PEG)20,5 fihn showed an average feature height of 498 i 84 nm of such sub-micron base pillars. On the other hand, PDAC/SPS forms relatively smooth films on planar substratesm, including PDMSm. On the PDMS substrate, the PAA/PEG film base pillars supported the continuous smooth sheet of PDAC/SPS. This is illustrated in Figure 8.1, where PDAC/SPS films are shown supported by the underneath discontinuous base pillars of PAA/PEG film. Figure 8.3b shows the surface morphology of (PDAC/SPS)n films assembled on top of (PAA/PEG)20.5 films i.e. the surface morphology of composite film (PAA/PEG)20,5(PDAC/SPS)n before peeling it off from PDMS, where n = 20, 60 and 80. 232 SI? '3'.) These composite fihns showed smooth surface morphology in some cases and rough morphology in other cases. (PDAC/SPS) multilayers alone on a planar substrate have smooth surface morphologym’ 20° , which is also observed for some n values in Figure 8.3b. The rough surface morphology, wherever observed, of these composite films confirms the presence of rough and disordered (PAA/PEG) films underneath (PDAC/SPS) films. The presence of base pillars facilitated the dry removal of smooth sheet from the PDMS substrate, and this process became easy with the increase in height of PAA/PEG base pillars (i.e. more number of PAA/PEG bilayers). Thus, higher the number of PAA/PEG bilayers, easier was the removal of composite films from PDMS. 233 Figure 8.3 AFM surface analysis of (a) (PAA/PEG)20_5 films formed on hydrophobic PDMS, (b) (PAA/PEG)20_5(PDAC/SPS)n multilayers formed on hydrophobic PDMS i.e. composite films before peeling them off from PDMS. 11 = 20, 60 and 80 for image bl, b2 and b3, respectively. (8) 51) "' Hill) (11m), 4110 ‘ F (111111 (11111) |llli ‘< lllllli 234 119,111 (b) Figure 8.3 continued (1)) 10 1112) 5 10 0 0 25 50 (um) Figure 8.4 shows the optical images (column 1) and corresponding AFM (amplitude mode) images (column 2) along with their section analysis (column 3) of the edges of composite films prior to their removal from PDMS substrates. Thickness of the composite films with minimum number of bilayers which were removable (i.e. 20.5 bilayers of PAA/PEG and 60 bilayers of PDAC/SPS) was found to be ~l urn. Thickness of 20 bilayers of PAA/PEG multilayers is about 400nm'4' 4', and thickness of 60 bilayers of PDAC/SPS (0.5M deposition salt) is about 450nm193 . Therefore, the thickness value of composite films obtained using AFM was on the order of expected value. The small 235 deviation from the expected thickness value can be explained as the experimental error associated with the sample preparation for AFM thickness measurement (see Materials and Methods section). Figure 8.4 Optical micrographs (leftmost column), AF M signal images (middle column), and AFM height analysis (rightmost column) of the edges of (PAA/PEG)20_5(PDAC/SPS)n composite films assembled 011 PDMS. 11 = 20, 60 and 80. Scale bar in optical micrographs is 100pm. temperance -i 10 v ‘. '. - .0‘ 4N” \h‘\&lI\l:‘Ll :lllL'l.llg‘\ (1311 WIN —‘ U i I) ll) :1) () 1() 201.1113“ llm ’ . 4“ Illll f ’ _"" ' '"i’ s .11) 2(1 (1 v H) -1(1(1(1 VV \\t-l.1;_1."ill.‘l\:ltx\ ll“ 1'°_llfil . () ll) 21) 3(1 41) (I ll) 31) 3(lpm—H) 11111 30 ) I . Y 1:“ H111 llYll \\ unite llllt'ltlIL‘» () ° W 211 311 21) 1111130 11111 -<)5(). 236 8.3.2.2 Surface Morphology of the SSC-PEM Figure 8.5a shows the SEM images of SSC-PEM i.e. after peeling the composite films from PDMS, with PDAC/SPS as the scanning surface. Surface roughness and t0pology of SSC-PEMs was found to be comparable with that of composite films i.e. regions of smooth morphology of the composite films before peeling them from the PDMS. The folds observed in SEM images could be due to uncontrolled wrapping of the SSC-PEM at the time of sample mounting during SEM analysis. Figure 8.5b shows the optical micrographs of SSC-PEMs with (PDAC/SPS) multilayers as the imaging surface. Although (PDAC/SPS) multilayers were observed to form smooth top surface of SSC- PEMs, however in certain film formations, top surface was also observed to be highly rough as shown in the bottom panel of Figure 8.5b. This could be due to different fields of imaging selected where the underneath (PAA/PEG) films could be uniform or rough. As discussed above, (PAA/PEG) formed a highly non-uniform multilayer with the pillar kind of structures, and therefore some regions of (PAA/PEG) film could be uniform and others as highly non-uniform. The surface morphology of (PDAC/SPS) was governed by the underneath (PAA/PEG) films. Overall this suggests that (PDAC/SPS) multilayers by themselves formed a uniform film component in SSC-PEM, and can yield a completely uniform film in aqueous phase when (PAA/PEG) film component gets dissolved at high pH conditions. However, in the dry state, SSC-PEM film formed a film where some of the top surface regions were uniform and other were non-uniform. 237 Figure 8.5 (a) SEM images of SSC-PEM (PAA/PEG)20_5(PDAC/SPS)30,5 (i.e. composite films after peeling-off from PDMS) with (PDAC/SPS) multilayers as the scanning surface, (b) optical micrographs of SSC-PEM (PAA/PEG)20,5(PDAC/SPS)30_5 with (PDAC/SPS) multilayers as the imaging surface. 0’) The surface morphology of PAA/PEG films played an important role in removal of these films from hydrophobic PDMS substrate, which in-turn depended on the thickness of 238 these films. A similar configuration of multilayers (as described for SSC-PEMs on PDMS), when fabricated on a planar hydrophilic substrate such as glass, did not yield any self-standing films. Therefore, it can be expected that the rough PAA/PEG films due to interaction with PDMS of low surface energies, facilitated the composite film removal from PDMS. Also, the surface morphology of a substrate is known to affect the cell adhesion response on a substrateu° . Therefore, it is important to assess the properties of films on PDMS as well as of SSC-PEMs in order to obtain better quality self-standing films and for their cell culture applications. 8.3.3 SSC-PEM Composition: XPS and FTIR XPS and FTIR spectra were obtained in order to confirm the presence of (PAA/PEG) multilayer component in SSC-PEM i.e. after peeling-off the composite fihns from PDMS substrate. The (PDAC/SPS) multilayers were the top component of composite fihns on PDMS, and therefore their presence in SSC-PEMs was not questionable. However, it was not sure whether (PAA/PEG) multilayer which were in the pillar kind of morphology directly on top of PDMS were peeled-off along with (PDAC/SPS) multilayers. Although the thickness measurements of composite films suggested that thick (PAA/PEG) multilayers were present underneath thin (PDAC/SPS) multilayers, yet it did not confirm that high thickness was not due to thick (PDAC/SPS) multilayers. Figures 8.6a and 8.6b show the XPS spectra of top component (PDAC/SPS multilayer on top of composite film) and bottom component (PAA/PEG multilayer at bottom of composite film) of SSC-PEM, respectively. Nitrogen and sulphur are the unique elements 239 representing PDAC and SPS, respectively, in SSC-PEM. Comparing the N/S ratios from both figures, the top of SSC-PEM has a N/S ratio of ~ 1 and bottom side has a ratio ~ 2. For PEMs, it is already known that there is an extensive interpenetration of polyelectrolytes among the neighboring layers, and the layering process is not discrete of one polyelectrolyte completely layering over another polyelectrolyte layer’g’ 193’ '95’ 198’ 247. Thus, the N/S ratio of top side of SSC-PEM suggested that topmost surface of SSC-PEM was a uniform intermixed layer of PDAC and SPS. Further, as discussed above, PAA/PEG multilayers formed a pillar kind of structure, and therefore these layers could be highly thick in some regions and thin in other regions. The first layer in the LbL deposition process after (PAA/PEG)20,5 was the PDAC layer. Thus, the N/S ratio of ~ 2 of bottom side of SSC-PEM suggested that (PAA/PEG) multilayers were indeed present underneath the (PDAC/SPS) films. The higher concentration of nitrogen as compared to sulphur could be due the thin covering of (PAA/PEG) underneath (PDAC/SPS) multilayers, thereby giving more access of photon beam in XPS to (PAA/PEG) layers and the first layer of PDAC than to next layer of SPS. 240 Figure 8.6 XPS spectra of SSC-PEM (PAA/PEG)20_5(PDAC/SPS)30_5: (a) top component of SSC-PEM i.e. PDAC/SPS multilayers as the surface analyzed, and (b) bottom component of SSC-PEM i.e. PAA/PEG multilayers as the surface analyzed. (a) x 104 [ OKLL Ols 1 l I 200 o 460 Binding Energy (eV) 1000 800 600 241 Figure 8.6 continued (b) x 104 12 1 I I I I I I 01s I 10 OKLL . I C13 | 8 CISLLNFLL FKLL ' Fls c/s l 6' le ‘ | 123 4. ' i - lSZs S21) C12p 2- I i 1000 L 800 L 600 L 400 L 200 ' 0 Binding Energy (eV) 242 Table 8.2 Atomic concentrations obtained from the XPS analysis of top component of (PAA/PEG)20,5(PDAC/SPS)30,5 SSC-PEM i.e. PDAC/SPS multilayers as the surface analyzed (corresponding to Figure 8.6a). C 1 s N 1 s Ols Si2p S2p C12p 64.46 2.60 23.41 5.85 2.65 1.03 Table 8.3 Atomic concentrations obtained from the XPS analysis of bottom component of (PAA/PEG)20_5(PDAC/SPS)80,5 SSC-PEM i.e. PAA/PEG multilayers as the surface analyzed (corresponding to Figure 8.6b). \ Cls \ le Ols Fls Si2p 82p C12p \61471 3.42 24.40 1.05 7.25 1.82 0.60 Figure 8.7 depicts the transmission FT IR spectrum of SSC-PEM. Signals from the sulfonates (960 to 1210 cm'1)249, C=O (1720 cm!)247 and OH of carboxylic acid (2500 to 3300 cm'l) are clearly observed. This suggests the presence of both protonated PAA i.e. (PAA/PEG) multilayers and SPS i.e. (PDAC/SPS) multilayers in SSC-PEM film. 243 Figure 8.7 Transmission F TIR spectrum of (PAA/PEG)20,5(PDAC/SPS)30,5 SSC-PEM. -SO3' Absorbance ...... 