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This is to certify that the
thesis entitled

MODIFICATION OF POLYLACTIDES FOR TISSUE GROWTH

presented by

Mei Wang

has been accepted towards fulfillment
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MODIFICATION OF POLYLACT IDES FOR TISSUE GROWTH

By

Mei Wang

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirement for the degree of

MASTER OF SCIENCE
Department of Chemistry

2004

ABSTRACT
MODIFICATION OF POLYLACTIDE COPOLYMERS FOR TISSUE GROWTH
By

Mei Wang

Polylactide (PLA) and its copolymers have been used as biomedical materials due
to their biocompatibility. In recent years, many studies have shown their application in
tissue engineering as a biocompatible and dissolvable scaffold. Designing scaffolds that
contain functionality is desirable for optimizing interaction between cell and surface. Our
group has worked out chemistry to synthesize functionalizable copolymers via bulk
copolymerization of L-lactide and diallylglycolide. Through hydroboration/oxidation, we
successfully converted the terminal double bond to terminal hydroxyl group and by
carbodiimide coupling reactions, we attached biological substrates such as fatty acids to
the copolymer covalently. These uniquely modified copolymers have also been evaluated
for their potential as biocompatible surfaces to direct cell growth and differentiation.

As for tissue growth on surfaces, cell geometry has been shown to be a critical
factor for death and growth. For functionalizing a biodegradable surface in a designed
and well controllable fashion for further growth studies, we also worked on patterning
polymer surfaces by photolabile protecting and deprotecting strategy. Anthraquinon-Z-
ylmethoxycarbonyl (Aqmoc) has been attached to our copolymer as the hydroxyl
protecting group, and was removed by irradiating with UV light (93> 350nm). The
synthesis and characterization of the functionalized copolymers, initial physiological

experiments results are reported.

To my family and parents

iii

ACKNOWLEDGMENTS

I would like to acknowledge my advisor Mitch Smith for his guidance during my
graduate study. I benefit tremendously from the training he has imparted to me, teaching
me to creatively think and effectively execute scientific research. I especially appreciate
his encouragement that made me realize my potentials and have the confidence in
pursuing a career as a scientist. I would also like to thank him for his understanding and
support in my life.

Secondly, I would like to thank Greg Baker for his guidance and help in my study
and research. I would also like to thank my committee member, William Wulff for his
helpful suggestions. I would also like to thank Professor Laura Mccabe and her group
members Moushumi Hossain and Katie Porter for their cooperation in the physiological
studies on the polymer surfaces.

Also, I would like to thank Christopher Radano for teaching me the basic research
skills, and other former group members Daniel Holmes, Jian-Yang Cho for their
friendship profitable scientific discussion. I would like to thank Abbas, Mike and J.T. Lee
for sharing lab duties and cooperation in everyday lab work. I would also like to
collectively thank the MSU faculty and staff for catering to my needs for three years.

Finally and most importantly, I would like to thank my family and friends for
their love, support and inspiration during my graduate study at Michigan State

University.

iv

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................................ vi
LIST OF TABLES ............................................................................................................. ix
LIST OF ABBREVIATIONS ............................................................................................. x
CHAPTER ONE ................................................................................................................. 1
1.1 Polylactide Applications as Biomedical Materials .............................................. 1
1.1.1 Polylactide Application in Drug Delivery ................................................... 1
1.1.2 Polylactide Application in Tissue Engineering ........................................... 2

1.2 Processing and Modification of Polylactide for Tissue Engineering .................. 5
1.2.1 Three Dimensional Structure Processing .................................................... 5
1.2.2 Surface Modification of PLA-PGA Polymers ............................................ 6
1.2.3 Synthesizing Novel PLA-PGA Based Polymers for Tissue Engineering 8

1.3 Cell Geometry Regulation on Material Surface ................................................ 10
1.3.1 Cell Geometry Control over Cell Function ............................................... 10
1.3.2 Micropatteming Material Surface for Cell Growth Studies ...................... 11
CHAPTER TWO ............................................................................................................... 17
2 Functionalization of Polylactide Based Copolymer .................................................. 17
2.1 Monomer Synthesis ........................................................................................... 18
2.2 Copolymer Synthesis and Characterization ...................................................... 20
2.3 Post-Polymerization Modification .................................................................... 22
2.3. 1 Hydroboration-Oxidation .......................................................................... 23
2.3.2 Side Chain Coupling Reactions ................................................................. 26

2.4 Cell Growth Studies on Fatty Acids Modified Poly(Lactic Acid) Surfaces 33
2.4.1 Copolymer Thin Films .............................................................................. 33
2.4.2 Osteoblast Cell Shape ................................................................................ 35
2.4.3 Osteoblast Proliferation ............................................................................. 36
2.4.4 Osteoblast Differentiation ......................................................................... 39

2.5 Conclusions ....................................................................................................... 43
CHAPTER THREE ........................................................................................................... 44
3 Photolabile Protection-Deprotection Strategy in Surface Pattering. ......................... 44
3.1 Photolabile Protecting Group Selection ............................................................ 45
3.2 Synthesis of Aqmoc Protected PLLA-g-HP ...................................................... 47
3.3 Photolysis of Aqmoc Protected PLLA-g-HP .................................................... 49
3.4 Conclusion ......................................................................................................... 52
CHAPTER FOUR ............................................................................................................. 54
4 Experimental Section. ............................................................................................... 54
4.1 General Considerations ..................................................................................... 54
4.2 Synthesis ............................................................................................................ 58
CHAPTER FIVE ............................................................................................................... 66
5 References ................................................................................................................. 66
Appendix A ....................................................................................................................... 71
Appendix B ....................................................................................................................... 79

LIST OF FIGURES

Figure l. Mechanism of delayed dissolution via biodegradable drug delivery system. 2

Figure 2. Schematic diagram of bone tissue regeneration via a cell-polymer biohybride

device. ......................................................................................................................... 3
Figure 3. Surface modification by alkali hydrolysis treatment. ......................................... 7
Figure 4. Copolymerization and modification of poly(lactic acid) using L-lysine and L-

Iactide cyclic comonomer ............................................................................................ 9
Figure 5. Amino acid containing cyclic comomomers. ................................................... 10

Figure 6. Diagram of substrates used to vary cell shape independently of the cell-ECM
contact area. Substrates were patterned with (A) a small single round island, (B)

small closely spaced circular islands and (C) a large single round island. ............... 11
Figure 7. Schematic Diagram of the Photoresist Surface Patterning Process. ................. 13
Figure 8. Schematic Diagram of the Microcontact Printing Process ............................... 14

Figure 9. Schematic Diagram of the Photografting Polymerization Process. (A) A
polymer substrate containing photoiniferter is prepared on silicon surface, (B)
additional monomer is added to the surface and exposed to UV light through a
photomask, (C) covalently-bound polymer is grown from the substrate surface in the

regions exposed to light ............................................................................................. 15
Figure 10. Synthetic route to 3,6-diallylglycolide ........................................................... 19
Figure 11. 1H NMR (300 MHz, CDC13) spectrum of 3,6-diallylglycolide. Hc* denotes the

R,R and 8,8 isomers, Hg” denotes the R,S isomer. ................................................. 20
Figure 12. Synthetic route to poly(L-lactide-co-diallylglycolide). .................................. 21

Figure 13. 1H NMR (300 MHz, CDC13) spectrum of poly(L-lactide-co-allylglycolide). 22
Figure 14. Mechanism of the hydroboration/oxidation reaction of terminal olefins. ...... 23

Figure 15. Synthetic route to poly(lactide-g-hydroxypropane) via hydroboration-
oxidation of poly(lactide-co-diallylglycolide). ......................................................... 24

Figure 16. 1H NMR (300 MHz,CDCl3) spectra of (A) PLLA-g—HP; poly(L—lactide-g-
hydroxypropane) and (B) PLLA-co-AG; poly(L-lactide-co-diallylglycolide). ........ 26

Figure 17. General synthetic route to polymer-bound esters using DCC coupling. ........ 27

Figure 18. Structure of phospholipid and cell membrane phospholipids bilayer .............. 28

vi

Figure 19. Synthetic route to saturated and unsaturated fatty acid-modified PLLA using
DCC coupling. ........................................................................................................... 29

Figure 20. 'H NMR (500 MHz, CDC13) spectra of (a ) PLLA—g-HP, (b) PLLA-g-MyPr,
and (c) PLLA-g-StPr. ................................................................................................ 31

Figure 21. 1H NMR (300 MHz, CDC13) spectra of (a) PLLA-g-OlPr; (b) PLLA-g-LnPr;
and (c) PLLA-g-ArPr. ............................................................................................... 32

Figure 22. Microscope images of osteoblasts cultured for 48 hours on (a) silicon oxide
and (b) PLLA-co-DAG (c) PLLA-g-HP (d) PLLA-g-MyPr (e) PLLA-g-StPr (f)
PLLA-g-OlPr (g) PLLA-g-LnPr (h) PLLA-g-ArPr. The cells are stained red and the
nuclei are stained blue. .............................................................................................. 36

Figure 23. Illustration of cell replication incorporating the BrdU fluorescent label. ....... 36

Figure 24. Digital photo images of fluorescent BrdU-labeled osteoblasts on (a) silicon
oxide, (b) PLLA-co-DAG (c) PLLA-g-HP (d) PLLA-g-MyPr (e) PLLA-g-StPr (f)
PLLA-g-OlPr (g) PLLA-g-LnPr (h) PLLA-g-ArPr. ................................................. 38

Figure 25. Result of osteoblast cell proliferation on (a) silicon oxide, (b) PLLA-co-DAG
(c) PLLA-g-HP (d) PLLA-g-MyPr (e) PLLA-g-StPr (f) PLLA-g-OlPr (g) PLLA-g-
LnPr (h) PLLA-g-ArPr. Averages are expressed as an n-fold change compared to
control, uncoated blank surfaces. .............................................................................. 39

Figure 26. Gene expression levels of osteocalcin and alkaline phosphatase as a function
of osteoblast culture time. ......................................................................................... 40

Figure 27. Result of osteoblast cell early and late stage differentiation on silicon oxide,
PLLA-co-DAG, PLLA-g-HP, PLLA-g-MyPr, PLLA-g-StPr, PLLA-g-OlPr, PLLA-
g-LnPr, and PLLA-g-ArPr. Averages are expressed as an n-fold change compared to
control, uncoated blank surfaces. .............................................................................. 41

Figure 28. Result of osteoblast cell early and late stage differentiation on silicon oxide,
PLLA-co-DAG, PLLA-g-HP, PLLA-g-MyPr, PLLA-g-StPr, PLLA-g-OlPr, PLLA-
g-LnPr, and PLLA-g-ArPr. Averages are expressed as a fold change compared to
control, uncoated blank surfaces. .............................................................................. 42

Figure 29. Photolabile protection-deprotection surface patterning for cell growth. (A)
Polymer thin film with a functionalized side chain protected by photolabile
protecting groups is exposed to UV light through a mask containing the desired
pattern, (B) after irradiation, protecting groups are removed in the exposed region,
(C) bioactive functional groups are incorporated in the deprotected region, (D) cells
are seeded on the polymer surface with desired geometry ........................................ 45

Figure 30. Photolabile polycyclic aromatic hydrocarbons bearing benzylic methylene
groups. ....................................................................................................................... 46

vii

Figure 31.
Figure 32.
Figure 33.
Figure 34.
Figure 35.

Figure 36.

Synthetic route of Aqmoc Protected PLLA-g-HP. ......................................... 47
N MR spectrum PLLA-g-AqmocPr ................................................................. 48
DMAP Catalyzed PLA Depolymerization. .................................................... 49
Photolysis of PLLA-g-AqmocPr. ................................................................... 49
UV-Vis spectra of PLLA-g-Aqmoc and PLLA-g-HP. ................................... 51
NMR spectra of PLLA-g-Aqmoc (a ) before and (b) after photolysis. .......... 52

viii

LIST OF TABLES

Table 1. Copolymerization of L-Lactide and 3,6-diallylglycolide. " Molar composition of
DAG determined by integration of the methylene resonance of the allyl repeat unit
compared to the methine resonance of the lactide repeat unit. b Molecular weight
determined by GPC. c PDI = Polydispersity index (MW/Mn). .................................. 21

Table 2. Saturated and unsaturated fatty acid-modified PLLA co lymers containing 8
mol % fatty acid. a Molecular weight determined by GPC. PDI = Polydispersity
index. c From differential scanning calorimetry of 8 mol % fatty acid-modified
copolymers. Date are from second heating scan at 10°C/min under helium. d
Hydroxylated copolymer starting material used to couple fatty acids. ..................... 30

Table 3. Thin films of copolymers spin-coated on l-inch diameter silicon wafers from a
1% (w/w) solution in toluene unless otherwise noted. Spin rate: 2500 rpm, spin
time: 60 sec. “ Mole percent loading determined by 1H NMR spectroscopy.
Determined by ellipsometry. C Samples were prepared by heating above the polymer
melting point and quenching to room temperature to obtain amorphous samples.
Static (65,), advancing (604,.) and receding (0,...) contact angles were measured at a
rate of 3 p.L/s up to a total volume of 30 ILL using Milli-Q grade water. ................. 34

Table 4. PDLLA stability under long and full range UV irradiation. " Molecular weight
determined by GPC. b Polydispersity index (MW/Mn). ............................................. 46

Table 5. PDLL-g-AqmocPr deprotection under UV irradiation. " Conversion is
determined by UV-vis spectroscopy. b Molecular weight determined by GPC. C
Polydispersity index (MW/Mn). .................................................................................. 50

Images in this dissertation are presented in color.

ix

Aqmoc
BBA
9-BBN
br

BrdU

DAG
DCC
dd
DMAP
DSC
ECM
GPC
HA
HPA

J

m

Mn

N MR
PDI
PDLLA
PDLLA-co-DAG

PDMS

LIST OF ABBREVIATIONS

Anthraquinon-Z-ylmethoxycarbonyl
4-tert-Butylbenzylalcohol
9-borabicyclononane

Broad

Bromodeoxyuridine

Doublet

3,6-Diallylglycolide
Dicyclohexylcarbodiimide

Doublet of Doublet

N ,N -Dimethyl-4-aminopyridine
Differential Scanning Calorimetry
Extracellular Matrix

Gel Permeation Chromatography
Hydroxyapatite
2-Hydroxypent-4-enoic acid
Coupling Constant

Multiplet

Number Average Molecular Weight
Nuclear Magnetic Resonance
Polydispersity Index

Racemic Poly(lactide)
Poly(D,L-lactide-co-diallylglycolide)

Polydimethylsiloxane

PEG
PGA
PLLA

PLLA-g-Aqmoc

PLLA-g-ArPr
PLLA-g-HP
PLLA-g-LnPr
PLLA-g-MyPr
PLLA-g-OlPr
PLLA-g-StPr
PTSA

q

RGD

1‘.I.

