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IMPLEMENTATION AND EVALUATION OF A MOLECULAR
STRATEGY FOR DELIVERY OF NUCLEIC ACID BASED
THERAPEUTICS

presented by
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IMPLEMENTATION AND EVALUATION OF A MOLECULAR STRATEGY FOR

DELIVERY OF NUCLEIC ACID BASED THERAPEUTICS

By

Felicia Codrea

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biochemistry and Molecular Biology

2005

ABSTRACT
IMPLEMENTATION AND EVALUATION OF A MOLECULAR STRATEGY FOR
DELIVERY OF NUCLEIC ACID BASED THERAPEUTICS
By
Felicia Codrea

Although “gene therapy” is a concept that was introduced more than there
decades ago, the successes of this method are below the expectations. The bottleneck of
gene therapy is gene delivery. The current non-viral methods used for transfection
possess low transfection efficiency and are not suitable for therapeutic use, showing high
toxicity.

A new macromolecular scaffold that combines the properties of cationic polymers
and cationic lipids was synthesized in only two steps. It contains a lipophilic part, like
lipids, and a charged amine moiety, like cationic polymers. A polymeric backbone was
synthesized through a Michael addition reaction, starting from 2(5H)-furanone and
cysteine. After purification through membrane fractionation, the backbone was
functionalized with N, N dimethylethylenediamine and octylamine. The ability of this
new carrier to mediate transfection of plasmid encoding for green fluorescent protein in
vitro was tested on COS 1 cells. The transfection conditions were optimized and an
efficiency of transfection of 6.59 % was achieved. The evaluation of the cytotoxicity of
this new carrier proved that the carrier is non-toxic. The cells proliferated up to
confluence even when the concentration was ten times higher than what was identified as

optimum for transfection.

ACKNOWLEDGEMENTS

This thesis tells the story of one chapter from the cutting edge research carried out
in Dr. Hollingsworth’s laboratory. One of the luckiest days of my life was in September
of 2002 when I met Dr. Rawle Hollingsworth. I will always be thankful for his time, the
most precious currency. I just hope that the future will prove the time that be invested in
me worthwhile. After spending three years in his laboratory, I have a different
perspective of “the chemistry of life”.

My deepest and sincere appreciation goes to Dr. John LaPres. His friendly attitude
encouraged me to speak openly about various ideas and concerns and his door was
always open for me. I would like to express my gratitude to Dr. Charles Hoogstraten, for
his guidance and suggestions that lead me on the right pathway. Dr. Shirley Owens from
the Confocal Laser Microscope facility was always there for me.

This thesis is dedicated to all my friends that help me to go through ups and
downs, here at MSU. I was always happy to count many smiles and encouraging words
around me. Dana was the “sister” I never had. Living together made us more than friends
— closer to family. Dr. Maria Zavodszky has been the Romanian island in the
department, a person who always understood and helped me with both personal and
professional issues.

Modern communication technology makes long distance relationship easier and
more effective. Throughout these years, I had constant encouragement from my brother

(the best gift my parents ever gave me). My parents are particularly thanked for their life-

iii

lasting support and for teaching me the ABCs of science. I wish I inherited some of my
grandmother’s genes, especially the ones related to the hard-working skills, strong will

and kindness.

iv

TABLE OF CONTENTS

Page
List of tables ..................................................................................................................... vii
List of figures ................................................................................................................... viii
List of abbreviations ........................................................................................................ xiii
Chapter 1 Introduction ...................................................................................................... l
1.1 Drug delivery ............................................................................................ l
1.2 Gene therapy ............................................................................................. 1
1.2.1 Nucleic acid based therapeutics ....................................................... 3
1.2.2 Nucleic acid delivery techniques ................................................... 11
1.2.2.1 Naked DNA ....................................................................... 11
1.2.2.2 Physical methods ............................................................... 12
1.2.2.3 Viral vectors ....................................................................... 13
1.2.2.4 Chemical methods ............................................................. 17
1.2.2.4.] Cationic lipids ................................................... 17
1.2.2.4.2 Cationic polymers ............................................. 28
1.3 Mechanism of uptake .............................................................................. 32
1.3.1 External barriers ............................................................................ 34
1.3.2 Internal barriers .............................................................................. 34
1.3.2.1 Endosomal escape .............................................................. 37
1.3.2.2 Nuclear entry ..................................................................... 41
Chapter 2 Design of novel carriers for nucleic acid delivery ......................................... 44
Chapter 3 Materials and Methods ................................................................................... 52
3.1 Synthesis of polymer I ............................................................................ 52
3.2 Synthesis of polymer II ........................................................................... 54
3.3 Labeling of polymer H with fluorescein isothiocyanate (FIT C) ............. 55
3.4 Cy5 labeled antisense oligonucleotide treatment .................................... 56
3.5 Evaluation of plasmid DNA transfection mediated by polymer II ......... 57
3.6 Effects of polymer H treatment on cells viability and proliferation ....... 58
3.7 Investigation of the uptake mechanism of FIT C labeled polymer II ...... 59
Chapter 4 Results and discussion ................................................................................... 61
4.1 Principle .................................................................................................. 61
4.2 Evaluation of the performance of polymers in vitro ............................... 67
4.2.1 Evaluation of the ability of polymer I to deliver
antisense oligonucleotide to MEF cells ......................................... 67