0.30 0.25 ~ -OH of carboxylic acid 0.20 ~ C=O 0.15- 4000 3500 3000 2500 2000 1500 1000 500 Wavenumbers (cm‘l) Since the (PAA/PEG) multilayers were present underneath the (PDAC/SPS) films in SSC-PEMs, therefore this in conjunction with discussion in previous sections, confirms that the high thickness of composite film of up to micron scale was due to thick non- degradable (PAA/PEG) multilayer component, and the non-degradable (PDAC/SPS) multilayers were only in nm scale thick in SSC-PEMs. 244 8.3.4 SSC-PEM in Cell culture Applications 8.3.4.1 SSC-PEM for 3-D Cell Culture SSC-PEMS were showed to develop a sandwich type 3-D culture. The advantages of using SSC—PEM over previous methodszs’ 29 of using LbL assembly process to create 3-D cellular scaffold are that: 1) the use SSC-PEM does not require the cells to be deprived of cell culture medium for PEM deposition. Depriving the cells of culture medium for long times can compromise the cells' viability. 2) Film properties can be tuned separately if harsh fabrication conditions, such as the use of non-physiological salt or pH, are required to alter the film properties for their better performance in 3-D culture. 3) Under the cell culture conditions, the H-bonded PAA/PEG multilayer component of these SSC-PEMs would eventually get dissolved at physiological pH conditions. However, the electrostatistically bonded non-degradable PDAC/SPS multilayers of SSC-PEM are the stable component of these composite films, and can be used for long term cell culture applications. 4) The H-bonded film used in this study consists of chemically unmodified biotolerated or biocompatible components, viz. PAA and PEG, and their release in solution post-degradation would not lead to adverse effect in vitro” 41. 5) the overall thickness of SSC-PEMs was significantly less than of H-bonded self-standing films reported previouslym’ 245, which should be favorable for the 3-D cell-cell contact and bi- directional diffusion of oxygen, nutrients and therapeutic molecules during cell culture. Therefore, this approach of fabricating SSC-PEMs offers the advantages in terms of cell culture applications over previous approaches of 3D cellular scaffold fabricationzs’ 29. 245 Plasma treated SSC-PEM was placed in contact with a layer of cells cultured on a (PDAC/SPS)10_5 film. Oxygen plasma treatment of SSC-PEMs enabled their attachment to underneath (PDAC/SPS)10,5 film forming a sheet cover on top of cultured cells. SSC- PEM adherence to the cell culture substrate was enhanced when the SSC-PEM was plasma treated in oxygen for 10 min, than a non-plasma-treated film. PDAC/SPS multilayer component of SSC-PEM was kept as the top multilayer which became the substrate for second layer of cells. Figure 8.8 illustrates the schematic of three dimensional sandwich co-culture. Figure 8.8 Schematic showing 3-D cell co-culture with an intermediate layer of SSC- PEM between two cell types. First layer of cells was cultured over a charged substrate, and SSC-PEM with bottom component as (PAA/PEG) multilayers was adhered over the cultured cells. Second layer of cells was cultured on top of (PDAC/SPS) component of SSC-PEM. Cell Culture Medium; Physiological pH Conditions \/\/\ \/\/\ \/\/\ «Self-standing Film \/\/\ \/\/\ a- Cell Type A \/\/\ 4—Base PEM m \/\/\\/\/\\/\/\ Figure 8.9a shows the 3-D co-culture using fibroblasts cells and SSC-PEM. A layer of fibroblasts was cultured on top on (PDAC/SPS)10_5 and plasma treated SSC-PEM was placed over cultured fibroblasts after aspirating the cell culture medium. Plasma treated SSC-PEM adhered to non-confluent layer of cells and (PDAC/SPS)10,5 films. The fresh 246 culture medium was added to cells irmnediately after placing the SSC-PEM on cells. Subsequently, a second layer of fibroblasts was cultured over the adhered sheet of SSC- PEM. The green areas in image shows the fibroblasts cultured underneath the SSC-PEM and red areas shows the fibroblasts cultured on top of SSC-PEM. Figure 8% shows the high magnification image where the fibroblasts cultured underneath the adhered SSC- PEM are clearly visible. This indicates that adherence of SSC-PEM on top of cultured film did not effect the cell morphology. Figure 8.9 (a) Three-dimensional cell co-culture showing two layers of fibroblasts with an intermediate layer of (PAA/PEG)20.5(PDAC/SPS)30.5 SSC-PEM which is adhered to the cells and substrate at bottom. Lower panel of images shows contrast modified images corresponding to the images in top panel. Red and green highlighted areas shows cells cultured above and below SSC-PEM, respectively (scale bar = 100 um). (b) High magnification image showing viable cells cultured below a (PAA/PEG)20,5(PDAC/SPS)30,5 SSC-PEM which is adhered to those cells and the exposed substrate. (a) 247 Figure 8.9 continued 0)) 10011111 Figure 8.10 shows the three dimensional co-culture of fibroblasts and HeLa cells. Fibroblast cells were cultured on (PDAC/SPS)10,5 film, plasma treated SSC-PEM was adhered over fibroblasts cells and subsequently, HeLa cells were cultured over the adhered sheet of SSC-PEM. Confocal z-series sections of 3-D co-culture of fibroblasts and HeLa cells are shown. 248 Figure 8.10 3-D cell co-culture of fibroblasts and HeLa cells using (PAA/PEG)20_5(PDAC/SPS)30,5 SSC-PEM as an intermediate layer, in the sequence of fibroblast, SSC-PEM, and HeLa cells from bottom to top. Confocal microscopy z-series analysis is shown. X Z = 0 (Top) 5011111 ( . Z = 4 lBottornl \ D \\\\\\\‘\\\\ \\\~\\\ \ \\\‘\\\\\\\‘\ \\\‘\‘ \\\ \ \\§\‘ _ cae- pm 5011111 2.; _ ..Y Z Zzllllm Zz9um G Zzl8llm Z227llm Zz36tlnl 249 8.3.4.2 SSC—PEM for Cell Sheet Culture Cell culture sheets hold importance in tissue engineering as they can be directly transplanted to host tissues or 3-D structures can be created via layering of two of more cell sheetszw. In a pioneering work, Okano, Sakurai and co-workers developed cell culture sheets which were made by harvesting cells on a temperature responsive polymer poly(N-isopropylacrylamide) (PIPAAm) conjugated to a surface and detaching the intact cultured cells sheets via a change in surface temperature25°’ 251. They demonstrated the use of these cell sheets in tissues reconstruction such as transplanting them directly to 252 253 cornea without the need of sutures with recovery of lost visual activity in humans . Cells sheets were also used to create 3-D tissues and organs structures, such as cardiac muscle via homotypic layering of cell sheetsz“, or kidney glomeruli and liver lobules via heterotypic stratification of cell sheetszss. Here, we used SSC-PEMs to create the free floating cell culture sheets (data not shown). Cells were cultured on top of floating SSC-PEMs i.e. without adhering SSC-PEM to base substrate, with PDAC/SPS component as the top multilayer and PAA/PEG component as the bottom multilayer. Figure 8.11 illustrates the schematic of a free floating culture sheet, where cells are shown cultured directly on top surface of SSC-PEM (i.e. PDAC/SPS), and SSC-PEM is kept floating in cell culture medium. A floating cell culture sheet using SSC-PEM was created in a similar way as shown Figure 8.9a, but in this case SSC-PEM was not adhered to base substrate and cells were directly cultured on top of floating SSC-PEM (data not shown). 250 Figure 8.11 Schematic showing SSC-PEM as a cell sheet. Cells can be cultured over the (PDAC/SPS) component of a floating SSC-PEM to create a floating cell sheet. <— Cells <— Self-standing Film 8.4 CONCLUSIONS Here, we show the fabrication of a self-standing polyelectrolyte multilayer film, which is a composite film of pH degradable H—bonded (PAA/PEG) multilayers and non- degradable electrostatistically bonded (PDAC/SPS) multilayers, termed SSC-PEMs. The thickness of SSC-PEMs was in the range of sub-microns. Formation of SSC-PEM was highly dependent on the number of bilayers, and there was a threshold value for the number of bilayers of (PAA/PEG) and (PDAC/SPS) multilayers both, below which SSC- PEMs cannot be obtained. SSC-PEM was used to create a 3-D cell co-culture. The use of SSC-PEM was advantageous over previous methods of 3-D cell culture as this process did not require the depletion of the cell culture medium for deposition of polyelectrolytes on top of cells. Further, the PEM conditions, such as thickness and ionic concentration can be tuned separately to make these films a better candidate for cell culture. The H- bonded film consists of chemically unmodified biocompatible components, viz. PAA and PEG, and their release in solution post-degradation would be a bioinert choice. Another cell culture application of SSC-PEM includes the formation a floating cell culture sheet. With further modifications, these films can be used for other applications such as with 251 drug incorporation in H-bonded degradable component; SSC-PEMs can also be used as drug eluting adhesive bandages for skin wound healing applications. 