SAM

Poly(ethylene glycol)

Poly(glycolide)

Poly(L-lactide)
Poly(L-lactide-g-Anthraquinon-2—ylmethy1 propyl
carbonate

Poly(L-lactide-g-Arachidonic acid propyl ester)
Poly(L-lactide-g-Hydroxypropane)
Poly(L-lactide-g-Linoleic acid propyl ester)
Poly(L-lactide-g-Myristic acid propyl ester)
Poly(L-lactide-g-Oleic acid propyl ester)
Poly(L—lactide-g-Stearic acid propyl ester)
p-Toluenesufonic acid

Quartet

Arginine-Gchine-Aspartic Acid

Room Temperature

Singlet

Self-Assembled Monolayer

Triplet

Crystallization Temperature

Tricalcium Phosphate

Glass Transition Temperature
Tetrahydrofuran

Melting Temperature

Ultraviolet—Visible

xi

CHAPTER ONE

1.] Polylactide Applications as Biomedical Materials

Polylactide was first used as absorbable suture material in the 19608. Numerous in
vivo and in vitro studies and its successful clinical use indicated satisfactory
biocompatibility and the absence of significant toxicity, which opened the door to
polylactide’s application as a biomedical material in the fields of drug delivery and tissue

engineering.1

1.1.1 Polylactide Application in Drug Delivery

Drug delivery systems can improve drug efficacy by two mechanisms. The first is
temporal control, where the effective drug concentration at the site of therapeutic action
is controlled by the drug delivery system. The second mechanism involves targeting a
specific therapeutic site. This maximizes drug concentration at the site of interest and
minimizes side effects at nontherapeutic sites. Many kinds of biodegradable polymers
have been investigated as drug carriers for controlled release. Ideal candidates allow for
modification of the pharmacokinetics of the encapsulated drug through
degradation/release kinetics and also should have good biocompatibility with the target
site.

Poly(lactide) (PLA) (including Poly(L—lactide) (PLLA), Poly(D-lactide) (PDLA)
and Poly(D,L-lactide (PDLLA)), poly(glycolide) (PGA), and their co-polymer,
poly(lactide-co-glycolide) (PLGA) have proven to be an effective system for drug
delivery, especially for prolonged delivery of peptides and proteins.2 The delivery is

usually achieved by a temporal release mechanism (Figure l).3 The therapeutic reagent is

either coated with a biodegradable polymer or incorporated in the polymer matrix. The
polymer degrades at a slower rate than the drug dissolution. Therefore the drug

concentration is maintained at a constant and effective level through the dosage period.

 

. Drug Molecules Retained Until
-. . P I D. l . . "x." Further Polymer Erosron
o ymer ISSO ve or , ..
.. . . . Degrades Slowly . 3 .. <:
1' C‘- -------- Released Dru Molecules

Figure 1. Mechanism of delayed dissolution via biodegradable drug delivery system.

The polymers are safe for in vivo use, because their degradation products are
corresponding hydroxy acids, which are natural metabolism products of the body. Even
though increased acidity during the polymer degradation might cause slight irritation at
the site of polymer employment, this problem can be solved by introduction of basic salts
to control the pH of the drug release environment.4 Moreover, since L-lactic acid is the
naturally occurring enantiomer of lactic acid, PLLA is considered to be more
biocompatible. It is widely applied as one of the major components of PLA based

biodegradable copolymers.

1.1.2 Polylactide Application in Tissue Engineering

Several years ago, scientists believed that human tissue could only be replaced
with direct transplants or fully artificial parts made of plastic. Although countless patients
benefited from these therapies, they are still not the perfect solution for millions of
patients suffering from tissue loss or organ failure. The number of organ donors is far

from enough to supply the increasing numbers of patients. Even for patients lucky to

have organ transplantation, a large percent of them experienced multiple side effects due
to rejection by the patients' immune system. Since fully artificial devices cannot perform
all of the functions of single organ, they are currently far from a perfect tissue or organ
substitutes‘6

In recent years, biohybrid organs (a combination of living cells and natural or
artificial polymers) have proven to be a feasible alternative to tissue or organ
replacement. Cells from the patient or from a suitable donor are harvested and seeded
into a three-dimensional scaffold of biodegradable polymers under appropriate in vitro
culture conditions. After cell proliferation, differentiation and initial tissue development
have taken place in vitro, the entire structure of cells and polymer scaffold is transplanted
into the appropriate site in the patient. The cells will further replicate, reorganize and
form new tissue. At the same time, the polymer scaffolds degrade into harmless small

molecules, leaving only the newly generated tissue (Figure 2).7'9

Three-dimensional Biohybrid Bone Regenerated
Biodegradable Scaffold Implant Bone Tissue

.93 —>

 

{ New Bone Grows to F ill the Space
I J“ between Two Bone Segments

 

.3 The Polymer Scaffold Dissolves r
in the Surrounding Environment

 

 

5

Figure 2. Schematic diagram of bone tissue regeneration via a cell-polymer biohybride device.

Designing biocompatible polymer scaffolds represents one of the major areas in
tissue engineering research. Materials now being used as tissue engineering scaffolds fall
into two major categories: natural materials such as collagen, and synthetic materials
such as PLA, PGA and their copolymers. The advantage of natural materials is that they
contain particular amino acid sequences that may facilitate cell attachment or guide cell
differentiation at different stages.‘0 However, natural materials typically have poor
physical properties, scale up difficulties, and show batch-to-batch variation. Synthetic
materials, on the other hand, present good physical strength and processibility as cell or
tissue supports. Their molecular weights and degradation rates can be well controlled
during large scale production.6'8’”

The ideal synthetic polymers for tissue engineering should meet the following
criteria: (1) The polymer surface should permit cell adhesion and growth, (2) both the
polymer and its degradation products should have low toxicity for in vivo application, (3)
the material should be processable into a three dimensional scaffold with high porosity to
provide sufficient surface area for cell attachment and extracellular matrix (ECM, an
insoluble aggregate of several large proteins and glycosoaminoglycans, which provide a
three dimensional environment to organize cells into tissue”) regeneration, (4) the three
dimensional scaffold must be strong enough to provide necessary support for the implant
until the newly generated tissue is fully developed, (5) the material should be
biodegradable, and the degradation rate should be adjustable to match the rate of tissue
regeneration.29

PLA, PGA and PLGA polymers are well characterized synthetic biomaterials.

Numerous studies have demonstrated their sufficient biocompatibility with cells, such as

bone and cartilage cells.”15 They present desirable physical properties to be processed
into three dimensional structures with high porosity and desired shapes.‘3 '16’” By
carefully manipulating monomer stereochemistry, comonomer ratio, molecular weight
and polydispersity, the polymer degradation rate can be tuned to match the rate of tissue

regeneration of different type of cells.”"20

1.2 Processing and Modification of Polylactide for Tissue Engineering

1.2.1 Three Dimensional Structure Processing

To provide high surface area for high density cell seeding, nutrient supply, and
ECM regeneration, the polymer scaffold must have high porosity (usually higher than
90%) and a large enough pore size.2| Many techniques have been successfully developed
for making PLA-PGA polymers into three dimensional scaffolds for tissue engineering.
Fiber bonding is one of the earliest techniques for developing PLA-PGA scaffolds The
three dimensional scaffold is developed by spraying PLLA or PLGA solution onto PGA
fibers, followed by solvent evaporation.22 Particulate leaching is the most common
method for creating porous polymer structures. A water soluble porogen, such as NaCl is
mixed with a polymer solution. After the polymer solvent is removed, the polymer/salt
composite is leached in water for two days to generate the porous structure.23 The
porosity of the resulting scaffold can be controlled by the amount of porogen added, and
the pore size can be controlled by the size of the porogen. Similar to the particulate
leaching methods, gas foaming techniques use gas as porogen to create porous scaffolds.
The absence of the solvent leaching step shortens the preparation time, however the
resulting pores are largely unconnected which might cause difficulties for sell seeding

and migration.24 Instead of using porogens, emulsification/freeze-drying and liquid-liquid

phase separation methods are based on the concepts of phase separation. In this method, a
polymer emulsion or a polymer-rich and polymer-poor phase are created by either adding
another immiscible solvent or cooling to a phase separation temperature. The polymer
solution is quenched below the freezing point, and then solvent is freeze—dried to produce
a porous structure. Due to the complexity of various thermodynamic and kinetic

parameters applying in this method, various porous structures can be easily obtained.”27

1.2.2 Surface Modification of PLA-PGA Polymers

Besides having a three dimensional macroporous structure that provides enough
surface area for cell seeding, nutrient supply, and tissue growth, the cell affinity of
biomaterials is a very important factor of successful tissue regeneration, because the
surface properties of the material can regulate how cell responds to the material. Many
studies have shown that cell attachment, growth, differentiation, and death is influenced
by hydrophilicity/hydrophobicity30, surface energy3 1, charge”33 and roughness34 of the
material surface.

PLA-PGA polymers have many advantages as synthetic biocompatible materials
being used in tissue engineering. However, the surface hydrophobicity, low surface
energy and lack of functional sites for cell recognition are potential problems for farther
tissue engineering applications. To solve these problems and improve their surface
interaction with cell cultures, much work has been done to improve the cell affinity of
PLA-PGA polymers.

Plasma (a partially or fully ionized gas containing electrons, ions, and neutral
atoms or molecules) treatment is one method used for improving cell affinity of PLA-

PGA polymer surfaces. Wang and coworkers demonstrated that O-containing and N-

containing groups such as hydroxyl and amine were incorporated onto the surface of
PDLLA films, improving the hydrophilicity of the surface and increasing the surface
energy. 35

Surface alkali hydrolysis treatment is also a convenient method to modify PLA-
PGA polymer surface. The polymer film or scaffolds were immersed into aqueous NaOH
or a mixture of aqueous NaOH and ethanol. The ester bonds at the surface can be
hydrolyzed, leaving carboxylic acid groups at the polymer surface. The resulting material
can be used to conjugate other bioactive molecules, such as Arg—Gly-Asp (RGD) peptide,

collagen, and biotin, which improve cell affiliation and reorganization (Figure 3). 36'”

 

Bioactive Substrates
I 7\

  

 

 

 

 

OH OH OH N N N
Surface Functional Grou
. O=< H O=< H O H P O=< H O=< H 0 H
Hydrolysrs I (1) Y 2% Attachment I 9 (P . 2%

Figure 3. Surface modification by alkali hydrolysis treatment.

PLA-PGA Polymer

Hybridization is another method to overcome the hydrophobicity and chemical
inertness of PLA-PGA polymers. PLA-PGA based polymers can be hybridized with
natural polymers such as collagen or bioactive ceramics such as tricalcium phosphate
(TCP), hydroxyapatite (HA), and selected compositions of commercially available
Bioglass®.3943 By either coating or mixing, hybride materials are developed with
improved bioactivity and optimized physical properties result.

Other approaches have been developed to coat a layer of a bioactive substrate or
another biocompatible polymer with functionalizable end groups on PLA-PGA polymer

surfaced by techniques such as electrostatic self-assembly44 and surface entrapment.” 47

ECM-like biomacromolecules were incorporated onto the polymer surface. In vitro cell
growth study proved the incorporation of the bioactive substrates using the above

methods has positive effects on cell attachment and growth.

1.2.3 Synthesizing Novel PLA-PGA Based Polymers for Tissue Engineering

Besides various techniques to modify PLA-PGA polymer surfaces, another
approach is to covalently attach bioactive substrates such as certain amino acid sequences
to the polymer chain. The hydroxyl end group on PLA-PGA polymer can be easily
functionalized with bioactive substrates such as amino acids to provide some degree of
functionality at the polymer surface. Langer and coworkers have developed a simple
method to synthesize block copolymers of poly(lactide-co-glycolic acid) and polylysine
by carbodiimide coupling reactions.48 This simple method covalently attaches bioactive
functionality to the polymer chain, however only the chain end is still limited to
incorporate enough functional groups without sacrificing the bulk physical properties of
the polymer.

Due to the limitation of end group functionalization, synthesizing novel PLA
based biodegradable polymers with biologically significant groups covalently grafted
onto the polymer back bone is a more desirable route for incorporating bioactive
functionality. The immobilized bioactive side chain groups can improve cell adhesion on
the polymer surface, and at the same time, it can also have specific interactions with cells
to promote cell proliferation, differentiation, and tissue regeneration.

Langer and coworkers developed a synthetic route to the cyclic dimer of L-lactic
acid and protected L-lysine. This cyclic dimer can be co-polymerized with L—lactide,

yielding a c0polymer of L-lactide with 1-5% of protected lysine units. After deprotection

of the lysine amino group, different bioactive substrates were attached to the polymer
side chain through coupling reactions (Figure 4).“9‘50 Other groups designed similar routes
to synthesize different amino acid containing copolymers that have different pendant
groups for further functionalization. Ouchi and coworkers synthesized the cyclic dimers
of glycolic acid with L-lysine, L-aspartic acid, and L-glutamic acid, and copolymerized
them with L-lactide (Figure 5). The surface wettability, cell attachment and proliferation
and material degradation during cell culture were analyzed. In general, the higher the
mole fraction of the amino acid unit in the polymer, the more hydrophilic the surface
becomes, and the higher degradation rate was observed. The amino acid containing
copolymers showed higher cell attachment efficiency but lower cell growth rate
compared with PLLA.5"52 The reason for the different results between cell attachment
and cell growth still remains unclear. However, these copolymers can be further modified

by coupling with other bioactive ligands such as the RGD cell adhesion peptide.

NHCbz

0 0 Random 0 0 o
.H . .
YKN + \OHKQ COPOIYmenzauon 0\/u\o/LWO\JLN °\,)Lo! :
MNHCbz TA _ Mi E O i {.1 0 § 0
O O

Deprotcction

o ioactive
molecules

NH

Coupling

NH;
0 o o R ‘t. o o o
MIGRAO/LKOE/ILN OJOW ea“ ”"5 “”IOT/lkO/Llfof/KN oi/lko/L )NM
5 o 5 H o 5 o i o i H o a 0

Figure 4. Copolymerization and modification of poly(lactic acid) using L-Iysine and L-lactide cyclic

 

comonomer.

O O 0
(KW (LKN’H o Aw”
OMNHCDZ ONOBZI OMOBZI

O O O O

Cyclo[Glc-L-Iys(Cbz)] Cyclo[Glc-L-Asp(OBzI)] Cyclo[GIc-L-Glu(Ole)]
Figure 5. Amino acid containing cyclic comomomers.

Amino acid containing copolymers provide a certain amount of reactive sites on
the polymer side chain for further functionaliztion. However the amino acid containing
cyclic dimers usually have lower polymerization reactivity which often results in lower
functional group density. The complex synthetic pathways often require protection and
deprotection at the side chain functional group, which also might cause polymer

molecular weight decrease and loss of functional groups.
1.3 Cell Geometry Regulation on Material Surface

1.3.1 Cell Geometry Control over Cell Function

Cell adhesion to surfaces plays a critical role in many cellular functions, such as
proliferation, apoptosis (programmed cell death), migration and differentiation. Ingber
and coworkers micropatterned planar surface to create cell adhesive islands that
contained ECM and observed that human and bovine capillary endothelial cells were
switched from growth to apoptosis by decreasing the cell attachment area (to about 400
um2 or smaller). To fully understand whether cell spreading or the total area of cell-ECM
contact leads to cell survival and growth, they evaluated cell growth and apoptosis on
multiple closely spaced adhesive islands. By changing the spacing between adhesive
islands, cell spreading can be increased 10 fold without significantly altering the total

cell-ECM contact area. They found that the extent of cell spreading, and not the area of

10

the adhesive contact, controlled cell life and apoptosis (Figure 6). These results showed
that cell shape appears to be the critical determinant that switches cells between life and
death and between proliferation and quiescence.53 Further more, they also observed that
intermediate cell spreading (area ~ 1000 umz) does not promote cell growth or death, but

often exhibits differentiation behavior.54

C) .1"; :1: /‘\
[ ApOptosis J I Growth .3 I -- Growth I

 

5 pm 50 um
fi: :33.
20 pm i‘ .10 pm
(A) (B) (C)

Figure 6. Diagram of substrates used to vary cell shape independently of the cell-ECM contact area.
Substrates were patterned with (A) a small single round island, (8) small closely spaced circular islands and

(C) a large single round island.

1.3.2 Micropatteming Material Surface for Cell Growth Studies

Based on the relationship between cell geometry and cell function, control of cell
geometry on material surfaces has become an important topic in tissue engineering.
However, the mechanisms of cell adhesion on specific material surfaces are still poorly
understood. Therefore, it is beneficial to design methods for organizing cells on material
surfaces, so cell-material and cell-cell interactions can be further studied. In recent
several years, there have been many studies in cell biology that took advantage of
patterned substrates. Healy and coworkers used patterned substrates to investigate gene
expression in the differentiation of bone cells.‘0 Nelson and Chen used patterned

substrates to confine pairs of contacting cells and demonstrated a novel proliferative

ll

signal that stems from a direct cell-cell contact.55 On the other hand, the cell biology
studies on patterned surfaces can also be used to analyze a materials’ ability to influence
cell adhesion, proliferation, apoptosis, migration, orientation and differentiation, which
leads to optimized biomaterial surface properties for tissue engineering applications.
Recent advances in pattering technology have generated a range of techniques to
immobilize biomolecules on material surface with micron scale dimensions. Most of
these different techniques begin with photolithography followed by another method such
as laser ablation, photoresist lift-off, photoresist protection, photopolymerization and
microcontact printing, etc. The micro scale pattern can be created by computer drawing
software and printed on glass plates as the photolithographic mask. Then the pattern is
transferred from the mask to the material surface.56
The simplest technique is laser ablation. The substrate coated with chemical
material is irradiated through a patterned mask. The material not protected by the pattern
is degraded leaving the patterned area with chemical material on the surface. This method
can be used to pattern protein materials such as polylysine.57
Photoresists are commonly used to transfer patterns to substrates. A
photodegradable material is first applied on the material surface, then placed in contact
with the patterned mask and exposed to UV light. After removal of the degraded material,
a photoresist copy of the pattern is produced. The area without photoresist pattern can be
used to incorporate cell adhesive material. Finally the photoresist is “lifted off” of the

material surface, leaving the cell adhesive material with designed pattern.”59 Similarly,

the photoresist can also be used to protect a material previously deposited on the

12

substrate. After patterning, the material at the unprotected area is removed from the

substrate by chemical process (Figure 7)."0

 

 

 

 

 

 

 

 

 

uv Light
I——— Mask
I I
Expose to UV Light Develop Photoresist
aflm 77*! mi 7/777 ' -—-———-r/77277
I I l
Remove Photoresist Deposit Cell Adhesive Material

Figure 7. Schematic Diagram of the Photoresist Surface Patterning Process.

Microcontact printing is suitable for patterning fragile molecules.6| The method
begins by using photolithography to mold a polydimethylsiloxane (PDMS) stamp.
Different strategies have been developed to transfer patterns from the PDMS stamp to a
substrate. A common example is to use alkanethiols, since the sulfur atoms in
alkanethiols exhibits a strong affinity for gold atoms and alkanethiols are also readily
absorbed into PDMS. Alkanethiols are stamped onto gold coated substrates with the
alkane groups away from the substrate for further protein adsorption and cell attachment.
The area without alkanethiol coating can be coated with polyethyleneglycol linked thiols

which resist adsorption of protein (Figure 8).“‘62

l i_j_j_i l a 3“

L— Mask
1 Seed with Cells

«2 Photoresist
Silicon Wafer .
Iithylcneglycol Terminated Surface
i
::I

Remove Stamp and Immerse in

1 Develop Photoresist
l Polyethyleneglycol Linked Thiols

.L 4.2.2.4 a

Silicon Wafer

   

Mold PDMS Stamp

 

‘ Stamp on Gold Surface

Peel off PDMS and
5—1 m.................. .
Alkanethiols

Figure 8. Schematic Diagram of the Microcontact Printing Process

The last example is the photografting polymerization technique, which usually
uses a photoiniferter such as dithiocarbamate derivative to prepare micropatterned
surface. Under UV irradiation, the photoiniferter can generate a reactive free radical that
will initiate radical polymerization. When the photoiniferter is chemically bound to a
surface with monomers deposited on it and irradiated through a photolithographic mask,

a pattern of graft polymer chain can be created (Figure 9)."3

Polymer , ’ Photoinifene

5 5 5 5 5 5 5 5 5 _5 5 , _ - /

55 .n <—-—" $ 2 5‘

Polymer Substrate with E‘m'” " "T” i ‘2 3 I: .' ;

(a) Photoinifcrter Group Incorporated Silicon Water ‘. ,1 ' 1‘ I

UV Light
1 l i i l i Monomers

f—Mask //o \.

/ O O \

Monomer—j , , .. .. ..|
O |
F” “”""“”*““‘—"*’1<—lv' 75.5 3. '5, ,_° 0 1
L. ..;.........w;’| \| r I:

(B) Photografting Polymerization Silicon Water l‘. (I, ’3 1, .’

Graft Polymer

;5f! \‘
2’15;

IIII IIIIE

p in
(C) Patterned Polymer Substrate Silicon Wafer

I
l
I
I
I

Figure 9. Schematic Diagram of the Photografting Polymerization Process. (A) A polymer substrate
containing photoiniferter is prepared on silicon surface. (8) additional monomer is added to the surface and
exposed to UV light through a photomask. (C) covalently-bound polymer is grown from the substrate
surface in the regions exposed to light.

The surface micropatterning techniques being used still have some limitations,
such as lengthy preparatory procedures and harsh chemical, photochemical and heating

conditions during the process. The substrate and functional patterns being incorporated

are also limited. Thus new simple and moderate micropatteming techniques need to be

designed for specific material surfaces and specific patterned functionalities.

l6

CHAPTER TWO

2 Functionalization of Polylactide Based Copolymer

PLA and its copolymers have been proven to be suitable biomedical materials for
drug delivery and tissue engineering applications. As synthetic biomaterials, they have
good mechanical strength, are easy manipulated into desired shapes, and have optimal
degradation kinetics. Through a variety of techniques, PLA copolymers can be made into
unique three-dimensional scaffolds with high porosity. The highly porous material can
provide enough surface area for cell seeding, nutrient supply, ECM regeneration and
tissue growth. However, their hydrophobic nature, lack of cell recognition signals and
poor surface chemistry for cell attachment, proliferation and differentiation could limit
their applications as ideal three-dimensional matrices for cells. It will be beneficial to
synthesize functionalized PLA based polymers that covalently incorporate bioactive
moieties for cell attachment and cell—surface interaction. Many research groups have
developed routes to synthesize lactide derivatives that have functionalizible side chains,
and then copolymerized them with lactide.”52 The copolymers synthesized have a
biodegradable ester backbone, and also present reactive side chain groups that can be
further functionalized through chemical reactions. These methods provided opportunities
to modify PLA polymer side chain for better cell surface interaction. However all of
these methods require complicated synthetic pathways for each monomer. Further more,
some reactive side chain functional groups are not compatible with the polymerization
conditions. Protection and deprotection processes are required during the polymer
synthesis, which might cause polymer molecular weight decrease or lose of functional

groups.

17

An alternative copolymer synthesis pathway is to synthesize a common
comonomer with a side group that is relatively inert under polymerization conditions, but
can be converted into different reactive functional groups after polymerization. By
applying this post polymerization modification strategy, protection-deprotection
processes are minimized. Only one kind of comonomer needs to be synthesized, and a
variety of functional groups can be attached to polymer side chain by a simple reaction

protocol.

2.1 Monomer Synthesis

The allyl substituted lactide comonomer, 3,6—diallylglycolide (DAG), was
synthesized using a general procedure for synthesizing lactide and a variety of alkyl
substituted glycolides.64 The metal-mediated allylation of glyoxylic acid monohydrate
using allyl bromide as the allyl source, zinc powder and bismuth trichloride for in situ
generation of the allylmetal species, gave 2-hydroxypent-4-enoic acid (2-HPA) in 92%
yield. The 2-HPA can be dimerized by acid catalyzed condensation in toluene at > 100
°C. The reaction usually takes 3 to 5 days, with the water produced from condensation
being removed using a Dean-Stark trap. The crude product mixture contained both
oligomers and the cyclic dimer. By distillation of the mixture over zinc oxide under
reduced pressure, the oligmers can be converted to the cyclic dimer, 3,6-diallylglycolide

(Figure 10).

18

0 o

H\n/LKOH + WEI ____>BICl3/Zn No.4

0 , H20 THF, 16 hrs OH

92% Yield

0 0
O W W V0“,
We»! __,1PFSA o + o + o
0 "WA 0 \

o
2 ZnO A om)...”
OH WK, W O A

O O

Meso (30%) L V J

Racemic (70%)

 

56% Yield
Figure 10. Synthetic route to 3,6-diallylglycolide.

The product contains 30% of the meso (RS) isomer and 70% of racemic (RR and
SS) isomers. The RR, SS, and RS stereoisomers can be distinguished by NMR
spectroscopy. This is mostly clearly seen in the methine region of the proton NMR
spectrum. These protons both are observed as doublets of doublets at ~~ 5.0 ppm (Figure

ll).