252 CHAPTER 9 CONCLUSIONS AND FUTURE DIRECTIONS First part of this thesis focus on the techniques to solve some of the challenging issues in the field of tissue engineering, viz. controlled drug delivery. Layer-by-layer (LbL) assembled multilayers, polymeric nanoparticles and microcontact printing (pCP) are shown as the contributing tools for drug delivery. Second part of this thesis focus on engineering the LbL assembled polyelectrolyte multilayers (PEMs) to create three- dimensional (3-D) cellular constructs to mimic the natural in-vivo environment. Optimization of PEM surface for controlled cell adhesion, PEM stamping in conditions conducive to cell culture, and fabrication of self-standing PEMs are shown as the few important pathways in generating the functional 3-D tissue engineered constructs. Chapter 2 demonstrate that pH-responsive H-bonded poly(ethylene glycol)(PEG)/poly(acrylic acid)(PAA)/protein hybrid layer-bylayer (LbL) thin fihns, when prepared over agarose, provided sustained release of protein under physiological conditions for more than four weeks. This is the first demonstration of month-long sustained protein release from an agarose hydrogel, whereby the drug/protein was loaded separately from the agarose hydrogel fabrication process. Chapter 3 describes the similar LbL multilayers controlled release of an anti-mitotic agent to control the formation of fibroblast reactive cell layer that forms post-implantation of agarose hydrogel scaffolds. Chapter 4 describes the gene therapy approach to knock-down the genes producing malfunctioning proteins inhibiting tissue regeneration. 25 kDa linear polyethylenimine (LPEI) was optimized as the siRNA transfection reagent, and a broad range of LPEI— siRNA nitrogen/phosphate (NIP) ratios (ranging from 5 to 90) was evaluated for the 253 relative amounts of siRNA incorporated into the nanoparticles, nanoparticle size and transfection efficiencies. Chapter 5 describes the fabrication of LPEI particles of novel shapes under high shear rate mixing conditions, coupled with some other parameters such as application of vacuum and high solution viscosity. Chapters 6, 7 and 8 discuss about the engineering of PEMs to modulate cell adhesion on films, stamping of PEMs for 3-D cell culture applications, and fabrication of thin self-standing PEMs, respectively. Subsequent sections of this chapter describe some of the future directions, which are the direct extensions of the work reported in this thesis. 9.1 FUTURE DIRECTIONS 9.1.1 Controlled Release of Growth Factor BDNF Neurotrophins such as nerve growth factor (N GF ), brain-derived neurotrophic factor (BDNF), and neurotrophin—3 (N T-3) are known to promote the neuron outgrowth and axonal regenerationz’ 6' 7. The technique explained in chapter 2, for controlled release of active lysozyme from agarose hydrogels, can further be extended for in-vitro or in-vivo investigations to provide sustained release of BDNF from the templated agarose scaffolds. However, the main constriction in using BDNF for investigations of sustained release is the high cost of the commercially available active BDNF. Currently our group has been focusing on large scale production of BDNF, following which the LbL approach can be applied to study the controlled release of BDNF from templated scaffolds. Impurity of the expressed BDNF obtained in large scale can be one of the main concerns for loading BDNF in scaffolds. However, the impure BDNF available in the presence of 254 other components at high concentrations could be better than pure BDNF available at very low concentrations. LbL formation is dependent on the concentration of polymer used to assemble multilayers, and very low concentrations of polymer do not yield the LbL multilayers. The impurities in BDNF supernatant would be useful to load BDNF during LbL formation, as the overall concentration including the other components would be higher than of BDNF alone, which can help in loading all of the components which are at low concentrations individually. Therefore, if the presence of other components is not harmful for axonal growth, then impure BDNF in large volumes can be used to form LbL over agarose directly and load BDNF in agarose. Following is the suggested list of tasks that can be performed: 1). Controlled release and activity of released lysozyme can be investigated using the “templated agarose scaffolds” instead of using the bulk agarose. Chapter 2 describes the work using bulk agarose i.e. large volumes of agarose, where the protein loading and releases were in micron scale. In the “templated agarose scaffolds”, the loading and release would be at nano-scales. 2). In order to increase the working pH for LbL fabrication (for minimal loss in BDNF activity due to reduction in pH), the lysozyme release can be tested in-vitro for a few LbL configurations (Schematic 2.1) using the sets of H-bonding polymers for which the critical pH values for film dissociation are higher. For example, it is known that the critical pH values for disintegration of H-bonded high molecular weight PEO/PAA films 255 is 3.6, for PRO/poly(methacrylic acid)(PMAA) films is 4.6, and for poly(vinylpyrrolidone)(PVPON)/PAA films is 6.9, depending on the system salt concentrations98. 3). If BDNF can retain significant activity at low pH conditions, the approach described in Chapter 2 of using only PEG and protein based multilayers (a completely biocompatible system) can be evaluated for controlled release of BDNF in-vitro and in— vivo to assess enhanced axonal growth. 4). The biocompatible polymers such as, poly(aspartic) acid, or poly(glutamic acid) can be tested in lieu of PAA to fabricate the LbL over agarose for controlled release of protein, to make the system completely biocompatible. 9.2 Drug Delivery to Control Reactive Cell Layer Chapter 3 describes the controlled release of AraC (an anti-mitotic agent) to control the reactive cell layer of fibroblasts formed in and around templated scaffold post- irnplantation. The released concentrations of AraC in this study were much below the concentrations that induce apoptosis in cerebellar and cortical neurons in culture12 1. This is good for in vivo nerve repair applications, where the released AraC at low concentrations would not impair the axonal growth. At the same time, the released AraC was shown to have a growth inhibitory effect on cultured fibroblasts, but only for the limited amount of time. The controlled release of AraC was not observed for very long periods. Therefore, one possible method to control reactive cell layer formation in vivo 256 could be to employ LbL multilayers based controlled release methodology using injectable agarose microspheres or other microstructures. The microstructures can be loaded with AraC using LbL method and injected at the site of injury after implantation (and not since beginning) when reactive cell layer is expected to start its formation on the scaffold. This way the AraC would be exposed to cells only whenever required and only for a limited time. 9.3 Application of Novel Shaped Particles for siRN A Delivery The LPEI-IPC micro- and name-particles of various shapes fabricated, as described in Chapter 5, can be further assessed for their cellular uptake by forming LPEI-IPC-siRNA complex particles for a more efficient siRNA delivery. The enhanced uptake of tapered shaped micron sized particles by the macrophages is already known“. The novel shaped particles fabricated in this study might be able to load more siRNA per particle as an effect of particle shape, and cellular uptake of particles with some unique shapes might be more efficient giving better silencing efficiency. 9.4 Selection of Suitable Polyelectrolytes for 3-D Cellular Constructs Chapters 7 and 8 describe the formation of 3-D cellular constructs, however with the limitations of low permeability of PDAC/SPS multilayers. Different sets of polyelectrolytes can be checked for multilayer deposition on top of the cell surface and their permeability for various molecules of different sizes, so that there would be sufficient transport of the molecules, like nutrients and oxygen, across the multilayer structure between two cell types. 257 APPENDIX A: AGAROSE HYDROGEL STAMPS FOR TIME AND SPACE CONTROLLED DRUG DELIVERY Chapter 2 and 3 discusses about the time controlled drug release from LbL multilayer functionalized agarose hydrogels. A patterned structure of agarose can be used as a stamp to transfer the drug of interest on a surface in a patterned and time dependent fashion, alter fimctionalizing the agarose with LbL multilayers. Figure A] shows the agarose stamps prepared using double replica molding process as described elsewhere256’ 257. These agarose stamps can be loaded with drugs using LbL multilayer process”, and drug patterns can be formed on a surface using microcontact printing (pCP) process°5’ 66. LbL functionalized agarose stamps can be used multiple times to form drug patterns on different surfaces, or the drug can be continuously delivered on to a single surface in a time dependent manner. E50011m Z 1 10011111 ‘ 2 ,"rr ‘t't MJs f2“; ' ‘ I ‘4 .r, .J'." 1.‘ l . ' 4 ' r‘ it H 100 um " M' ~~i 100 rm . . I ‘ vi . f! ’2 ., ' I: I , - . -, ‘3. u . V ‘1 r 5 3‘, "' ’3 - x 9m, era» .., 2,: ,. n . ,. , . ... ..., . . - .12; ._1_ 4;; :.. ... . .9; '.: ..: g . " " ““ ‘ l- ! D .u: .."i .e.. . . . ‘3 21:34 {3&5 are ”aim sea. titte- liaise . -. H . kn i4. ‘1'. r 5» i ,. l» 31 ‘ r . 4 .‘. 1' ' a4 . 1. _‘1. s n}. '7‘ . ,. _ '- ’k‘ 131 I 1 s Figure A.l Micro-pattemed agarose hydrogel stamps. 258 Figure A.2 shows the fibroblast cells stamped on to a glass surface using pCP process and agarose hydrogel as the stamps. UV sterilized agarose stamps were soaked with a high density suspension of fibroblast cells, and hydrated stamps were kept in contact with surface for at least 30 minutes. Figure A.2 Fibroblast cells stamped on a glass surface using microcontact printing (pCP) process and agarose hydrogel stamps. 