19

He. Hd

Hb

 

 

 

 

ivjrvvilviv

’ I l ' ' ' I—r I I ‘ I ' ' ' l ' ' ' ’ l ‘ ' ' ' ' ' ' ' l .
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
Figure 11. 1H NMR (300 MHz, CDCI3) spectrum of 3,6—diallylglycolide. H¢* denotes the R,R and 5,5

isomers, H," denotes the R,S isomer.

2.2 Copolymer Synthesis and Characterization

The copolymer poly(L-lactide-co-diallylglycolide), PLLA-co-DAG was
synthesized by copolymerization of PLLA and DAG at 145 °C for 20 minutes, using
tin(II)—2-ethylhexanoate as the catalyst and 4-tert—butylbenzylaleohol (BBA) as the
initiators (Figure 12). The molecular weight can be controlled by varying the initiator and
monomer ratio (M/I). Usually the copolymer is designed to contain 7-13% of DAG unit

and have 3 Mn of 20,000-40,000 (Table l).

20

Mm + 5:1 “3:22th were 55015055" 35 am

 

 

o o
94% Yield
Figure 12. Synthetic route to poly(L-|actide-c0-diallylglycolide).
Monomer Feed Monomer/Innitiator C2011}; 00’: ""2220 M /gm0f’ (PDI)b'C
Ratio (LLA:DAG) Ratto ([M]:[I]) (LLA:DAG)
90 : 10 300:1 92 : 8 33,900 (1.3)
85 : 15 300:1 86.5 : 13.5 20,800 (1.5)

 

Table l. Copolymerization of L-Lactide and 3,6-diallylglycolide. " Molar composition of DAG determined
by integration of the methylene resonance of the allyl repeat unit compared to the methine resonance of the

lactide repeat unit. b Molecular weight determined by GPC. C PDI = Polydispersity index (MW/Mn).

The copolymers were also characterized by 1H NMR spectroscopy (Figure 13).
The multiplet at 5.62- 5.81ppm indicates the presence of the internal olefin proton, while
the resonance of the terminal olefin protons appears at 5.10-5.17, overlapping the
methine proton resonance of both lactide and diallylglycolide units. Also, the methylene
resonances of the pendant allyl group are observed between 2.5-2.8 ppm. The allyl
monomer composition can be calculated by the integration of the methylene resonance
vs. the integration of methyl resonance of the lactide unit. The molecular weights of the
copolymers were determined by gel permeation chromatography (GPC). Based on the
Fox equation, the thermal analysis previously done in our laboratory suggested the
presence of a random copolymer.65 The randomly distributed side chain olefins along the

backbone are now ready to undergo functional group transformation.

21

 

 

 

 

 

 

 

 

 

 

 

Figure 13. lH NMR (300 MHz, CDCl;) spectrum of poly(L~lactide-c0-allylglycolide).

2.3 Post-Polymerization Modification

Our goal for the post polymerization modification is to link the polymer side
chain with molecules with biological functions. The terminal olefin by itself doesn’t
provide enough available chemistry to achieve our desired transformation. However, the
terminal olefin can be modified into other functional groups such as hydroxyl, carboxylic
acid, etc, which would allow us to explore various types of coupling chemistry for
attachment of bioactive substrates to the side chain. Alcohols are a particularly useful
functional group for tethering substrates through reactions such as esterification or
etherification, as well as carbonate and carbamate linkages. The best known

transformation for converting olefins to alcohols is hydroboration followed by oxidation.

22

2.3.1 Hydroboration-Oxidation

Hydroboration followed by oxidation is a very useful method to convert terminal
olefins into primary alcohol groups. In a typical hydroboration-oxidation reaction, a
borane molecule is added to the olefin double bond in an anti-Markovnikov fashion. The
resulting organoborane is then treated with hydrogen peroxide under basic conditions to
afford the primary alcohol (Figure 14). Similar to small molecule cases, hydroboration-
oxidation reactions can also be applied on polymer substrates with an olefin group either
in the polymer back bone or pendant from the polymer.“67 The successful transformation
provides a new method for synthesizing polymers with reactive side groups starting from

simple, relatively inert monomers.

JH \ B/ R2 H IR?
I -——-—> Bx
R1/\/—R2 FIZJV R2

Step 1. Boron adds to the less substituted carbon of the double bond.

 

 

/‘\/B N \ - 1 l - I

Step 2. Oxidation of the trialkylborate followed by migration of the alkyl group to the adjacent oxygen.

No, ,0. OH
81 I? R2 H20 H /\/OH + 53 + 2 RZOH
0 ‘ Ho’ ‘OH

 

R2/

Step 3. Hydroxide attacks boron to give a primary alcohol and boronic acid.
Figure 14. Mechanism of the hydroboration/oxidation reaction of terminal olefins.

For our case, successful transformation of terminal olefin side group on the
PLLA-co-DAG copolymer into a terminal hydroxyl side group provides us the
opportunity to further incorporate biologically significant ligands onto our polymer side

chain using simple organic methodology. This post—polymerization modification method

23

also avoids the synthesis of complex monomers, and subsequent protection and

deprotection schemes before and after polymerization.

2.3.1.1 Synthesis and Characterization
The hydroxylated copolymer, poly(lactide-g-hydroxypropane) (PLLA-g-HP) was
synthesized by the hydroboration of PLLA-co-DAG followed by oxidation using aqueous

sodium acetate and hydrogen peroxide (Figure 1515).

 

 

 

 

 

H 9 H P H P H ..
53315359333... -0
9H2 cH2 ' ’ ’ 2 2’ L cH2 9H2
pH pH 9H2 c522
CH2 CH2 9H2 9H2
OH OH
PLLA-co-DAG PLLA-g-HP

W W0”

Figure 15. Synthetic route to poly(lactide-g-hydroxypropane) via hydroboration-oxidation of poly(lactide—
co-diallylglycolide).

The first step of the hydroboration was carried out in the glove box. The polymer
solution was stirred at room temperature for 2 hours using 9-borabicyclononane (9-BBN)
as the borane source. The reaction can be followed by HB NMR, with a peak centered at
88 ppm indicating formation of the trialkyl-borane. In our first attempt, once the
hydroboration was determined to be complete, the polymer was treated with 1.5 to 2.0
equivalents of aqueous sodium acetate and hydrogen peroxide. Boron was incompletely
removed even when we increased the reaction time for the second step to 16 hours. A
resonance at ~ 34 ppm in the 11B NMR spectrum was observed indicating residual
boronic acid on the polymer side chain. Repetition of the oxidation step was needed to

fully remove the boron from the polymer. However, when we increased the amount of

24

H202 to three equivalents based on the 9-BBN added in the hydroboration step, the boron
was removed completely after 4 hours and a second oxidation was not needed. After
reprecipitation, no boron resonance can be observed in 11B NMR spectrum and in lH
NMR, a new peak at 3.62 ppm can be clearly seen indicating the successful
hydroxylation of the olefin double bond.

After hydroxylation, the polymer molecular weight was unchanged (Mn = 20,800
gmol'l PDI = 1.50 for PLLA-co-AG vs. Mn = 19,200 gmol'l PDI = 1.55 for PLLA-g-HP)
showing that no significant polymer degradation or crosslinking during the
hydroboration-oxidation steps. The lH NMR spectrum of the copolymer shows the
disappearance of the allyl methylene resonances and the appearance of the methylene
next to the primary alcohol at 3.62 ppm. Also, the alkyl methylene resonances of the

hydroxypropane group are seen between 1.65-2.04 ppm (Figure 16).

25

 

 

 

 

 

 

 

 

o5 0)
CH3 c,c' a.a'OH Cfls
b,b'
(A)
ll .1
i a,a’ c,c’ HI
._J L L _J\ L W Kb
0
H H
W095 w/CH‘
CH3 egg
c,c' a.a'
W
b
(B) .
lit
9 i If
aif L C,C,
b " ”L4
AJJ LL _/ c._____.__
Tsioifi'vsis sio 4i5 4i0 3i5 ' 3:0 215 2T0 iris I110 T7

Figure 16. 'H NMR (300 MHz,CDCl3) spectra of (A) PLLA-g-HP; poly(L-lactide-g-hydroxypropane) and
(B) PLLA-co-AG; poly(L-lactide-co-diallylglycolide).

The hydroxylated copolymer can be used directly as one kind of functional
biomaterial for cell growth studies. On the other hand, the primary alcohol group on the
polymer side chain can also be further functionalized with other bioactive molecules

through simple transformations.

2.3.2 Side Chain Coupling Reactions

The primary alcohol group at the polymer side chain provides various methods for
coupling the polymer side chain with other molecules through ester, carbonate or
carbamate bonds. Since the polymer backbone contains ester bonds, harsh acidic or basic

reaction conditions will cause polymer degradation. Therefore further side chain

26

modification reactions must be carried out under mild conditions. Many mild coupling
reaction routes have been developed for coupling polymer chains or coupling a polymer

chain with small molecules, such as dicyclohexyl carbodiimide (DCC) couplingm’m'75

and 1.1’-Carbonyldiimindazole (CDI) coupling.“69

2.3.2.1 DCC Coupling

DCC coupling is a convenient synthetic route to connect alcohols with carboxylic
acids by forming ester bonds. The reaction can be carried out at room temperature with a
catalytic amount of N,N-dimethyaminopyridine (DMAP) as the catalyst. The insoluble
dicyclohexyl urea byproduct can be removed by filtration. Due to the mild reaction
conditions and easy product purification, DCC coupling is applied in peptide

72‘” and for coupling substrates to polymers.”75 DCC

synthesis,70'7| dendrimer chemistry,
coupling of our hydroxylated copolymer with a variety of carboxylic acids provides us a
simple way to covalently attach bio-significant molecules that have carboxylic acid

functional groups though an ester linkage (Figure 17). The newly modified copolymer

might have improved biocompatibility as a material in tissue regeneration.

 

o DCC, DMAP (0.2 eq) JOL
WOH + HOAR CHZClz, RT 16 hrs.’ We a

O
k = Carboxylic Acids
HO R

Figure 17. General synthetic route to polymer-bound esters using DCC coupling.

2.3.2.2 Side-Chain Esterification of Fatty Acids

Fatty acids are important biomolecules in cell biology. The basic structural unit of

virtually all biomembranes is the phospholipid bilayer. In most common phospholipids,

27

glycerol forms the backbone of the molecules, with two hydroxyl groups linking to fatty
acid residues to form the hydrophobic interior, and a third hydroxyl group linking to a
bridging phosphate group to form the hydrophilic exterior (Figure 18). Besides being an
important component of cell membrane, fatty acids have also been reported to influence
the biology of bone tissue growth.76‘77

o-(‘ijg-o’VNHZ

r(9

Integral Protein
0 O —i
O 0

WE? iiifififlwmme ,

Phospholipid Bilayer

 
   
  
 

Figure 18. Structure of phospholipid and cell membrane phospholipids bilayer.

We chose myristic, stearic, oleic, linoleic and arachidonic acid to establish DCC
coupling as a viable method for functionalizing PLLA-g-HP (Figure 19). Myristic acid
and stearic acid are the most common saturated fatty acids, and cis unsaturated fatty acids
are more common in cells than the trans form. The pendant hydroxyl groups reacted with
the fatty acids in the presence of DCC and a catalytic amount of DMAP to incorporate
fatty acids on our copolymer. Due to the biological significance of fatty acids in cell
membrane structure and bone tissue growth, we expect that modified PLLA copolymers

should show improved bone cell growth.

28

 

o DCC, DMAP (0.2 eg) it
W0” + /U\ CH2C12,R.T.16hr§. W0 R

O
PLLA-g-Mypr = WOW

Poly(L-lactide-g-myristic acid propyl ester)

W
O/\/\O

PLLA-g-StPr =
Poly(L-lactide-g-stearic acid propyl ester)
0
PLLA—g-OlPr = WOWVVW
Poly(L-lactide-g-oleic acid propyl ester)
0
PLLA-g-LnPr = WOWW
Poly(L-lactide-g-linoleic acid propyl ester)
0
PLLA-g-ArPr ___ WOWWW

Poly(L-lactide-g-arachidonic acid propyl ester)

Figure 19. Synthetic route to saturated and unsaturated fatty acid-modified PLLA using DCC coupling.

The reactions were carried out at room temperature in CH2C12 for 20 hours. After
the desired reaction time, the insoluble dicyclohexyl urea was removed by filtration, and
the polymer solution was concentrated, and precipitated twice from methanol. Since no
harsh acidic or basic conditions are required for DCC coupling reactions, high yield and
full conversion were achieved with no molecular weight lost for all of the above fatty
acid coupling reactions. Thermal analysis of the copolymers using DSC showed the Tg of
the materials decreased from the original PLLA-g-HP (51°C) upon esterification of the
fatty acids (Table 2). The DSC results of linoleic acid and the arachidonic acid-modified

copolymers showed higher Tgs compares with saturated fatty acid and the oleic acid

29

modified copolymers we prepared, and no Tc and Tm were observed indicating oxidative

and thermally-induced cross-linking occurred while the polymers were exposed to air.65

 

 

Fatty Acid M,,/gmor’(PDI)“-” T, C/ ”C T, ‘/ ”C T,,, ‘/ "C % Yield
PLLA-co-DAG 20,800 (1.50) 52 -- 141 94
PLLA-g-HP" 24,900 ( 1 .26) 5 1 122 147 85
Myristic 25,500 (1.29) 25 81 1 18 85
Stearic 28,900 (1.26) 37 1 12 141 86
Oleic 19,500 (1.35) 30 89 142 88
Linoleic 24,300 (1.40) 45 -- -- 71
Arachidonic 25,600 (1.41) 45 -- -- 78

 

Table 2. Saturated and unsaturated fatty acid-modified PLLA copolymers containing 8 mol % fatty acid. "
Molecular weight determined by GPC. b PDI = Polydispersity index. C From differential scanning
calorimetry of 8 mol % fatty acid-modified copolymers. Date are from second heating scan at 10°C/min

under helium. d Hydroxylated copolymer starting material used to couple fatty acids.

The products were also characterized by NMR. Quantitative incorporation was
achieved for all coupling reaction with the above fatty acids. The methylene proton
resonance at 3.62 ppm in PLLA-g-HP shifted to 4.04 ppm after CDD coupling indicating
the complete conversion of fatty acids to their polymer-bound esters (Figure 20, 21). For
the unsaturated fatty acid modified copolymers, olefinic proton resonances can be
observed near 5.3 ppm. The olefinic proton integration increases with the increasing
number of cis double bonds. PLLA-g-LnPr and PLLA—g-ArPr both have bisallylic
methylene protons appearing at 2.7-2.8 ppm. The integration of the bisallylic methylene

protons in PLLA—g-ArPr is about 3 times as much as in PLLA-g-LnPr.

3O

 

 

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32

2.4 Cell Growth Studies on Fatty Acids Modified Poly(Lactic Acid) Surfaces

2.4.1 Copolymer Thin Films

In order to evaluate the properties of the modified materials for tissue engineering
applications, thin films were prepared by spin coating the polymers on freshly cleaned
silicon wafers. Osteoblasts were seeded on the polymer-coated wafers and their
proliferation, differentiation, and shape during the culturing process were assessed.65

Osteoblasts are important cells in bone physiology. They are responsible for the
formation and organization of the extracellular matrix of bone and its subsequent
mineralization. When seeded onto a surface, the osteoblasts first proliferate until the
surface is covered completely by cells, then under the right conditions, they will undergo
progressive differentiation and secrete a collagen rich substance essential for later
mineralization of hydroxyapatite and other crystals.84 We used osteoblast growth on our
hydroxylated and fatty acids modified copolymer surfaces to evaluate the influence of our
functional copolymer on cell attachement, shape, growth and differentiation.

To determine the polymer thin film thickness and whether or not our polymer
surfaces possessed any functionalities, we characterized the surfaces of thin films using
ellipsometry and contact angle measurements (Table 3). All of the films prepared were
spin-coated from a 1% (w/w) solution of the polymer in toluene. The film thicknesses
were measured using ellipsometry and had thicknesses generally around 30 i 0.2 nm.
The thicknesses of the films were uniform on a given substrate, but varied slightly

between copolymers.

33

 

 

 

Copolymer Loading 0 Thickness/ nm b 65, C 60d, 6 6,“ C
PDLLA -- 30 i 0.3 74.1 :1: 5.2 72.8 i 3.3 65.3 :1: 2.1
PLLA -- l9 1 0.1 74.1 :1: 3.1 77.4 :1: 4.4 65.6 :1: 4.8
PLLA-co-DAG 8% 27 t 0.3 74.7 :t 2.2 79.2 :1: 1.3 62.8 i: 1.8
PLLA—g-HP 8% 25 i 0.1 62.0 t 2.0 72.1 :1: 4.2 56.1 :1: 3.6
LLA-g-MyPr 8% 29 t 0.3 87.8 i 1.5 88.0 :1: 1.2 75.2 i 0.7
PLLA-g-StPr 8% 29 i 0.3 86.1 i 1.5 91.9 :1: 1.8 77.1 i 1.8
PLLA-g-OlPr 8% 30 :1: 0.2 84.2 :1: 2.2 89.0 :1: 2.7 75.6 :1: 1.1
PLLA-g-LnPr 8% 27 i 0.2 73.6 1- 1.2 82.4 :1: 1.2 68.9 :1: 1.5
PLLA-g-ArPr 8% 29 i 0.1 73.2 t 1.8 80.7 :1: 1.8 70.6 :1: 1.9

 

Table 3. Thin films of copolymers spin-coated on l-inch diameter silicon wafers from a 1% (w/w)
solution in toluene unless otherwise noted. Spin rate: 2500 rpm, spin time: 60 sec. " Mole percent loading
determined by 1H NMR spectroscopy. b Determined by ellipsometry. ‘ Samples were prepared by heating
above the polymer melting point and quenching to room temperature to obtain amorphous samples. Static
(6"), advancing (6m) and receding (19,“) contact angles were measured at a rate of 3 11115 up to a total
volume of 30 1.1L using Milli-Q grade water.

The wettability of the polymer surface was monitored by recording the static,
advancing and receding contact angles of Milli-Q grade water. The polymer samples
were all heated to 160°C under nitrogen, and then immediately cooled to room
temperature and placed on a clean surface at room temperature in order to ensure a
common sample history. Six separate contact angle measurements were taken on each
polymer-coated silicon wafer, with the error set as one standard deviation from the mean.
The surface wettability for PLLA, PDLLA and PLLA-co-DAG are similar. However,
compared to PLLA, the contact angles for PLLA-g-HP is significantly smaller (by about
10 degrees), suggesting that the hydroxyl side groups are present near the surface of the
film. Compared to PLLA, the contact angles for saturated fatty acid modified polymers

are significantly higher (by about 12 degrees), indicating the highly hydrophobic alkyl

34

ester side chains are present near the surface of the film. The contact angles for
unsaturated fatty acids modified copolymers are between those of saturated fatty acids

modified copolymers and PLLA.

2.4.2 Osteoblast Cell Shape

Cell shape has been demonstrated to have important effect on cell growth, death
or differentiation. Ingber and coworkers’ research on cell geometry control over cell
function showed that cells grow when spread, die when fully retracted, and differentiate
if maintained at a moderate degree of extension.53 By studying the osteoblast cell shape
on different modified polymer surfaces we hope to obtain some information on
biocompatibility of our polymers for bone tissue growth.

MC3T3-El osteoblasts were seeded at a concentration of 4400 cells/cmz, cultured
for 2 days in OL-MEM (minimal essential medium) containing 2 mM B-glycerolphosphate
and 25 ug/mL ascorbic acid, after which they were stained and visualized in order to
observe their appearance on different polymer surfaces. The differences in morphology of
these sets of cells showed how the osteoblasts spread on each of the surfaces (Figure 22).
Cells on different polymer surfaces showed different degrees of spreading, providing
positive evidence that the different functionalized polymer surfaces influence the
conformation of the cells and therefore, may be able to influence osteoblast proliferation

and differentiation.

(a) Silicon Oxide (b) PLLA-co-DAG (c) PLLA-g-HP (d) PLLA-g-MyPr

 

35

(e) PLLA-g—StPr (f) PLLA-g-OlPr (g) PLLA-g-LnPr (h) PLLA-g-ArPr

 

Figure 22. Microscope images of osteoblasts cultured for 48 hours on (a) silicon oxide and (b) PLLA-co-
DAG (c) PLLA-g-HP (d) PLLA-g—MyPr (e) PLLA-g-StPr (I) PLLA—g—OlPr (g) PLLA-g-LnPr (h) PLLA-g-

ArPr. The cells are stained red and the nuclei are stained blue.

2.4.3 Osteoblast Proliferation

Osteoblast proliferation was quantified by visualizing the DNA of dividing cells.
This information was obtained by incubating the cultured osteoblast cells with the
fluorescent label bromodeoxyuridine (BrdU). During the process of DNA replication, the
BrdU label gets taken up by proliferating cells, and makes them visible as green
fluorescent nuclei under UV light. The total number of nuclei is determined by propidium
iodide staining which stains all nuclei red. The number of green nuclei was calculated as

a percentage of the total nuclei to quantify osteoblast proliferation (Figure 23).

mamam

O = Adenosine
C = Thymidine
O = Bromodeoxyuridine

Figure 23. Illustration of cell replication incorporating the BrdU fluorescent label.

Osteoblasts were cultured on polymer-coated silicon discs. Afier 48 hours they
were incubated for 2 hours with BrdU. Then, the cells were stained with propidium

iodide. Digital pictures of cultured cells and cells under proliferation were taken and the

36

number of cells was counted respectively (Figure 24). The percentage of dividing cells on
different surfaces was calculated and the numbers were normalized to the silicon oxide
surface.

Nuclei Undergoing

Total Nuclei DNA Replication

(a) Silicon Oxide

(b) PLLA-co-DAG

W

(C) PLLA-g-HP

o/\/\OH

(d) PLLA-g-MyPr

Woke”

 

37

(e) PLLA-g-StPr

O
Woke”

(f) PLLA-g-OlPr

:
i

(g) PLLA-g-LnPr

3
i

01) PLLA-g-ArPr

3,
g

 

Figure 24. Digital photo images of fluorescent BrdU-labeled osteoblasts on (a) silicon oxide, (b) PLLA-
co-DAG (c) PLLA—g-HP (d) PLLA-g—MyPr (e) PLLA-g-StPr (f) PLLA—g—OlPr (g) PLLA-g-LnPr (h)
PLLA-g-ArPr.

Three sets of BrdU growth studies were carried out for each modified copolymer
surface. The results indicated that osteoblasts placed on modified polymer surfaces

showed different degrees of proliferation. Cells placed on PLLA-g-HP surfaces showed

38

the highest increase of growth by 3 fold which might be related to the hydrophilicity of

the polymer surfaces (Figure 25).

Ilflla

Q\ ‘5 $
s s
6‘0 9?? $6 :5 ,e‘ 53‘ 858‘ )8st
V ,% Y'l% A7

Dmdmg Nuclel
(Normalized to Blank)
N

 

 

Figure 25. Result of osteoblast cell proliferation on (a) silicon oxide, (b) PLLA-co-DAG (c) PLLA-g-HP
(d) PLLA-g-MyPr (e) PLLA-g—StPr (f) PLLA-g-OlPr (g) PLLA-g-LnPr (h) PLLA-g—ArPr. Averages are

expressed as an n-fold change compared to control, uncoated blank surfaces.

2.4.4 Osteoblast Differentiation

After osteoblasts proliferate to a critical point, bone tissue formation can start
through cell differentiation. To differentiate, cells must change the types of proteins they
make. A hierarchy exists in the cell to control these changes. Genes/DNA provide the
blueprint for mRNA which provides the blueprint for new proteins to be made. When
studying osteoblast differentiation, levels of mRNA for alkaline phosphatase and
osteocalcin are measured, which indicate levels of osteoblast differentiation in different

stages (Figure 26). Elevated runx2 mRNA is another marker that indicates differentiation

39

since the runx2 protein goes into the nucleus and turns on genes associated with

. . . i-
differentiations‘ 87

Osteoblast Developmental Time

   
       
   

 

 

 

101 I’dv—~___ ‘ runx
g I, " — Alkaline Osteocalcin
'2 7 ‘ Phosphatas
E
E 5
T:
.E 2 -
i?
N
E: 0 I '
\
° 0 7 l4 2] 28
— - —
Days in culture
Proliferation (cell division) Mineralization (black)

 

Figure 26. Gene expression levels of osteocalcin and alkaline phosphatase as a function of osteoblast
culture time.

Osteoblasts were grown on the polymer surfaces for 14 days under conditions that
enhance differentiation. RNA was extracted from the cells and levels of specific mRNAs
were determined by standard quantitative RT-PCR. Elevated levels of alkaline
phosphatase mRNA demonstrate that an osteoblast is in a early-middle stage of
differentiation, while elevated levels of osteocalcin mRNA demonstrate that osteoblasts
are more mature in late stage of differentiation. Data were collected from 6 sets of

experiments and referenced to the result on a blank silicon oxide surface (Figure 27).

40

.N
M
1

 

N
1

   

 

in
l

_.
1

(Early Differentiation marker)

.0
LA
1

AlkalinePhosphate mRNA

 

o
1

 

Osteocalcin mRNA

(Late differentiation marker)

Figure 27. Result of osteoblast cell early and late stage differentiation on silicon oxide, PLLA-co-DAG,
PLLA-g-HP, PLLA~g—MyPr, PLLA-g-StPr. PLLA-g-OlPr. PLLA-g-LnPr, and PLLA-g-ArPr. Averages are

expressed as an n-fold change compared to control, uncoated blank surfaces.

41

Runx 2 is a transcription factor, which functions as a switch to turn on genes (and
increase mRNA levels) associated with osteoblast differentiation. RNA of the cells was
sampled and monitored for runx2 levels after 14 days of culturing. Data were collected
from 6 sets of experiments on different polymer surfaces and referenced to the result on

blank silicon oxide surface (Figure 28).

 

Runx 2 mRNA

(Transcription Factor for osteoblast Differentiation)

‘1‘

 

Figure 28. Result of osteoblast cell early and late stage differentiation on silicon oxide, PLLA-co-DAG,
PLLA-g-HP, PLLA-g-MyPr, PLLA-g-StPr, PLLA-g-OlPr, PLLA-g-LnPr, and PLLA-g-ArPr. Averages are
expressed as a fold change compared to control, uncoated blank surfaces.