259 APPENDIX B: LIST OF PUBLICATIONS The following manuscripts were published in peer-reviewed journal articles, or are under preparation based on the work reported in this thesis. Journal Articles: 1. Mehrotra S, Hunley S C, Pawelec K M, Zhang L, Lee 1, Back S, Chan C, Cell adhesive behavior on thin polyelectrolyte multilayers: cells attempt to achieve homeostasis of its adhesion energy, Langmuir, 26, 12794-12802, 2010. Mehrotra S, Lynam D, Maloney R, Pawelec K M, Tuszynski M, Lee 1, Chan C, Sakamoto J. Time controlled protein release from layer-by-layer assembled multilayer functionalized agarose hydrogels. Advanced Functional Materials, 20, 247-258, 2010. . Mehrotra S, Lee 1, Chan C. Multilayer mediated forward and patterned siRNA transfection using linear-PEI at extended N/P ratios. Acta Biomaterialia, 5, 1474- 1488, 2009. ' Mehrotra S. et al. Time Controlled Release of Cytosine Arabinofuranoside (AraC) from Agarose Hydrogels (Journal of Controlled Release, Submitted) Manuscripts in Preparation: 1. Mehrotra S. et al. Polyelectrolyte Multilayer Transfer in Aqueous Phase and Non-Contact Mode (In preparation, Industrial & Engineering Chemistry Research). Mehrotra S. et al. Fabrication and Characterization of Thin Self-Standing Composite Polyelectrolyte Multilayers (In preparation, Langmuir). 3. Mehrotra S. et al. Fabrication of Linear-PEI Nanoparticles under High Shear Rate Mixing Conditions (In preparation, Macromolecular Rapid Communications). Mehrotra S. et al. Microscopy analysis of H-bonded poly(ethylene glycol) (PEG) and poly(acrylic acid) (PAA) multilayers. (In preparation). 260 [l 10. 11. 12. BIBLIOGRAPHY Langer, R. & Vacanti, J. P. Tissue Engineering. Science 260, 920-926 (1993). Stokols, S. et al. Templated agarose scaffolds support linear axonal regeneration. Tissue Engineering 12, 2777-2787 (2006). Tuszynski, M. H. et al. Nerve growth factor delivery by gene transfer induces differential outgrowth of sensory, motor, and noradrenergic neurites after adult spinal cord injury. Experimental Neurology 137, 157-173 (1996). Moore, M. J. et al. Multiple-channel scaffolds to promote spinal cord axon regeneration. Biomaterials 27, 419-429 (2006). Prang, P. et al. The promotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels. Biomaterials 27, 3560- 3569(2006) Schmidt, C. E. & Leach, J. B. Neural tissue engineering: Strategies for repair and regeneration. Annual Review of Biomedical Engineering 5, 293-347 (2003). Stokols, S. & Tuszynski, M. H. The fabrication and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury. Biomaterials 25, 5839- 5846 (2004). Stokols, S. & Tuszynski, M. H. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials 27, 443-451 (2006). Yang, Y. et al. Multiple Channel Bridges for Spinal Cord Injury: Cellular Characterization of Host Response. Tissue Engineering Part A 15, 3283-3295 (2009) Bunge, M. B. Bridging areas of injury in the spinal cord. Neuroscientist 7, 325- 339 (2001). Tong, X. J. et al. Sciatic-Nerve Regeneration Navigated by Laminin-Fibronectin Double Coated Biodegradable Collagen Grafts in Rats. Brain Research 663, 155- 162 (1994). Doolabh, V. B., Hertl, M. C. & Mackinnon, S. E. The role of conduits in nerve repair: A review. Reviews in the Neurosciences 7, 47—84 (1996). 261 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. F oumier, E., Passirani, C., Montero-Menei, C. N. & Benoit, J. P. Biocompatibility of implantable synthetic polymeric drug carriers: focus on brain biocompatibility. Biomaterials 24, 3311-3331 (2003). Mehrotra, S. et a1. Time Controlled Protein Release from Layer-by-Layer Assembled Multilayer F unctionalized Agarose Hydrogels. Advanced Functional Materials 20, 247-258 (2010). Bouhadir, K. H., Kruger, G. M., Lee, K. Y. & Mooney, D. J. Sustained and controlled release of daunomycin from cross-linked poly(aldehyde guluronate) hydrogels. Journal of Pharmaceutical Sciences 89, 910-919 (2000). Hoare, T. R. & Kohane, D. S. Hydrogels in drug delivery: Progress and challenges. Polymer 49, 1993-2007 (2008). Nuttehnan, C. R., Tripodi, M. C. & Anseth, K. S. Dexamethasone-fimctionalized gels induce osteogenic differentiation of encapsulated hMSCs. Journal of Biomedical Materials Research Part A 76A, 183-195 (2006). Richardson, T. P., Peters, M. C., Ennett, A. B. & Mooney, D. J. Polymeric system for dual growth factor delivery. Nature Biotechnology 19, 1029-1034 (2001). Sutter, M., Siepmann, J., Hennink, W. E. & Jiskoot, W. Recombinant gelatin hydrogels for the sustained release of proteins. Journal of Controlled Release 119, 301-312 (2007). De Geest, B. G. et al. Self-rupturing microcapsules. Advanced Materials 17, 2357-2361 (2005). Matsusaki, M., Sakaguchi, H., Serizawa, T. & Akashi, M. Controlled release of vascular endothelial growth factor from alginate hydrogels nano-coated with polyelectrolyte multilayer films. Journal of Biomaterials Science, Polymer Edition 18, 775-783 (2007). Lynch, 1., de Gregorio, P. & Dawson, K. A. Simultaneous release of hydrophobic and cationic solutes from thin-film "plum-pudding" gels: A multifunctional platform for surface drug delivery? Journal of Physical Chemistry B 109, 6257- 6261(2005) Nakamae, K., Nishino, T., Kato, K., Miyata, T. & Hoffman, A. S. Synthesis and characterization of stirnuIi-sensitive hydrogels having a different length of ethylene glycol chains carrying phosphate groups: loading and release of lysozyme. Journal of Biomaterials Science, Polymer Edition 15, 1435-1446 (2004) 262 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. Park, H., Temenoff, J. 8., Holland, T. A., Tabata, Y. & Mikes, A. G. Delivery of TGF-beta l and chondrocytes via injectable, biodegradable hydrogels for cartilage tissue engineering applications. Biomaterials 26, 7095-7103 (2005). Peppas, N. A., Hilt, J. Z., Khademhosseini, A. & Langer, R. Hydrogels in biology and medicine: From molecular principles to bionanotechnology. Advanced Materials 18, 1345-1360 (2006). Peppas, N. A. & Leobandung, W. Stimuli-sensitive hydrogels: ideal carriers for chronobiology and chronotherapy. Journal of Biomaterials Science, Polymer Edition 15, 125-144 (2004). Tuszynski, M. H. Personal Communication. Rajagopalan, P. et al. Polyelectrolyte nano-scaffolds for the design of layered cellular Architectures. Tissue Engineering 12, 1553-1563 (2006). Matsusaki, M., Kadowaki, K., Nakahara, Y. & Akashi, M. Fabrication of cellular multilayers with nanometer-sized extracellular matrix films. Angewandte Chemie-Intemational Edition 46, 4689-4692 (2007). Germain, M. et a1. Protection of mammalian cell used in biosensors by coating with a polyelectrolyte shell. Biosensors & Bioelectronics 21, 1566-1573 (2006). Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811 (1998). Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 4-11, 494-498 (2001). Cheema, S. K., Chen, E., Shea, L. D. & Mathur, A. B. Regulation and guidance of cell behavior for tissue regeneration via the siRN A mechanism. Wound Repair and Regeneration 15, 286-295 (2007). Ryther, R. C. C., Flynt, A. 8., Phillips, J. A. & Patton, J. G. siRNA therapeutics: Big potential from small RNAs. Gene Therapy 12, 5-11 (2005). Aigner, A. Delivery systems for the direct application of siRNAs to induce RNA interference (RNAi) in vivo. Journal of Biomedicine and Biotechnology (2006). Zhang, S. B., Zhao, B., Jiang, H. M., Wang, B. & Ma, B. C. Cationic lipids and polymers mediated vectors for delivery of siRNA. Journal of Controlled Release 123, 1-10 (2007). Pack, D. W., Hoffman, A. S., Pun, S. & Stayton, P. S. Design and development of polymers for gene delivery. Nature Reviews Drug Discovery 4, 581-593 (2005). 263 38. 39. 40. 41. 42. 43. 45. 46. 47. 48. Mehrotra, S., Lee, I. & Chan, C. Multilayer mediated forward and patterned siRNA transfection using linear-PEI at extended N/P ratios. Acta Biomaterialia 5, 1474-1488 (2009). Decher, G. Fuzzy nanoassemblies: Toward layered polymeric multicomposites. Science 277, 1232-1237 (1997). Decher, G., Hong, J. D. & Schmitt, J. Buildup of Ultrathin Multilayer Films by a Self-Assembly Process .3. Consecutively Alternating Adsorption of Anionic and Cationic Polyelectrolytes on Charged Surfaces. Thin Solid Films 210, 831-835 (1992) Ono, S. S. & Decher, G. Preparation of ultrathin self-standing polyelectrolyte multilayer membranes at physiological conditions using pH-responsive film segments as sacrificial layers. Nano Letters 6, 592-598 (2006). Schlenoff, J. B., Dubas, S. T. & Farhat, T. Sprayed Polyelectrolyte Multilayers. Langmuir 16, 9968-9969 (2000). Tang, Z. Y., Wang, Y., Podsiadlo, P. & Kotov, N. A. Biomedical applications of layer-by-layer assembly: From biomimetics to tissue engineering. Advanced Materials 18, 3203-3224 (2006). Lynn, D. M. Peeling back the layers: Controlled erosion and triggered disassembly of multilayered polyelectrolyte thin fihns. Advanced Materials 19, 4118-4130 (2007). De Geest, B. G., Sanders, N. N., Sukhorukov, G. B., Demeester, J. & De Smedt, S. C. Release mechanisms for polyelectrolyte capsules. Chemical Society Reviews 36, 636-649 (2007). Neu, M., Fischer, D. & Kissel, T. Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. Journal of Gene Medicine 7, 992-1009 (2005). Merdan, T., Kopecek, J. & Kissel, T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Advanced Drug Delivery Reviews 54, 715-758 (2002). Abdallah, B. et al. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: Polyethylenirnine. Human Gene Therapy 7, 1947-1954 (1996) 264 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. Boussif, O. et al. A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in-Vivo - Polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America 92, 7297-7301 (1995). Choosakoonkriang, S., Lobo, B. A., Koe, G. S., Koe, J. G. & Middaugh, C. R. Biophysical characterization of PEI/DNA complexes. Journal of Pharmaceutical Sciences 92, 1710-1722 (2003). Gary, D. J., Purl, N. & Won, Y. Y. Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery. Journal of Controlled Release 121, 64-73 (2007). Grayson, A. C. R., Doody, A. M. & Putnam, D. Biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro. Pharmaceutical Research 23, 1868-1876 (2006). Grzelinski, M. et al. RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts. Human Gene Therapy 17, 751-766 (2006) Kunath, K. et al. Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular—weight polyethylenimine. Journal of Controlled Release 89, 113-125 (2003). Ogris, M. et al. The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Therapy 5, 1425-1433 (1998). Thomas, M. et al. Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proceedings of the National Academy of Sciences of the United States of America 102, 5679-5684 (2005). Urban-Klein, B., Werth, S., Abuharbeid, S., Czubayko, F. & Aigner, A. RNAi- mediated gene-targeting through systemic application of polyethylenimine (PEI)- complexed siRNA in vivo. Gene Therapy 12, 461-466 (2005). Yamauchi, F ., Kato, K. & Iwata, H. Layer-by-layer assembly of poly(ethyleneirnine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer. Langmuir 21, 8360- 8367(2005) Spagnou, S., Miller, A. D. & Keller, M. Lipidic carriers of siRNA: Differences in the formulation, cellular uptake, and delivery with plasmid DNA. Biochemistry 43, 13348-13356 (2004). 265 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. Champion, J. A., Katare, Y. K. & Mitragotri, S. Making polymeric micro- and nanoparticles of complex shapes. Proceedings of the National Academy of Sciences of the United States of America 104, 11901-11904 (2007). Champion, J. A. & Mitragotri, S. Role of target geometry in phagocytosis. Proceedings of the National Academy of Sciences of the United States of America 103, 4930-4934 (2006). Bonadio, J ., Smiley, E., Patil, P. & Goldstein, S. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nature Medicine 5, 753-759 (1999). De Laporte, L. & Shea, L. D. Matrices and scaffolds for DNA delivery in tissue engineering. Advanced Drug Delivery Reviews 59, 292-307 (2007). Kumar, A. & Whitesides, G. M. Features of Gold Having Micrometer to Centimeter Dimensions Can Be Formed through a Combination of Stamping with an Elastomeric Stamp and an Alkanethiol Ink Followed by Chemical Etching. Applied Physics Letters 63, 2002-2004 (1993). Xia, Y. N. & Whitesides, G. M. Soft lithography. Angewandte Chemic- Intemational Edition 37, 551-575 (1998). Xia, Y. Soft lithography and the art of patterning - A tribute to Professor George M. Whitesides. Advanced Materials 16, 1245-1246 (2004). Kumar, A., Biebuyck, H. A. & Whitesides, G. M. Patterning Self-Assembled Monolayers - Applications in Materials Science. Langmuir 10, 1498-1511 (1994). Ruiz, S. A. & Chen, C. S. Microcontact printing: A tool to pattern. Soft Matter 3, 168-177 (2007). Kidambi, S., Chan, C. & Lee, I. S. Selective depositions on polyelectrolyte multilayers: Self-assembled monolayers of m-dPEG acid as molecular template. Journal of the American Chemical Society 126, 4697-4703 (2004). Jiang, X., Zheng, H., Gourdin, S. & Hammond, P. T. Polymer-on-Polymer Stamping: Universal Approaches to Chemically Patterned Surfaces. Langmuir 18, 2607-2615 (2002). Blesch, A. & Tuszynski, M. H. Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination. Journal of Comparative Neurology 467, 403-417 (2003). 266 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. Bradbury, E. J., Khemani, S., King, V. R., Priestley, J. V. & McMahon, S. B. NT- 3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord. European Journal of Neuroscience 11, 3873-3883 (1999) Menei, P., Montero-Menei, C., Whittemore, S. R., Bunge, R. P. & Bunge, M. B. Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord. European Journal of Neuroscience 10, 607-621 (1998). Fatin-Rouge, N., Milon, A., Buffle, J ., Goulet, R. R. & Tessier, A. Diffirsion and partitioning of solutes in agarose hydrogels: The relative influence of electrostatic and specific interactions. Journal of Physical Chemistry B 107, 12126-12137 (2003) Liang, S. M. et al. Protein diffusion in agarose hydrogel in situ measured by improved refractive index method. Journal of Controlled Release 115, 189-196 (2006) Maaloum, M., Pemodet, N. & Tinland, B. Agarose gel structure using atomic force microscopy: Gel concentration and ionic strength effects. Electrophoresis 19, 1606-1610 (1998). Pemodet, N., Maaloum, M. & Tinland, B. Pore size of agarose gels by atomic force microscopy. Electrophoresis 18, 55-58 (1997). Gutenwik, J., Nilsson, B. & Axelsson, A. Coupled diffusion and adsorption effects for multiple proteins in agarose gel. Aiche Journal 50, 3006-3018 (2004). Xiong, J. Y. et a1. Topology evolution and gelation mechanism of agarose gel. Journal of Physical Chemistry B 109, 5638-5643 (2005). Blake, C. C. F. et al. Structure of Hen Egg-White Lysozyme - a 3-Dimensional Fourier Synthesis at 2a Resolution. Nature 206, 757-761 (1965). Donath, E., Sukhorukov, G. B., Caruso, R, Davis, S. A. & Mohwald, H. Novel hollow polymer shells by colloid-templated assembly of polyelectrolytes. Angewandte Chemie-Intemational Edition 37, 2202-2205 (1998). Sukhorukov, G. B. et al. Layer-by-layer self assembly of polyelectrolytes on colloidal particles. Colloids and Surfaces a-Physicochemical and Engineering Aspects 137, 253-266 (1998). Antipov, A. A., Sukhorukov, G. B., Donath, E. & Mohwald, H. Sustained release properties of polyelectrolyte multilayer capsules. Journal of Physical Chemistry B 105, 2281-2284 (2001). 267 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. Berg, M. C., Zhai, L., Cohen, R. E. & Rubner, M. F. Controlled drug release from porous polyelectrolyte multilayers. Biomacromolecules 7, 357-364 (2006). Chung, A. J. & Rubner, M. F. Methods of loading and releasing low molecular weight cationic molecules in weak polyelectrolyte multilayer films. Langmuir 18, 1176-1183 (2002). Qiu, X. P., Donath, E. & Mohwald, H. Permeability of ibuprofen in various polyelectrolyte multilayers. Macromolecular Materials and Engineering 286, 591- 597 (2001). Shi, X. Y. & Caruso, F. Release behavior of thin-walled microcapsules composed of polyelectrolyte multilayers. Langmuir 17, 2036-2042 (2001). Sukhorukov, G. B., Antipov, A. A., Voigt, A., Donath, E. & Mohwald, H. pH- controlled macromolecule encapsulation in and release from polyelectrolyte multilayer nanocapsules. Macromolecular Rapid Communications 22, 44-46 (2001) Sukhorukov, G. B., Brumen, M., Donath, E. & Mohwald, H. Hollow polyelectrolyte shells: Exclusion of polymers and donnan equilibrium. Journal of Physical Chemistry B 103, 6434-6440 (1999). Kim, B. S., Park, S. W. & Hammond, P. T. Hydrogen-bonding layer-by-layer assembled biodegradable polymeric micelles as drug delivery vehicles from surfaces. Acs Nano 2, 386-392 (2008). Picart, C. et al. Controlled degradability of polysaccharide multilayer films in vitro and in vivo. Advanced Functional Materials 15, 1771-1780 (2005). Serizawa, T., Yamaguchi, M. & Akashi, M. Time-controlled desorption of ultrathin polymer films triggered by enzymatic degradation. Angewandte Chemic- Intemational Edition 42, 1 1 15-1118 (2003). Wood, K. C., Boedicker, J. Q., Lynn, D. M. & Hammond, P. T. Tunable drug release from hydrolytieally degradable layer-by-layer thin films. Langmuir 21, 1603-1609 (2005). Wood, K. C., Chuang, H. F., Batten, R. D., Lynn, D. M. & Hammond, P. T. Controlling interlayer diffusion to achieve sustained, multiagent delivery from layer-by-layer thin films. Proceedings of the National Academy of Sciences of the United States of America 103, 10207-10212 (2006). 268 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. Stockton, W. B. & Rubner, M. F. Molecular-level processing of conjugated polymers .4. Layer-by-layer manipulation of polyaniline via hydrogen-bonding interactions. Macromolecules 30, 2717-2725 (1997). Wang, L. Y. et al. A new approach for the fabrication of an alternating multilayer film of poly(4-vinylpyridine) and poly(acrylic acid) based on hydrogen bonding. Macromolecular Rapid Communications 18, 509-514 (1997). Sukhishvili, S. A. & Granick, S. Layered, erasable, ultrathin polymer films. Journal of the American Chemical Society 122, 9550-9551 (2000). Sukhishvili, S. A. & Granick, S. Layered, erasable polymer multilayers formed by hydrogen-bonded sequential self-assembly. Macromolecules 35, 301 -3 10 (2002). DeLongchamp, D. M. & Hammond, P. T. Highly ion conductive poly(ethylene oxide)-based solid polymer electrolytes from hydrogen bonding layer-by-layer assembly. Langmuir 20, 5403-5411 (2004). Kharlampieva, E. & Sukhishvili, S. A. Release of a dye from hydrogen-bonded and electrostatically assembled polymer films triggered by adsorption of a polyelectrolyte. Langmuir 20, 9677-9685 (2004). De Geest, B. G. et al. Intracellularly degradable polyelectrolyte microcapsules. Advanced Materials 18, 1005-1009 (2006). Koennings, S., Sapin, A., Blunk, T., Menei, P. & Goepferich, A. Towards controlled release of BDNF - Manufacturing strategies for protein-loaded lipid implants and biocompatibility evaluation in the brain. Journal of Controlled Release 119, 163-172 (2007). Brinker, C. J. & Scherer, G. W. Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing (Academic Press, Boston, 1990). Selsted, M. E. & Martinez, R. J. A Simple and Ultrasensitive Enzymatic Assay for the Quantitative-Determination of Lysozyme in the Picogram Range. Analytical Biochemistry 109, 67-70 (1980). Hollis, E. R., Jamshidi, P., Low, K., Blesch, A. & Tuszynski, M. H. Induction of corticospinal regeneration by lentiviral trkB-induced Erk activation. Proceedings of the National Academy of Sciences of the United States of America 106, 7215- 7220(2009) Izumrudov, V. A., Kharlampieva, E. & Sukhishvili, S. A. Multilayers of a globular protein and a weak polyacid: Role of polyacid ionization in growth and decomposition in salt solutions. Biomacromolecules 6, 1782-1788 (2005). 269 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. Izumrudov, V. A. & Lim, S. H. Controlled phase separations in solutions of poly(methacrylate) anion complexes with globular proteins. Polymer Science Series A 44, 484-490 (2002). Jiang, X. P., Clark, S. L. & Hammond, P. T. Side-by-side directed multilayer patterning using surface templates. Advanced Materials 13, 1669-1673 (2001). Gros, T., Sakamoto, J ., Blesch, A., L., H. & Tuszynski, M. H. Templated Agarose Scaffolds Orient and Guide Regenerating Long-Tract Axons Through Sites of Spinal Cord Injury. (2010;Submitted). Kidambi, S. et al. Cell adhesion on polyelectrolyte multilayer coated polydimethylsiloxane surfaces with varying topographies. Tissue Engineering 13, 2105-2117 (2007). Kidambi, S., Chan, C. & Lee, I. Tunable resistive m-dPEG acid patterns on polyelectrolyte, multilayers at physiological conditions: Template for directed deposition of biomacromolecules. Langmuir 24, 224-230 (2008). Zhang, M. Q., Desai, T. & Ferrari, M. Proteins and cells on PEG immobilized silicon surfaces. Biomaterials 19, 953-960 (1998). Krogman, K. C., Zacharia, N. S., Schroeder, S. & Hammond, P. T. Automated process for improved uniformity and versatility of layer-by-layer deposition. Langmuir 23, 3137-3141 (2007). Mehrotra, S. et al. Cell Adhesive Behavior on Thin Polyelectrolyte Multilayers: Cells Attempt to Achieve Homeostasis of Its Adhesion Energy. Langmuir 26, 12794-12802 (2010). Capizzi, R. L. Curative chemotherapy for acute myeloid leukemia: The development of high-dose ara-C from the laboratory to bedside. Investigational New Drugs 14, 249-256 (1996). Plagemann, P. G. W., Marz, R. & Wohlhueter, R. M. Transport and Metabolism of Deoxycytidine and l-Beta-D-Arabinofuranosy1cytosine into Cultured Novikoff Rat Hepatoma-Cells, Relationship to Phosphorylation, and Regulation of Triphosphate Synthesis. Cancer Research 38, 978-989 (1978). Wiley, J. S., Jones, S. P., Sawyer, W. H. & Paterson, A. R. P. Cytosine- Arabinoside Influx and Nucleoside Transport Sites in Acute-Leukemia. Journal of Clinical Investigation 69, 479-489 (1982). Young, J. D. & Jarvis, S. M. Nucleoside Transport in Animal-Cells. Bioscience Reports 3, 309-322 (1983). 270 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. Major, P. P., Egan, E. M., Herrick, D. J. & Kufe, D. W. Effect of Ara-C Incorporation on Deoxyribonucleic-Acid Synthesis in Cells. Biochemical Pharmacology 31, 2937-2940 (1982). Fram, R. J. & Kufe, D. W. Inhibition of DNA Excision Repair and the Repair of X-Ray-Induced DNA Damage by (bitosine-Arabinoside and Hydroxyurea. Pharmacology & Therapeutics 31, 165-176 (1985). Dessi, F., Pollard, H., Moreau, J ., Benari, Y. & Charriautmarlangue, C. Cytosine- Arabinoside Induces Apoptosis in Cerebellar Neurons in Culture. Journal of Neurochemistry 64, 1980-1987 (1995). Geller, H. M. et al. Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside. Journal of Neurochemistry 78, 265- 275 (2001). Wallace, T. L. & Johnson, E. M. Cytosine-Arabinoside Kills Postrnitotic Neurons - Evidence That Deoxycytidine May Have a Role in Neuronal Survival That Is Independent of DNA-Synthesis. Journal of Neuroscience 9, 115-124 (1989). Tomkins, C. E., Edwards, S. N. & Tolkovsky, A. M. Apoptosis Is Induced in Postrnitotic Rat Sympathetic Neurons by Arabinosides and Topoisomerase-Ii Inhibitors in the Presence of Ngf. Journal of Cell Science 107, 1499-1507 (1994). Blanco, M. D., Trigo, R. M., Teijon, C., Gomez, C. & Teijon, J. M. Slow releasing of ara-C from poly(2-hydroxyethyl methacrylate) and poly(2- hydroxyethyl methacrylate-co-N-vinyl-Z-pyrrolidone) hydrogels implanted subcutaneously in the back of rats. Biomaterials 19, 861-869 (1998). Kwong, Y. L., Yeung, D. Y. M. & Chan, J. C. W. Intrathecal chemotherapy for hematologic malignancies: drugs and toxicities. Annals of Hematology 88, 193- 201 (2009). Keime-Guibert, F., Napolitano, M. & Delattre, J. Y. Neurological complications of radiotherapy and chemotherapy. Journal of Neurology 245, 695-708 (1998). Glantz, M. J. et al. Randomized trial of a slow-release versus a standard formulation of cytarabine for the intrathecal treatment of lymphomatous meningitis. Journal of Clinical Oncology 17, 3110-3116 (1999). Gomez, C. et al. Cytarabine release from comatrices of albumin microspheres in a poly(lactide-co-glycolide) film: in vitro and in vivo studies. European Journal of Pharmaceutics and Biopharrnaceutics 57, 225-233 (2004). 271 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. Esposito, E. et al. Controlled-Release of 1-Beta-D-Arabinofiuanosylcytosine (Ara-C) from Hydrophilic Gelatin Microspheres - in-Vitro Studies. International Journal of Pharmaceutics 117, 151-158 (1995). Sastre, R. L., Blanco, M. D., Gomez, C., del Socorro, J. M. & Teijon, J. M. Cytarabine trapping in poly(2-hydroxyethyl methacrylate-co-acrylamide) hydrogels: drug delivery studies. Polymer International 48, 843-850 (1999). Schiavon, O. et al. PEG-Ara-C conjugates for controlled release. European Journal of Medicinal Chemistry 39, 123-133 (2004). Neumann, M. G. & Tiera, M. J. Photochemical determination of the interactions between surfactants and polyelectrolytes. Pure and Applied Chemistry 69, 791- 795 (1997). Elbert, D. L., Herbert, C. B. & Hubbell, J. A. Thin polymer layers formed by polyelectrolyte multilayer techniques on biological surfaces. Langmuir 15, 5355- 5362 (1999). Picart, C. et al. Buildup mechanism for poly(L-lysine)/hyaluronic acid films onto a solid surface. Langmuir 17, 7414-7424 (2001). Picart, C. et al. Molecular basis for the explanation of the exponential grth of polyelectrolyte multilayers. Proceedings of the National Academy of Sciences of the United States of America 99, 12531-12535 (2002). Ren, K. F ., Ji, J. & Shen, J. C. Construction and enzymatic degradation of multilayered poly-L-lysine/DNA films. Biomaterials 27, 1152-1159 (2006). Manakova, S., Puttonen, K. A., Raasmaja, A. & Mannisto, P. T. Ara-C induces apoptosis in monkey fibroblast cells. Toxicology in Vitro 17, 367-373 (2003). Calderon-Martinez, D., Garavito, Z., Spinel, C. & Hurtado, H. Schwann cell- enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C). Journal of Neuroscience Methods 114, 1-8 (2002). Clark, S. L., Montague, M. F. & Hammond, P. T. Ionic effects of sodium chloride on the templated deposition of polyelectrolytes using layer-by-layer ionic assembly. Macromolecules 30, 7237-7244 (1997). Houchin-Ray, T., Whittlesey, K. J. & Shea, L. D. Spatially patterned gene delivery for localized neuron survival and neurite extension. Molecular Therapy 15, 705-712 (2007). 272 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. Pannier, A. K., Anderson, B. C. & Shea, L. D. Substrate-mediated delivery from self-assembled monolayers: Effect of surface ionization, hydrophilicity, and patterning. Acta Biomaterialia 1, 511-522 (2005). Wheeler, D. B., Carpenter, A. E. & Sabatini, D. M. Cell microarrays and RNA interference chip away at gene function. Nature Genetics 37, S25-S30 (2005). Ziauddin, J. & Sabatini, D. M. Microarrays of cells expressing defined cDNAs. Nature 411,107-110(2001). Diaz-Mochon, J. J ., Tourniaire, G. & Bradley, M. Microarray platforms for enzymatic and cell-based assays. Chemical Society Reviews 36, 449-457 (2007). Kong, H. J. et al. Non-viral gene delivery regulated by stiffness of cell adhesion substrates. Nature Materials 4, 460-464 (2005). Hammond, P. T. Form and function in multilayer assembly: New applications at the nanoscale. Advanced Materials 16, 1271-1293 (2004). Jewell, C. M., Zhang, J. T., Fredin, N. J. & Lynn, D. M. Multilayered polyelectrolyte films promote the direct and localized delivery of DNA to cells. Journal of Controlled Release 106, 214-223 (2005). Jessel, N. et al. Multiple and time-scheduled in situ DNA delivery mediated by beta-cyclodextrin embedded in a polyelectrolyte multilayer. Proceedings of the National Academy of Sciences of the United States of America 103, 8618-8621 (2006) Meyer, F., Ball, V., Schaaf, P., Voegel, J. C. & Ogier, J. Polyplex-embedding in polyelectrolyte multilayers for gene delivery. Biochimica Et Biophysica Acta- Biomembranes 1758, 419-422 (2006). Shim, M. S. & Kwon, Y. J. Controlled delivery of plasmid DNA and siRNA to intracellular targets using ketalized polyethylenimine. Biomacromolecules 9, 444- 455 (2008). Mendelsohn, J. D., Yang, S. Y., Hiller, J., Hochbaum, A. I. & Rubner, M. F. Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. Biomacromolecules 4, 96-106 (2003). Ogris, M., Brunner, S., Schuller, S., Kircheis, R. & Wagner, E. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Therapy 6, 595-605 (1999). 