The hydroxylated copolymer PLLA-g-HP increases osteocalcin mRNA levels
while decreasing alkaline phosphatase mRNA levels. This suggests that PLLA-g-HP
promotes late stage differentiation of osteoblasts. Other surface coatings promoted
changes (increase or decrease) in early differentiation marker alkaline phosphatase

mRNA levels. The significance of these effects is not yet understood.

42

The cell growth studies on our modified polymer surfaces indicate that PLLA-g-
HP surface stimulates osteoblast proliferation and promotes late stage differentiation,
which is consistent with the findings that hydrophilic surface could improve cell
attachment and tissue growth.30 PLLA-g-HP copolymer might be an interesting synthetic

implant material for osteoblast growth and bone formation.

2.5 Conclusions

Copolymerization of L—lactide and diallylglycolide followed by hydroboration-
oxidation have been successfully performed. Different fatty acids have been grafted to
the copolymer side chain through DCC coupling reactions. Thin films of PLA and its
functionalized derivatives were prepared on silicon wafers for studying the physiological
response of osteoblast cultured on the polymer surfaces. Cell shape, proliferation and
differentiation were analyzed. The distinct enhancement of cell proliferation and
differentiation on PLLA—g-HP surface proved that surface hydrophilicity is an important
factor for cell attachment and growth. The different responses of osteoblast to the
modified copolymer surfaces provide us a direction of screening biopolymer substrate for
improved tissue engineering applications.

The post polymerization modification has proven to be an effective way to graft
functional or biosignificant side chain units on biodegradable polymers such as PLLA.
The mild synthetic conditions, high yield and conversion, and the commercial availability
and diversity of the functional side chains make this route widely applicable for

biopolymer modifications.

43

CHAPTER THREE
3 Photolabile Protection-Deprotection Strategy in Surface Pattering.

As for tissue growth on surfaces, cell geometry has been shown to be a critical
factor for death and growth.”78 In order to control cell geometry on surfaces, strategies
are needed for patterning surfaces with biosignificant functionalities. Many methods have
been developed for patterning surfaces for cell growth studies, such as photolithographic
laser ablation, photopolymerization and microcontact printing, etc. To functionalize our
biodegradable polymer surface in a designed and well controllable fashion for further cell
growth studies, we attempted to use a photolabile protection and deprotection strategy to
pattern our modified PLA copolymer substrates.

Photolabile protecting groups have great potential in both synthetic and biological
chemistry. The importance of this type of protection has been growing because of recent
advances in solid phase synthesis in combinatorial chemistry and the need to
photochemically removable probe molecules in cell biology.”80 These photolabile
groups can be cleaved photochemically without harsh acidic or basic conditions. We
think that photolabile protecting groups can also be applied to patterning polymer
surfaces with functional groups which will be beneficial to control cell geometry on
polymer surfaces. A polymer that has functionalized side chains protected by photolabile
groups is spin coated on a silicon wafer. Then, the surface is exposed to UV light through
a mask containing the desired pattern. After irradiation, the protecting groups are
removed at the exposed region, and therefore the surface is patterned with functional
groups for further biosignificant molecule incorporation. Cells can then be placed on

polymer surfaces with certain geometries to optimize cell surface interaction (Figure 29).

44

Photolabile Mask

Protecting Group /

Functionalized ifiewifiw _> rwWwe—z—i
Polymer Surface .5' . - ms!” .. . . , v ,.....'-.
(.4) (B)
Cell . .
\ /'\ B1oact1ve Groups
1’ 7 (A5.
a «$1 ; ‘imé- gl‘U" "HI I " meLx «LI
iSi W _ £1.5qu .. . H...” ”w T .. "i
(1)) (C)

Figure 29. Photolabile protection-deprotection surface patterning for cell growth. (A) Polymer thin film
with a functionalized side chain protected by photolabile protecting groups is exposed to UV light through
a mask containing the desired pattern. (8) after irradiation. protecting groups are removed in the exposed
region, (C) bioactive functional groups are incorporated in the deprotected region, (D) cells are seeded on

the polymer surface with desired geometry.

3.] Photolabile Protecting Group Selection

To choose a suitable photolabile protecting group for our copolymer substrates,
the PDLLA was tested for stability under short and long wavelength UV light. The
polymer was dissolved in dioxane, and then the same volume of H20 was added into the
solution. The polymer suspension was transfered into quartz and Pyrex tubes
respectively. After degassing, the samples were irradiated under UV light. The quartz
tube allowed the full range of UV light to pass though while the Pyrex tube cut off UV
light with wavelengths shorter than 290 nm. After irradiation, the polymers were
recovered and the molecular weight was measured by GPC. The PDLLA in the quartz

tube had a large molecular weight decrease after being irradiated for 22 hours. The

45

molecular weight of the PDLLA in the Pyrex tube did not decrease even after irradiation

for 6 days, and instead increased (Table 4).

 

 

Mn /gmol-1 Mn /gmol-1
Polymer ,1 (nm) Time (hrs) Solvent (PDI)“'b Before (PDI)"'b After

Irradiation Irradiation
PDLLA Full range 22 1:1 Dioxane/l-IZO 33,300 (1.48) 4,900 (1.24)
PDLLA > 290 144 1:1 Dioxane/HZO 41,600 (1.16) 64,700 (1.13)

 

Table 4. PDLLA stability under long and full range UV irradiation. " Molecular weight determined by

GPC. ” Polydispersity index (MW/Mn).

The above results showed that the PLA based polyester degraded when irradiated
at shorter wavelength, but no polymer degradation when irradiated at k > 290 nm.
Therefore, an appropriate photolabile protecting group should be cleaved by irradiation at
k>290 nm with a high quantum yield. Polycyclic aromatic hydrocarbons bearing
benzylic methylene groups are of good photolabile protecting groups for alcohols.“
Recently the anthraquinon-Z-ylmethoxycarbonyl (Aqmoc), Pyren-l-ylmethoxycarbonyl
(Pmoc), and 7-methoxycoumarin-4-ylmethoxycarbonyl (Mcmoc) groups have be
synthesized and tested (Figure 30). They showed high reactivity at 350 nm, which is
within the wavelength safe for our polymer substrates.

0 0 . 0 on
k 06 ° r
0 on
00 0A0... \ o
MeO O

O

Aqmoc Pmoc Mcmoc

Figure 30. Photolabile polycyclic aromatic hydrocarbons bearing benzylic methylene groups.

46

3.2 Synthesis of Aqmoc Protected PLLA-g-HP

Aqmoc was linked to the hydroxyl group on PLLA-g-HP through a carbonate
bond. The synthesis was based on a literature procedure.82 A solution of PLLA-g-HP in
CH2C12 was added dropwise into a CHzClz solution of 4-nitropheyl chloroformate and
DMAP. After stirring at room temperature for 6 hours, the polymer intermediate was
isolated by precipitation in methanol, and then redissolved in 01sz for the next step.
One equivalent of DMAP and 2-hydroxymethyl anthraquinone was added into the
polymer solution and the solution was stirred at room temperature for another 6 hours.
The final product PLLA-g—AqmocPr was precipitated in methanol for two times. The two

step reaction gives a 73% overall yield (Figure 31).

0

° 0"“ *w
H
(:le i ONOZ O i o
WOH CH2C162,hDMAP We 0 CH3C12. DMAP W0 O 0.0
L!- 0UT5 r.t.6hours

O
90% Yield 73% Overall Yield

Figure 31. Synthetic route of Aqmoc Protected PLLA-g-HP.

The protected polymer, PLLA-g-AqmocPr, was characterized by 1H NMR
spectroscopy. Nearly complete conversion of the side chain alcohol to the carbonate was
indicated by the disappearance of the methylene resonance at 3.62 ppm and the

appearance of a methylene resonance at 4.18 ppm (Figure 32). The polymer is stable in

solid form at room temperature under visible light.

47

 

 

 

 

 

C,g,h,1 f,j,k dl C a b
i t (9 J}

T" F’Yfr'rrr TY—TTTtrrrTrr

FWW l— v r l T r r r v r r 1 I v r r T l r
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Figure 32. NMR spectrum PLLA-g-AqmocPr.

 

 

The molecular weights of the polymers before and after the protecting reactions
were measured by GPC. The Mn for the starting PLLA-g-HP was 19,200 and the PDI was
1.55. After the protecting reaction, The Mn of the resulting PLLA-g-AqmocPr dropped to
11,000 and the PDI increased to 1.90. The molecular weight decrease indicates chain
scission occurred during the coupling reactions in the presence of DMAP. Similar base
catalyzed transesterification of PLA has been previously reported by Hedrick and

coworkers (Figure 33).83

48

O

O OJYYXWY‘LLO

. f

O /
N —N®
o %_ o\
O
0N @0199»
0 o

—- /
J
NQ—N\ / “‘0”
OH 0

o
o \(‘ko’n
We)? * ”5%on
o 0

Figure 33. DMAP Catalyzed PLA Depolymerization.

 

3.3 Photolysis of Aqmoc Protected PLLA-g-HP

O O
Wo/ko hv 99350 g
. 1:1 THF/H20 mi WOH + Photolysis byproduct
O

 

Figure 34. Photolysis of PLLA-g-AqmocPr.

The photolysis of PLLA-g—AqmocPr was carried out in 1:1 THF/HZO solution.
After degassing, the sample was irradiated with a high pressure mercury arc lamp (Figure
34). Initially, we used a Pyrex reaction vessel to limit the wavelength to above 290 nm.
However after 6 hours, polymer gelation indicating that the deprotected PLLA-g-HP
might be crosslinked under the photolysis conditions. To solve this problem, we filtered
out UV light < 350 nm, and carried out the photolysis under the same conditions. After 6

hours, the sample was immediately worked up and reprecipitated with cold methanol. No

49

gelation was observed and the polymer molecular weight remained constant after

irradiation (Table 5).

 

 

Time Conversio Mn /gmol-I (PDI)”" Mn /gmoI—I (PDI)”"
’1 (nm) (hrs) Solvent n( %)" Before Irradiation After Irradiation
> 290 6 1:1 THF/HZO -- 11,000 (1.90) l3,000(1.91)
> 350 2.5 1:1 THF/HzO 60.7 8,900 (1.50) 9,950 (1.75)
> 350 6 1:1 THF/HZO 83.6 8,900 (1.50) 9,688 (1.84)

 

Table 5. PDLL-g-AqmocPr deprotection under UV irradiation. “ Conversion is determined by UV-vis

spectroscopy. b Molecular weight determined by GPC. C Polydispersity index (MW/Mn).

Quantification of the photochemical conversion of PLLA-g-AqmocPr to PLLA-g-
HP was carried out by UV-Vis and 1H NMR spectroscopy. GPC was used not only for
measuring the polymer molecular weight before and after the photolysis reaction, but also
to quantitate the photochemical deprotection conversion using ultraviolet-visible
spectroscopy. Since GPC is a chromatographic separation technique, we measured the
absorbance of the polymer using the UV-Vis detector and correlated it to the
concentration of the chromophore using a calibration curve. The prerequisite for reliable
UV—Vis quantification is that the side chain chromophore strongly absorbs at a
wavelength where the polymer backbone is transparent. In our case, 70m is 234 nm for
PLLA—g-HP, while the km,“ for the Aqmoc moiety is 327 nm (Figure 35). Therefore, we
were able to quantify the amount of conversion using UV-Visible spectroscopy. The
model compound anthraquinon-Z-ylmethyl butylcarbonate was synthesized and solutions
of different concentrations were injected into GPC. By plotting absorbance at 325 nm vs.
sample concentration, a calibration curve was generated. The chromophore

concentrations on our polymer substrates before and after photolysis were compared to

50

determine the percent conversion of the photolysis reactions. When the sample was
irradiated for 2.5 hours, 61% deprotection of the Aqmoc group was observed. When the
irradiation time was increased to 6 hours under the same condition, deprotection

increased to 84%.

 

 

 

 

 

 

1.00 f PLLA-g-AqmocPr
0.80 .3
<0 :
O ‘2
g :
.9 0.60 _‘
,_ -
o :
m -t
"D 7:
<5 :
0.40 —_
030 j PLLA-g-HP
0.00 E _._.._,._ __ _ -_
250 300 350 400 450
Wavelength (nm)

Figure 35. UV-Vis spectra of PLLA-g-Aqmoc and PLLA-g-HP.

lH NMR analysis was performed by comparing the integration of the resonances
between photochemically deprotected polymer and the unreacted polymer (Figure 36).
After photolysis, the aromatic proton resonances at 8.29 ppm and 7.78 ppm, and the
methylene proton resonance at 5.15 ppm decreased indicating the cleavage of Aqmoc

group. A peak that appeared at 3.63 indicated the recovery of PLLA-g-HP. The percent

51

conversion determined by 1H NMR integration compared well with result obtained from

UV-Vis analysis.

 

 

 

 

 

 

 

 

 

 

 

 

0
WM
0 CH
H J
0
Art 0 0
CH3 a c A d e h
OM" ° 0.0
b
i k
9 O I
(a)

. . , l
e,g,h,1 f,_],l( ail c a b ,
Most AW _ ML.

a C
0M0”
b
(b)
c a bl
L- s - 1 W AW_
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 15 1.0

Figure 36. NMR spectra of PLLA-g-Aqmoc (a ) before and (b) after photolysis.