273 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. Bishop, N. E. An update on non-clathrin-coated endocytosis. Reviews in Medical Virology 7, 199-209 (1997). Pataer, A. et al. Melanoma differentiation-associated gene-7 protein physically associates with the double-stranded RNA-activated protein kinase PKR. Molecular Therapy 11, 717-723 (2005). Dalby, B. et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods 33, 95-103 (2004). Perrine, T. D. & Landis, W. R. Analysis of Polyethylenimine by Spectrophotometry of Its Copper Chelate. Journal of Polymer Science Part a-l- Polymer Chemistry 5, 1993-2003 (1967). Bertschinger, M., Chaboche, S., Jordan, M. & Wurm, F. M. A spectrophotometric assay for the quantification of polyethylenimine in DNA nanoparticles. Analytical Biochemistry 334, 196-198 (2004). Link, S., Wang, Z. L. & El-Sayed, M. A. Alloy formation of gold-silver nanoparticles and the dependence of the plasmon absorption on their composition. Journal of Physical Chemistry B 103, 3529-3533 (1999). Mallik, K., Manda], M., Pradhan, N. & Pal, T. Seed mediated formation of bimetallic nanoparticles by UV irradiation: A photochemical approach for the preparation of "core-shell" type structures. Nano Letters 1, 319-322 (2001). Chen, H. M., Liu, R. S., Jang, L. Y., Lee, J. F. & Hu, S. F. Characterization of core-shell type and alloy Ag/Au bimetallic clusters by using extended X-ray absorption fine structure spectroscopy. Chemical Physics Letters 421, 118-123 (2006) Koyama, Y., Yamashita, M., Iida-Tanaka, N. & Ito, T. Enhancement of transcriptional activity of DNA complexes by amphoteric PEG derivative. Biomacromolecules 7, 1274-1279 (2006). Pannier, A. K., Wieland, J. A. & Shea, L. D. Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes. Acta Biomaterialia 4, 26-39 (2008). Mao, S. R. et al. Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft- poly(ethylene glycol) block copolymer/SiRNA polyplexes. Bioconjugate Chemistry 17, 1209-1218 (2006). 274 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. Svintradze, D. V. & Mrevlishvili, G. M. Fiber molecular model of atelocollagen- small interfering RNA (siRNA) complex. International Journal of Biological Macromolecules 37, 283-286 (2005). Chatani, Y., Tadokoro, H., Saegusa, T. & Ikeda, H. Structural Studies of Poly(Ethylenimine) .1. Structures of 2 Hydrates of Poly(Ethylenimine) - Sesquihydrate and Dihydrate. Macromolecules 14, 315-321 (1981). Chatani, Y., Kobatake, T. & Tadokoro, H. Structural Studies of Poly(Ethylenimine) .3. Structural Characterization of Anhydrous and Hydrous States and Crystal-Structure of the Hemihydrate. Macromolecules 16, 199-204 (1983) Ye, K. Q., Malinina, L. & Patel, D. J. Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426, 874-878 (2003). Pergushov, D. V. et al. Novel water-soluble micellar interpolyelectrolyte complexes. Journal of Physical Chemistry B 107, 8093-8096 (2003). Dragan, E. S. & Schwarz, S. Polyelectrolyte complexes. VI. Polycation structure, polyanion molar mass, and polyion concentration effects on complex nanoparticles based on poly(sodium 2-acrylamido-2-methylpropanesulfonate). Journal of Polymer Science Part a-Polymer Chemistry 42, 2495-2505 (2004). Mihai, M., Dragan, E. S., Schwarz, S. & Janke, A. Dependency of particle sizes and colloidal stability of polyelectrolyte complex dispersions on polyanion structure and preparation mode investigated by dynamic light scattering and atomic force microscopy. Journal of Physical Chemistry B 111, 8668-8675 (2007) Zhunuspayev, D. E., Mun, G. A., Hole, P. & Khutoryanskiy, V. V. Solvent Effects on the Formation of Nanoparticles and Multilayered Coatings Based on Hydrogen-Bonded Interpolymer Complexes of Poly(acrylic acid) with Homo- and Copolymers of N-Vinyl Pyrrolidone. Langmuir 24, 13742-13747 (2008). Bailey, F. E., Callard, R. W. & Lundberg, R. D. Some Factors Affecting Molecular Association of Poly(Ethylene Oxide) + Poly)Acrylic Acid) in Aqueous Solution. Journal of Polymer Science Part a-General Papers 2, 845-851 (1964). Ikawa, T., Abe, K. Honda, K. & Tsuchida, E. Interpolymer Complex between Poly(Ethylene Oxide) and Poly(Carboxylic Acid). Journal of Polymer Science Part a-Polymer Chemistry 13, 1505- 1514 (1975). Thiinemann, A. F., Muller, M., Dautzenberg, H., Joanny, J. F. & Lowen, H. Polyelectrolyte complexes. Polyelectrolytes with Defined Molecular Architecture 275 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. II (Advances in Polymer Science); M.Schmidt, Springer-Verlag Berlin Heidelberg, Germany 166, 113-171 (2004). Sukhishvili, S. A., Kharlampieva, E. & Izumrudov, V. Where polyelectrolyte multilayers and polyelectrolyte complexes meet. Macromolecules 39, 8873-8881 (2006). ' Dragan, E. S. & Schwarz, S. Polyelectrolyte complexes. VII. Complex nanoparticles based on poly(sodium 2-acrylamido-2-methylpropanesulfonate) tailored by the titrant addition rate. Journal of Polymer Science Part a-Polymer Chemistry 42, 5244-5252 (2004). Srivastava, D. Fabrication of nanostructures and nanostructure based interfaces for biosensor application. A Dissertation, Doctor of Philosophy, 88-136 (2008). Galindo-Rodriguez, S., Allemann, E., Fessi, H. & Doelker, E. Physicochernical parameters associated with nanoparticle formation in the salting-out, emulsification-diffusion, ’ and nanoprecipitation methods. Pharmaceutical Research 21, 1428-1439 (2004). Desgouilles, S. et al. The design of nanoparticles obtained by solvent evaporation: A comprehensive study. Langmuir 19, 9504-9510 (2003). Weyts, K. F. & Goethals, E. J. Back Titration of Linear Polyethylenimine - Decrease of Ph by Addition of Sodium-Hydroxide. Makromolekulare Chemic- Rapid Communications 10, 299-302 (1989). Cheng, N. S. Formula for the viscosity of a glycerol-water mixture. Industrial & Engineering Chemistry Research 47, 3285-3288 (2008). Brunner, T., Cohen, S. & Monsonego, A. Silencing of proinflammatory genes targeted to peritoneal-residing macrophages using siRNA encapsulated in biodegradable microspheres. Biomaterials 31, 2627-2636 (2010). Sagvolden, G., Giaever, I., Pettersen, E. O. & Feder, J. Cell adhesion force microscopy. Proceedings of the National Academy of Sciences of the United States of America 96, 471-476 (1999). Kidambi, S., Lee, I. & Chan, C. Controlling primary hepatocyte adhesion and spreading on protein-free polyelectrolyte multilayer films. Journal of the American Chemical Society 126, 16286-16287 (2004). Engler, A. et al. Substrate compliance versus ligand density in cell on gel responses. Biophysical Journal 86, 617-628 (2004). 276 187. 188. 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. Engler, A. J., Richert, L., Wong, J. Y., Picart, C. & Discher, D. E. Surface probe measurements of the elasticity of sectioned tissue, thin gels and polyelectrolyte multilayer films: Correlations between substrate stiffness and cell adhesion. Surface Science 570, 142-154 (2004). Pelham, R. J. & Wang, Y. L. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proceedings of the National Academy of Sciences of the United States of America 94, 13661-13665 (1997). Swiston, A. J. et al. Surface Functionalization of Living Cells with Multilayer Patches. Nano Letters 8, 4446-4453 (2008). Richert, L., Engler, A. J., Discher, D. E. & Picart, C. Elasticity of native and cross-linked polyelectrolyte multilayer films. Biomacromolecules 5, 1908-1916 (2004) Thompson, M. T., Berg, M. C., Tobias, 1. S., Rubner, M. F. & Van Vliet, K. J. Tuning compliance of nanoscale polyelectrolyte multilayers to modulate cell adhesion. Biomaterials 26, 6836-6845 (2005). Shiratori, S. S. & Rubner, M. F. pH-dependent thickness behavior of sequentially adsorbed layers of weak polyelectrolytes. Macromolecules 33, 4213-4219 (2000). Schlenoff, J. B. & Dubas, S. T. Mechanism of polyelectrolyte multilayer growth: Charge overcompensation and distribution. Macromolecules 34, 592-598 (2001). Dubas, S. T. & Schlenoff, J. B. Swelling and smoothing of polyelectrolyte multilayers by salt. Langmuir 17, 7725-7727 (2001). Dubas, S. T. & Schlenoff, J. B. Factors controlling the growth of polyelectrolyte multilayers. Macromolecules 32, 8153-8160 ( 1999). Kohler, K., Shchukin, D. G., Sukhorukov, G. B. & Mohwald, H. Drastic morphological modification of polyelectrolyte microcapsules induced by high temperature. Macromolecules 37, 9546-9550 (2004). von Klitzing, R. Internal structure of polyelectrolyte multilayer assemblies. Physical Chemistry Chemical Physics 8, 5012-5033 (2006). Schlenoff, J. B., Ly, H. & Li, M. Charge and mass balance in polyelectrolyte multilayers. Journal of the American Chemical Society 120, 7626-7634 (1998). Yoo, D., Shiratori, S. S. & Rubner, M. F. Controlling bilayer composition and surface wettability of sequentially adsorbed multilayers of weak polyelectrolytes. Macromolecules 31, 4309-4318 (1998). 277 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. Jaber, J. A. & Schlenoff, J. B. Mechanical properties of reversibly cross-linked ultrathin polyelectrolyte complexes. Journal of the American Chemical Society 128, 2940-2947 (2006). Collin, D. et al. Mechanical properties of cross-linked hyaluronic acid/poly-(L- lysine) multilayer films. Macromolecules 37, 10195-10198 (2004). Hubsch, E. et al. Controlling the growth regime of polyelectrolyte multilayer films: Changing from exponential to linear growth by adjusting the composition of polyelectrolyte mixtures. Langmuir 20, 1980-1985 (2004). Porcel, C. et al. Influence of the polyelectrolyte molecular weight on exponentially growing multilayer films in the linear regime. Langmuir 23, 1898- 1904 (2007). Wu, Z. R., Ma, J ., Lin, B. F., Xu, Q. Y. & Cui, F. Z. Layer-by-layer assembly of polyelectrolyte films improving cytocompatibility to neural cells. Journal of Biomedical Materials Research Part A 81A, 355-362 (2007). Hillberg, A. L., Holmes, C. A. & Tabrizian, M. Effect of genipin cross-linking on the cellular adhesion properties of layer-by-layer assembled polyelectrolyte films. Biomaterials 30, 4463-4470 (2009). McAloney, R. A., Sinyor, M., Dudnik, V. & Goh, M. C. Atomic force microscopy studies of salt effects on polyelectrolyte multilayer film morphology. Langmuir 17, 6655-6663 (2001). Sen, S., Engler, A. J. & Discher, D. E. Matrix Strains Induced by Cells: Computing How Far Cells Can Feel. Cellular and Molecular Bioengineering 2, 39-48 (2009). Engler, A. J. et a1. Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. Journal of Cell Biology 166, 877-887 (2004). Richert, L. et al. Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking. Biomacromolecules 5, 284-294 (2004). Dubreuil, F ., Elsner, N. & Fery, A. Elastic properties of polyelectrolyte capsules studied by atomic-force microscopy and RICM. European Physical Journal E 12, 215-221 (2003). Pavoor, P. V., Bellare, A., Strom, A., Yang, D. H. & Cohen, R. E. Mechanical characterization of polyelectrolyte multilayers using quasi-static nanoindentation. Macromolecules 37, 4865-4871 (2004). 278 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222. 223. Mueller, R., Kohler, K., Weinkamer, R., Sukhorukov, G. & Fery, A. Melting of PDADMAC/PSS capsules investigated with AFM force spectroscopy. Macromolecules 38, 9766-9771 (2005). Nolte, A. J ., Rubner, M. F. & Cohen, R. E. Determining the young's modulus of polyelectrolyte multilayer films via stress-induced mechanical buckling instabilities. Macromolecules 38, 5367-5370 (2005). Salomaki, M., Laiho, T. & Kankare, J. Counteranion-controlled properties of polyelectrolyte multilayers. Macromolecules 37, 9585-9590 (2004). Vinogradova, O. I., Andrienko, D., Lulevich, V. V., Nordschild, S. & Sukhorukov, G. B. Young's modulus of polyelectrolyte multilayers from microcapsule swelling. Macromolecules 37, 1113-1 117 (2004). Gao, C. Y., Leporatti, S., Moya, S., Donath, E. & Mohwald, H. Stability and mechanical properties of polyelectrolyte capsules obtained by stepwise assembly of poly(styrenesulfonate sodium salt) and poly(diallyldirnethyl ammonium) chloride onto melamine resin particles. Langmuir 17, 3491-3495 (2001). Oommen, B. & Van Vliet, K. J. Effects of nanoscale thickness and elastic nonlinearity on measured mechanical properties of polymeric films. Thin Solid Films 513, 235-242 (2006). de Hemptinne, 1., Verrneiren, C., Maloteaux, J. M. & Hermans, E. Induction of glial glutamate transporters in adult mesenchymal stem cells. Journal of Neurochemistry 91, 155-166 (2004). Seglen, P. O. in Methods in Cell Biology (ed. Prescott, D. M.) 29—83 (Academic, New York, 1976). Dunn, J. C. Y., Tompkins, R. G. & Yarrnush, M. L. Long-Terrn Invitro Function of Adult Hepatocytes in a Collagen Sandwich Configuration. Biotechnology Progress 7, 237-245 (1991). Sui, Z. J., Salloum, D. & Schlenoff, J. B. Effect of molecular weight on the construction of polyelectrolyte multilayers: Stripping versus sticking. Langmuir 19, 2491-2495 (2003). Jaber, J. A. & Schlenoff, J. B. Dynamic viscoelasticity in polyelectrolyte multilayers: Nanodamping. Chemistry of Materials 18, 5768-5773 (2006). Miller, M. D. & Bruening, M. L. Correlation of the swelling and permeability of polyelectrolyte multilayer films. Chemistry of Materials 17, 5375-5381 (2005). 279 224. 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. Gao, C. Y., Leporatti, S., Moya, S., Donath, E. & Mohwald, H. Swelling and shrinking of polyelectrolyte microcapsules in response to changes in temperature and ionic strength. Chemistry-a European Journal 9, 915-920 (2003). Jaber, J. A. & Schlenoff, J. B. Counterions and water in polyelectrolyte multilayers: A tale of two polycations. Langmuir 23, 896-901 (2007). Yang, S. Y., Mendelsohn, J. D. & Rubner, M. F. New class of ultrathin, highly cell-adhesion-resistant polyelectrolyte multilayers with micropatteming capabilities. Biomacromolecules 4, 987-994 (2003). Benhabbour, S. R., Sheardown, H. & Adronov, A. Cell adhesion and proliferation on hydrophilic dendritically modified surfaces. Biomaterials 29, 4177-4186 (2008). Steele, J. G. et al. Attachment of Human Bone-Cells to Tissue-Culture Polystyrene and to Unmodified Polystyrene - the Effect of Surface-Chemistry Upon Initial Cell Attachment. Journal of Biomaterials Science-Polymer Edition 5, 245-257 (1993). Kneser, U. et al. Long-term differentiated function of heterotopically transplanted hepatocytes on three-dimensional polymer matrices. Journal of Biomedical Materials Research 47, 494-503 (1999). Mooney, D. J. et al. Long-term engraftment of hepatocytes transplanted on biodegradable polymer sponges. Journal of Biomedical Materials Research 37, 413-420 (1997). Park, J ., F ouche, L. D. & Hammond, P. T. Multicomponent patterning of layer- by-layer assembled polyelectrolyte/nanoparticle composite thin films with controlled alignment. Advanced Materials 17, 2575-2579 (2005). Park, J. & Hammond, P. T. Multilayer transfer printing for polyelectrolyte multilayer patterning: Direct transfer of layer-by-layer assembled micropattemed thin films. Advanced Materials 16, 520-525 (2004). Dunn, J. C. Y., Yarmush, M. L., Koebe, H. G. & Tompkins, R. G. Hepatocyte Function and Extracellular-Matrix Geometry - Long-Term Culture in 3 Sandwich Configuration. Faseb Journal 3, 174-177 (1989). Berthiaurne, F., Moghe, P. V., Toner, M. & Yarmush, M. L. Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: Hepatocytes cultured in a sandwich configuration. F aseb Journal 10,1471-1484(1996). 280 235. 236. 237. 238. 239. 240. 241. 242. 243. 244. 245. 246. Kidambi, S. et al. Patterned co-culture of primary hepatocytes and fibroblasts using polyelectrolyte multilayer templates. Macromolecular Bioscience 7, 344- 353 (2007). Bhatia, S. N., Balis, U. J., Yarmush, M. L. & Toner, M. Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. Faseb Journal 13, 1883-1900 (1999). Hong, S. U., Malaisamy, R. & Bruening, M. L. Separation of fluoride from other monovalent anions using multilayer polyelectrolyte nanofiltration membranes. Langmuir 23, 1716-1722 (2007). Malaisamy, R. & Bruening, M. L. High-flux nanofiltration membranes prepared by adsorption of multilayer polyelectrolyte membranes on polymeric supports. Langmuir 21, 10587-10592 (2005). Miller, M. D. & Bruening, M. L. Controlling the nanofiltration properties of multilayer polyelectrolyte membranes through variation of film composition. Langmuir 20, 11545-11551 (2004). Lee, I., Ahn, J. S., Hendricks, T. R., Rubner, M. F. & Hammond, P. T. Patterned and controlled polyelectrolyte fractal growth and aggregations. Langmuir 20, 2478-2483 (2004). Bhatia, S. N., Balis, U. J ., Yarmush, M. L. & Toner, M. Probing heterotypic cell interactions: Hepatocyte function in microfabricated co-cultures. Journal of Biomaterials Science-Polymer Edition 9, 1137-1160 (1998). Bhatia, S. N., Balis, U. J., Yarmush, M. L. & Toner, M. Microfabrication of hepatocyte/fibroblast co-cultures: Role of homotypic cell interactions. Biotechnology Progress 14, 378-387 (1998). Lutkenhaus, J. L., Hrabak, K. D., McEnnis, K. & Hammond, P. T. Elastomeric flexible free-standing hydrogen-bonded nanoscale assemblies. Journal of the American Chemical Society 127, 17228-17234 (2005). Podsiadlo, P. et al. Exponential grth of LBL films with incorporated inorganic sheets. Nano Letters 8, 1762-1770 (2008). Seo, J ., Lutkenhaus, J. L., Kim, J ., Hammond, P. T. & Char, K. Development of surface morphology in multilayered films prepared by layer-by-layer deposition using poly(acrylic acid) and hydrophobically modified poly(ethylene oxide). Macromolecules 40, 4028-4036 (2007). Mamedov, A. A. & Kotov, N. A. Free-standing layer-by-layer assembled films of magnetite nanoparticles. Langmuir 16, 5530-5533 (2000). 281 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. 257. Dubas, S. T., Farhat, T. R. & Schlenoff, J. B. Multiple membranes from "true" polyelectrolyte multilayers. Journal of the American Chemical Society 123, 5368— 5369 (2001). Hendricks, T. R. & Lee, I. Wrinkle-free nanomechanical film: Control and prevention of polymer film buckling. Nano Letters 7, 372-379 (2007). Farhat, T., Yassin, G., Dubas, S. T. & Schlenoff, J. B. Water and ion pairing in polyelectrolyte multilayers. Langmuir 15, 6621-6623 (1999). Yang, J. et al. Cell sheet engineering: Recreating tissues without biodegradable scaffolds. Biomaterials 26, 6415-6422 (2005). Kushida, A. et a1. Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature- responsive culture surfaces. Journal of Biomedical Materials Research 45, 355- 362 (1999). Nishida, K. et al. Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsieve cell culture surface. Transplantation 77, 379-385 (2004). Nishida, K. et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. New England Journal of Medicine 351, 1187-1196 (2004). Shimizu, T. et al. Fabrication of pulsatile cardiac tissue grafts using a novel 3- dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces. Circulation Research 90, E40-E48 (2002). Harimoto, M. et al. Novel approach for achieving double-layered cell sheets co- culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes. Journal of Biomedical Materials Research 62, 464-470 (2002). Stevens, M. M. et a1. Direct patterning of mammalian cells onto porous tissue engineering substrates using agarose stamps. Biomaterials 26, 7636-7641 (2005). Mayer, M., Yang, J., Gitlin, 1., Gracias, D. H. & Whitesides, G. M. Micropatterned agarose gels for stamping arrays of proteins and gradients of proteins. Proteomics 4, 2366-2376 (2004). 282