3.4 Conclusion

The Aqmoc group is a good photolabile protecting group for the side chain
hydroxyl group of PLLA-g-HP. It has been successfully linked to PLLA-g-HP through a
carbonate bond by using 4—nitropheyl chloroformate as coupling reagent.
Transesterification side reaction occurred in the presence of DMAP, which decreased the
polymer molecular weight. However, by increasing the molecular weight of the starting
PLLA-g-HP, one can still expect to synthesize PLLA-g-AqmocPr with reasonable

molecular weights. Photolysis of PLLA-g—AqmocPr was carried out with UV light (71. >

52

350 nm). The hydroxyl group is successfully deprotected with high conversion. And the
polymer molecular weight remained constant before and after irradiation. The above
experiment suggests that Aqmoc group can be an efficient photolabile group for
protecting the side chain hydroxyl group of PLLA-g-HP during chemical reactions, and it

can also be applied in PLLA-g-HP coated surface photolithographic patterning.

53

CHAPTER FOUR

4 Experimental Section.

4.1 General Considerations

1H NMR spectra were recorded on Varian Gemini—300 and Inova—300 (299.9
MHz) and Varian Unity-500 (499.9 MHz) spectrometers and referenced to residual
proton solvent signals. '3 C NMR spectra were recorded on Varian Gemini—300 and
Inova—300 (75.4 MHz) and Varian Unity-500 (125.7 MHz) spectrometers and referenced
to residual solvent signals. l1B NMR spectra were recorded on a Varian VXR—300
(160.6 MHz) and referenced using BF3.OEt2 as the external standard. Differential
Scanning Calorimetry (DSC) analyses of the polymers were performed under a helium
atmosphere at a heating rate of 10°C/min on a Perkin-Elmer DSC 7, with the temperature
calibrated using an indium standard. The reported DSC curves are second heating scans,
taken after an initial heating scan to erase thermal history, and a slow cooling scan to -
50°C. Molecular weights of the polymers were determined by gel permeation
chromatography (GPC) Waters instrument, equipped with a Waters 410 differential
refractometer detector and a Waters 960 photodiode array detector. Measurements were
obtained in THF with two PLgel 5 11 Mix columns (Polymer Laboratories) at a flow rate
of 1.0 mL/min at 35°C. Results were calibrated with monodisperse polystyrene
standards. Polished silicon wafers purchased from WaferWorldTM were cleaned by
immersion in a 70:30 volume ratio solution of concentrated H2804 (98% by mass) and
H202 (30% by mass) and rinsed in deionized water. Polymers were dissolved in toluene
(1% w/w), filtered two times through 0.2 11m PTFE filters and spin-coated onto polished

silicon wafers (1” diameter) at a spin rate of 2500 rpm for 60 s. Ellipsometric

54

measurements were obtained with a rotating analyzer ellipsometer (model M-44, J .A.
Woollarn) using WVASE32 software. The angle of incidence was 75° for all
measurements. A film refractive index of 1.50 was used for the calculation of the film
thickness.

Contact Angle Measurements. After spin-coating the polymers onto silicon
wafers, the wafers were held under vacuum at 40°C for 12 hours to ensure that all solvent
was removed from the wafers. The polymer-coated wafers were kept at room
temperature for 1 hour under a constant purge of nitrogen. Samples were heated under a
nitrogen atmosphere at a rate of 10°C/min to 160°C (15°C above the melting point of the
all polymer samples) and held for 1 minute. The samples were immediately removed
from the oven and placed on a clean surface at room temperature. Advancing and
receding contact angles were measured by dispensing a droplet of Milli-Q grade water
(18 M9 cm) at a rate of 3 “Us to a total volume of 30 11L on the surface of polymer-
coated silicon wafers. Contact angles were measured by capturing the image as a movie
with a Sanyo® B/W CCD camera (model VC8 3512). The angles were measured using
the PTAZOO software.

Cell Growth Study. The osteoblast growth studies were done at the department
of physiology by Dr. Laura McCabe group members.

Cell Culture. MC3T3-E1 osteoblasts (OBS) were plated on silicon discs
(WaferWorldTM) at a concentration of 4,400 cells/cmz. These were either cultured for 2
days for the proliferation studies or 14 days for the differentiation studies, where at this

stage OBS exhibit maximal levels of alkaline phosphatase and mineralization. Cells were

55

 

fed every two days with a—MEM containing 2mM B-glycerolphosphate (Sigma, St.
Louis, MO) and 25pg/ml ascorbic acid (Sigma).

Cell Growth and Shape. After plating for 48 hours, osteoblasts were incubated
with 10 11M BrdU (5—Bromo—2’Deoxyuridine) (Sigma, St. Louis, M0) for two hours.
Subsequently, cells were fixed with 70% ethanol. The cell culture plates were then spun
at 1500 rpm for 5 minutes to ensure that all cells remained at the bottom of the plate,
followed by a quick rinse with lXphosphate buffered solution (lXPBS). Cells were
permealized with 4 N HCl in 1XPBS, and subsequently washed gently with 1 mL of
lXPBS until the PBS in the wells reached pH=7. Osteoblasts were further incubated in
20% anti-BrdU (BD Biosciences, San Jose, CA) antibody solution, in 1XPBS, 0.5%
BSA, 0.1% Tween-20, for 45 minutes at 37°C under humid conditions. Brdu antibodies
were removed and cells further introduced to propidium iodide stain at 200 ug/mL PBS
(BD Biosciences, San Jose, CA) immediately after which nuclei of the cells were
visualized by fluorescence microscopy and recorded with a digital camera. BrdU stains
the nuclei at the S phase of cell cycle, i.e. when cells are dividing, and therefore, was
used to determine cell growth. Propidium iodide stains all nuclei.

Actin and Hoechst Stain. Osteoblasts were fixed with 3.7% formaldehyde for
10 minutes at room temperature, 2 days after seeding, and subsequently permealized for 5
minutes with 0.1% Triton X-100 in 1XPBS. Cells were blocked with 1% bovine serum
albumin for 30 minutes and subsequently incubated for 20 minutes with rhodamine-
pahlloidin (Molecular Probes Inc. Euegene, OR) and further incubated with at 10 11M

stain in deionized H20, for 30 minutes. Actin localization and Hoechst stained

56

(Molecular Probes Inc. Eugene, OR) nuclei were visualized by fluorescence microscopy
and recorded with a digital camera.

RNA Analysis. RNA was extracted, on day 14, using TRI Reagent RNA
isolation reagent (Molecular Research Center, Inc., Cincinnati, OH). Integrity of the RNA
was verified by formaldehyde-agarose gel electrophoresis. Two-step quantitative RT-
PCR was performed to verify gene expression. First strand cDNA was synthesized by
reverse transcription of 2 ug RNA, using the Superscript II Kit and oligo d(T12-18)
primers (Invitrogen Life Technologies, Carlsbad, CA). 1 ul of cDNA was amplified by
Real Time PCR, by using the SYBR Green Core Reagents (PE Biosystems, Warrington,
UK) and Taq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA) with a final
volume of 25 pl. Genes associated with osteoblast differentiation, alkaline phosphatase,
osteocalcin, and runx2, were analyzed using cyclophilin as the internal control. Real time
PCR was carried out and analyzed for 40 cycles, using iCycler software (Biorad, Hecules,
CA). Gel electrophoresis and melting curves were used to verify the integrity of a single
PCR product (amplicon).

Photolysis. The photolysis samples were prepared in 1:1 THF/1120 solution in
Pyrex tubs, and degassed by purging with N2. The irradiation was carried out by using
high pressure mercury arc lamp with a 350 nm uranium glass filter.

Solvents. Toluene, tetrahydrofuran, diethyl ether, and pentane were pre—dried
over sodium and distilled from sodium/benzophenone ketyl. Dichloromethane was
distilled from calcium hydride (CaHg). All other reagents were used as received unless

otherwise noted.

57

4.2 Synthesis

0
N011

OH
2-Hydroxypent-4-enoic acid (2-HPA).240 Glyoxylic acid monohydrate (9.60g,
0.104mol) and freshly distilled allyl bromide (18.8g, 0.156mol) were added to a stirred
suspension of zinc powder (14.2g, 0.217mol), and BiCl3 (49.2g, 0.156mol) in 120mL
tetrahydrofuran. The reaction was initially kept at 0°C and allowed to warm to room
temperature while stirring under N2 for 16h. After 16h, the reaction was quenched with
80mL of 1M HCl, and the product was extracted with diethyl ether (3x150mL). The
organic phases were decanted from the solid, combined, and washed with saturated
NaCl(aq) until the ether solution became colorless (5x20mL). The ether layer was then
dried over MgSO4, and the solvent removed to give a colorless oil which crystallized
under vacuum. Yield: 11.1 g (92%). IH NMR (300 MHz, CDCl3, 5): 5.74-5.85 (m, 1H, -
CHCHzCHzCHg), 5.16-5.28 (m, 2H, ~CHCH2CH=CH2), 4.24 (dd, 1H,
HOZCCH(OH)CH2CH=CH2), 2.42-2.66 (m, 2H, —CHCH2CH=CH2). l3C NMR (75.0

MHz, CDCl3, 5): 178.5, 131.9, 119.3, 69.8, 38.3.

WU

O

3,6-Diallylglycolide (DAG). 2-HPA (7.8g, 0.0672mol) was dissolved in 672mL
toluene (0.1M), and a catalytic amount (0.895g, 0.0047mol) of p-toluenesulfonic acid
(stOH) was added into the solution. The mixture was refluxed for 3 days with the water
removed by Dean-Stark trap. After 3 days the mixture was cooled, washed 3 times with

20mL of saturated aqueous NaHC03 and the resultant oil distilled over ZnO under high

58

vacuum at 80°C/0.01mmHg. Gas chromatography data indicates a statistical mixture of
S/S, R/S, and R/R isomers of the diallylglycolide. Yield: 3.68g (56%). 1H NMR
(300MHz, CDC13, 5): 5.70-5.83 (m, 1H, -CHCH2CH=CH2), 5.18-5.27 (m, 2H, -
CHCHgCHzCHz), 5.00 (dd, 1H, -CHCH2CH=CH2, = 6.3 Hz), 4.93 (dd, 2H, *-
CHCHZCHzCHZ, J = 7.2 Hz), 2.68-2.79 (m, 4H, -CHCH2CH=CH2). 13C NMR (75
MHz, CDCl3, 5): *166.2, 164.8, *130.6, 129.8, 121.3, *120.0, 75.8, *75.0, 36.3, *34.0.
LRMS (E1, m/z): 197 (MH+), 98, 81, 54, 41. Anal. Calcd for C10H1204: C, 61.21; H,
6.18. Found: C, 61.29; H, 6.17. *Indicates RR/SS isomer. (Note: Using a combination
of L-lactic acid and 2-HPA in the desired ratio under identical reaction conditions and
workup results in a statistical mixture of L-lactide, 3-ally1-6-methylglycolide, and 3,6-
diallylglycolide and appropriate stereoisomers.)
W

Poly(L-lactide-co-diallylglycolide) (PLLA-co-DAG). In a typical synthesis of
PLLA-co-AG, freshly sublimed L-lactide (4.16g, 28.9mmol) and distilled
diallylglycolide (DAG) (1.0g, 5.10mmol) were combined in a glass tube with tin(II)-2-
ethylhexanoate (0.068mmol) and 4-tert-butylbenzylalcohol (BBA) (0.068mmol) as the
coinitiators. The reaction tube was flame-sealed under vacuum and placed in an oil bath
at 145°C. After 20 minutes, the sample was removed from the oil bath, cooled, and
monitored for conversion. lH NMR of the crude product revealed nearly complete
conversion (>90%). Polymers were dissolved in a minimal amount of CHzClz, and
precipitated into cold methanol two times. The copolymer has 13.1% diallylglycolide
units (percentage can vary according to percentage of diallylglycolide co-monomer)

Yield: 94%. lH NMR (300 MHZ, CDCl3, 5): 5.62- 5.81 (m, 1H, -CHCH2CH=CH2),

59

 

5.10-5.17 (q, *-CH(CH3), -CHCH2CH=CH2), 2.5-2.8 (m, 2H, -CH2CH=CH2), 1.5-1.7 (d,
*-CH(CH3)). 13C NMR (75 MHz, CDCl3, 5): *169.6, 168.2, 131.4, 119.2, 72.0, *69.0,

35.1, * 16.6. *PLLA.

Won

PLLA-g-HP (HP = Hydroxypropane) In a round bottom flask, PLLA-co-AG
(1.5 g, 1.0 mmolg", 1.49 mmol) was dissolved in 50 mL of dry THF. 9-BBN solution in
THF (4.16 mL, 0.50 M, 2.08 mmol) was slowly added to the polymer solution via syringe
and stirred at room temperature for 2 h. The reaction was then cooled to 0°C and a 1 M
aqueous solution of sodium acetate (6.24 mL, 6.24 mmol) and a 30% solution of H202
(0.71 g, 6.24 mmol) were added. The mixture was stirred for 4 h. The reaction was
quenched with 2 M HCl (3.12 ml, 6.24 mmol) and the solvent were reduced by rotary
evaporation. The polymer was then precipitated from methanol. The polymer was
filtered and dried under vacuum. Yield: 85%. 1H NMR (300 MHz, CDCl3, 5): 5.08-5.20
(q, *-CH(CH3), -CHCH2CH2CHgOH), 3.62 (m, 2H, -(CH2)2CH20H), 2.04 (m, 2H, —
CH2(CH2)2OH), 1.65 (m, 2H, -CH2CH2CHZOH), 1.54-1.62 (d, *-CH(CH3)). '3 C NMR
(75 MHz, CDCl3, 5): *169.6, 168.7, 72.5, *69.0, 61.8, 27.7, 27.5, *16.6. * PLLA. Note:
the base catalyzed oxidation can be monitored by 11B NMR, the disappearance of peak at
34 ppm indicates complete conversion. Repetition of the oxidation step can fully remove

the boronic acid residue if the first oxidation does not achieve 100% conversion.

0

)K/VVWW
We

PLLA-g-MyPr (MyPr = Myristic acid propyl ester). PLLA-g-HP (0.352 g,

0.950 mmolg", 0.333 mmol), myristic acid (0.114 g, 0.50 mmol), and DMAP (8.0 mg,

60

0.066 mmol) were dissolved in 15 ml CH2C12 in a small round bottom flask. 1,3-
Dicyclohexylcarbodiimide (DCC) (0.50 mL, 1.0 M, 0.50 mmol) was then added dropwise
via syringe and the reaction mixture was stirred under N2 at room temperature. (Note:
After 15 minutes, the colorless, homogeneous solution became cloudy, indicative of
esterification). After 12 hours, the reaction mixture was filtered to remove the insoluble
dicyclohexyl urea byproduct. The filtrate was then concentrated and the polymer was
precipitated twice into methanol. The polymer was filtered and dried under vacuum.
Yield: 85%. 1H NMR (500 MHz, CDCl3, 5): 5.15-5.08 (q, *-CH(CH3), -
CH(CH2)3CO2C13H27), 4.04 (m, 2H, -CH2CO2C13H27), 2.26-2.23 (m, 2H, -
CO2CH2CI2H25), 1.95 (m, 2H, -CH2(CH2)2CO2C.3H27), 1.75-1.63 (m, 2H, -
CH2CH2CH2CO2C13H27), l.60-1.50 (d, *-CH(CH3)), 1.28 (m, 2H, -CO2CH2CH2C11H23),
1.22 (m, 20H, -(CH2)10CH3), 0.84 (t, 3H, -(CH2)12CH3). 13C NMR (75 MHz, CDC13, 5):
173.7, *169.6, 72.1, *69.0, 63.2, 34.1, 33.9, 31.9, 29.6, 29.4, 29.3, 29.25, 29.1, 27.6, 24.9,

24.2, 22.6, *16.6, 14.1. * PLLA.

0

WOW

PLLA-g-StPr (StPr = Stearic acid propyl ester). The polymer—bound DCC
coupling procedure was identical to that for PLLA-g-MyPr. Yield: 86%. 1H NMR (500
MHz, CDC13, 5): 5.18-5.5.09 (q, *-CH(CH3), -CH(CH2)3CO2C17H35), 4.05 (m, 2H, -
CH2CO2CI7H35), 2.27 (m, 2H, -CO2CH2C16H33), 1.96 (m, 2H, -CH2(CH2)2CO2C.7H35),
1.73 (m, 2H, -CH2CH2CH2CO2C.3H37), 1.60-1.51 (d, *-CH(CH3)), 1.29 (m, 2H, -

CO2CH2CH2C15H31), 1.22 (m, 28H, -(CH2).4CH3), 0.85 (t, 3H, ~(CH2)16CH3). 13C NMR

61

 

(75 MHz, CDC13, 5): 173.7, *169.6, 72.1, *69.0, 63.2, 34.2, 33.9, 31.9, 29.7, 29.5, 29.3,

29.2, 29.1, 27.6, 24.9, 24.7, 24.2, 22.7, *l6.6, 14.1. * PLLA.
WOW—W

PLLA-g-OlPr (OlPr = Oleic acid propyl ester). The polymer-bound DCC
coupling procedure was identical to that for PLLA-g-MyPr. Yield: 88%. 1H NMR (300
MHz, CDC13, 5): 5.32 (m, 2H, -CH=CH), 5.18-5.10 (q, *-CH(CH3), -
CH(CH2)3CO2C.7H33), 4.05 (m, 2H, -CH2CO2C17H33), 2.26 (m, 2H, -CO2CH2C.6H31),
1.99 (m, 6H, -CH2CH2CH2CO2C17H33, -(CH2)(CH=CH)(CH2)), 1.76 (m, 2H, -
CH2CH2CH2CO2C17H33), 1.60-1.52 (d, *-CH(CH3)), 1.45 (m, 2H, -CO2CH2CH2C.5H29),
1.28-1.25 (m, 20H, -(CH2)4(CH2)(CH=CH)(CH2)(CH2)6CH3), 0.86 (t, 3H, -(CH2)6CH3).
13C NMR (75 MHz, CDC13, 5): 173.8, *169.6, 130.0, 129.7, 72.2, *69.0, 63.2, 34.2,

31.9, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 27.2, 27.1, 24.9, 22.6, *16.6, 14.1. * PLLA.

JK/VVW
0M0

PLLA-g-LnPr (LnPr = Linoleic acid propyl ester). The polymer-bound DCC
coupling procedure was similar to that used to prepare PLLA-g-MyPr. This polymer has
been reported to be sensitive to oxygen.65Therefore the reaction was carried out in a
glove box under nitrogen, and then worked up quickly in air. The product was kept under
nitrogen. Yield: 71%. 1H NMR (300 MHz, CDC13, 5): 5.32 (m, 4H, -CH=CH-(CH2)-
CH=CH), 5.18-5.10 (q, *-CH(CH3), -CH(CH2)3CO2C17H31), 4.05 (m, 2H,
CH2CO2C17H3I), 2.78 (t, 2H, =CH-(CH2)-CH), 2.4-2.2 (m, 2H, -CO2CH2C.6H29), 2.03
(m, 6H, -CH2CH2CH2CO2C17H31, -CH2(C5H6)CH2), 1.76 (m, 2H, -

CH2CH2CH2CO2C17H31), 1.60-l.52 (d, *-CH(CH3)), 1.45 (m, 2H, -CO2CH2CH2C15H27),

62

1.27 (m. 14H. -(CH2)4<CH2)(C5H6>(CH2)<CH2>3CH3). 0.86 (t. 3H. -<CH2>6CH3>. 13C
NMR (75 MHz, CDCl3, 8): 173.7, *169.6, 130.2, 130.0, 128.0, 127.8, 72.2, *69.0, 63.2,
34.1, 33.8, 31.5, 29.7, 29.6, 29.3, 29.5, 29.3, 29.1, 29.0, 27.6, 27.2, 25.6, 24.9, 24.7, 24.2,

22.5, *16.6, 14.1. * PLLA.

0

W0 — — — —

PLLA-g-ArPr (ArPr = Arachidonic acid propyl ester). The polymer-bound
DCC coupling procedure was similar to that used to prepare PLLA-g-LnPr. This
polymer has been reported to be sensitive to oxygen.65 Therefore the reaction was carried
out in a glove box under nitrogen, and then worked up quickly in air. The product was
kept under nitrogen. Yield: 78%. 1H NMR (300 MHz, CDC13, 5): 5.34 (m, 8H, -
(CH=CH-CH2)3CH=CH), 5.18-5.10 (q, *-CH(CH3), -CH((CH2)3CO2C19H31). 4.05 (m,
2H, -CH2CO2C19H3.), 2.79 (m, 6H, -(CH=CH-CH2)3), 2.4-2.2 (m, 2H, -CO2CH2C.3H29),
2.10-1.90 (m, 6H, -CH2CH2CH2CO2C19H31, -CH2(CH=CH-CH2)3(CH=CH)CH2), 1.8-1.6
(m, 4H, -CO2CH2CH2C17H27, -CH2CH2CH2CO2C19H31), 1.60—1.52 (d, *-CH(CH3)), 1.26
(m, 6H, -(CH2)3CH3), 0.86 (t, 3H, -(CH2)4CH3). l3c NMR (75 MHz, CDC13, 6): 173.8,
*169.6, 130.45, 128.85, 128.55, 128.18, 128.17, 128.12, 127.80, 127.48, 72.1, *69.0,

63.3, 33.8, 33.51, 31.47, 29.28, 27.63, 27.17, 26.49, 25.56, 24.70, 24.17, 22.54, *16.6,

0
aw.”
O O

PLLA-g-prPr (prPr = p-nitro-phenyl propyl carbonate). To a stirred a

14.0. * PLLA.

solution of 4-dimethylaminopyridine (0.0914g, 0.748mmol) and 4-nitropheyl

63

chloroformate (0.172g, 0.855mmol) in CH2Cl2 (6mL), a 5mL solution of PLLA-g-HP
(0.4109g, 0.713mmol) was added dropwise. The reaction mixture was stirred for 5 hours
at room temperature. The solution was then concentrated, and the polymer was
precipitated in cold methanol. Yield: 90%. 1H NMR (300 MHz, CDC13, 5): 8.25 (d, 2H, -
Hm), 7.36 (d, 2H, H”), 5.15 (q, 11 H, *-CHCO2-), 4.30 (br, 2H, -CH2CH2CH2CO2-),
2.17 — 2.0 (br, 2H, -CH2CH2CH2CO2-), 1.82-1.98 (br, 2H, -CH2CH2CH2CO2-), 1.56 (d,
33H, *CH3CH-)

PLLA-g-AqmocPr (AqmocPr = Anthraquinon-Z-ylmethyl propyl carbonate)
The isolated polymer, PLLA-g-prPr was dissolved in CH2C12, and one equivalent of 4-
dimethylaminopyridine (0.078g, 0.642mmol) and 2-hydroxymethyl anthraquinone
(0.168g, 0.71mmol) were added. After stirring for 12 hours the solution was concentrated
and the polymer was precipitated in methanol Yield: 81%. 1H NMR (300 MHz, CDC13,
5): 8.29 (br, 4H, Hm), 7.78 (br, 3H, Hm), 5.15 (br, 2H, HNCH2OCO2-), 5.14 (q, 12H, *-
CHCO2-), 4.18 (br, 2H, -CO2OCH2CH2CH2-), 2.02 (br, 2H, -CO2OCCH2H2CH2-) 1.83
(br, 2H, -CO2OCH2CH2CH2-) 1.56 (d, 33H, *CH3CH). 13C NMR (75 MHz, CDC13, 5):
182.53, *169.46, *169.25, 154.71, 141.72, 134.11, 133.53, 133.29, 133.10, 132.82,
127.65, 127.14, 126.05, *72.09, *68.88, 68.14, 67.35, 66.57, 27.37, 24.07, *16.52. Note:

* From Polymer backbone.

O
“Om/OW
O O

Anthraquinon-Z-ylmethyl butyl carbonate. To a stirred a solution of 4-

dimethylaminopyridine (0.134g, 1.1mmol) and 4-nitropheyl chloroformate (0.222g,

1.1mmol) in CH2C12 (6mL), l-butanol (0.148g, 0.18mL) was added dropwise. The
reaction mixture was stirred for 5 hrs at room temperature. Thin layer chromatography
indicated the formation of intermediate 4-nitrophenyl carbonate was complete. Then, one
equivalent of 2-hydroxymethyl anthraquinone (0.238g, Immol) and an additional
equivalent of 4-dimethylaminopyridine were added in the reaction mixture. After 12
hours, the mixture was diluted with CHC13, washed with sat. NaHCOg, 1M HCl, and
dried over MgSO4. The product was purified by column chromatography. Yield: 72%. 1H
NMR (300 MHz, CDC13, 5): 8.31-8.28 (m, 4H, -HAr). 7.80-7.77 (m, 3H, HA”), 5.29 (s,
2H, -CH2OCOOCH2-), 4.17 (t, 2H, -CH2OCOOCH2-, J = 6.6 Hz), 1.69-1.66 (m, 2H, -
OCH2CH2CH2CH3), 1.41-1.38 (m, 2H, -OCH2CH2CH2CH3), 0.92 (t, 3H, -
OCH2CH2CH2CH3, J = 7.2 Hz). 13C NMR (75 MHz, CDC13, 5): 182.72, 182.64, 155.00,
142.03, 134.18, 134.14, 133.38, 133.17, 132.88, 127.72, 127.24, 127.22, 126.23, 68.42,
68.07, 30.58, 18.86, 13.59.

Photolysis of PLLA-g-AqmocPr. In a Pyrex tube, PLLA-g-AqmocPr (0.107g,
0.938mmolg", 0.1mmol Aqmoc) was dissolved in 25ml THF (no stabilizer included).
Then, 25mL deionized water was added dropwise to the polymer solution (the polymer
solution became cloudy after adding water). After the mixture was degassed by purging
with N2 for 15 minutes, the sealed sample tube was irradiated at >350 nm using a high
pressure mercury lamp for 5 hours. After irradiation, the solvent was immediately
removed by rotovap and the polymer was isolated by centrifugation followed by
reprecipitation in cold methanol. Yield: 79%. Conversion: 76% (by NMR), 83.6% (By

UV-Vis).

65

 

CHAPTER FIVE

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70

Appendix A

DSC Data for:

PLLA-co-DAG
PLLA-g-HP
PLLA-g-MyPr
PLLA-g-StPr
PLLA-g-OlPr
PLLA-g-LnPr
PLLA-g-ArPr

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78

Appendix B

TGA Data for:

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