4.2.2 Evaluation of plasmid DNA transfection mediated by

polymer H ...................................................................................... 71
4.2.3 Effects of polymer II treatment on cells viability and
proliferation ................................................................................... 82
4.3 Characterization of the mechanism of uptake ......................................... 92
4.4 Conclusions ............................................................................................. 98
Bibliography ................................................................................................................... 100

vi

LIST OF TABLES

Table 1. Cationic lipids classified according to structural properties ......................... 24

Table 2. Examples of combinations of polymer Ilzplasmid ratios ............................... 71

vii

Figure 1.

Figure 2.

Figure 3.

Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.

Figure 9.

Figure 10.

Figure 11.
Figure 12.
Figure 13.

Figure 14.

Figure 15.
Figure 16.
Figure 17.

Figure 18.

LIST OF FIGURES

A schematic representation of plasmid with sequences needed for
effective expression and replication. ................................................................ 4

Mechanism of action of antisense oligonucleotides ........................................ 6

Triplex forming oligonucleotides binding in the major groove of the

duplex ................................................................................................................ 8
Diagrammatic structure of RN A/DNA chimera ............................................... 8
Overview of RNAi pathway. .......................................................................... 10
Gene silencing by RNA interference (RNAi). ................................................ 11

Introduction of foreign DNA into the HSV-l genome by recombination. ..... 14

Structures of cationic lipids used as materials for gene therapy. .................... 19
Structures of neutral lipids used as materials for gene therapy. ..................... 20
Structures and compositions of some commercially available cationic

lipids formulations for nucleic acid transfer Lipofectin, Lipofectamine ....... 21
Graphic representation of cationic lipids. ....................................................... 22
Liposome structure .......................................................................................... 24
Schematic representation of four major liposome types ................................ 27
Immobilization of antibody on PEG-liposomes by (a) direct coupling to

the liposome surface and (b) coupling to the terminal ends of the PEG

chains. ............................................................................................................ 28
Cationic polymers used for gene delivery polylysine, polyethyleneimine 30
Cationic polymers - polyamidoarnine structures. ........................................... 31
Structures of chitin and chitosan. .................................................................... 32

The dilemma of the DNA — polymer complex: how tightly should DNA
be complexed? ................................................................................................ 33

viii

Figure 19.
Figure 20.

Figure 21.

Figure 22.
Figure 23.

Figure 24.

Figure 25.

Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.

Figure 31.

Figure 32.

Figure 33.

Figure 34.

Figure 35.
Figure 36.

Figure 37.

Cell entry mechanisms for cationic liposome/DMA complex ........................ 35
Phase change of lipid structure from lamellar phase to hexagonal phase ....... 38
Condensed multilamellar lipids incorporating DNA and hexagonal

arrangement of lipids with DNA ..................................................................... 38
Mechanism of uptake and release of plasmid DNA from the complex. ......... 39
The PEI proton sponge hypothesis ................................................................. 40
BNL.Cl-2 hepatocytes transfected with nuclearLacZ/PEI appear as

twins ................................................................................................................ 42
Hypothetical mechanism of nuclear import of plasmid DNA by importin

transport receptors ........................................................................................... 43
Overview of vectors used in clinical trials in October 2000. .......................... 44
Overview of vectors used in clinical trials in October 2005 ........................... 45
Transmitted images of MEF cells (a) control, (b) treated with SuperFect. 46
Schematic illustration of the EPR effect principle .......................................... 48
General representation of a cationic lipid. ...................................................... 49
Model for the general platform that allows incorporation of hydrophobic

chain, and charged groups in the same molecule ............................................ 51
pEGFP-C2 encodes a red-shifted variant of wild-type GFP ........................... 57
Polymerization of cysteine with furanone via a Michael addition leading

to synthesis of the backbone polymer ........................................................... 62
The 1H NMR spectra of the cysteine and furanone mixture before

polymerization ................................................................................................ 63
The 1H NMR spectra of the polymeric backbone ........................................... 64
Functionalization of the free carboxylic groups .............................................. 65
Functionalization of the polymeric backbone by coupling amines to free

carboxylic groups ............................................................................................ 66

ix

Figure 38.
Figure 39.
Figure 40.

Figure 41.

Figure 42.

Figure 43.

Figure 44.

Figure 45.

Figure 46.

Figure 47.

Figure 48.

Figure 49.

Figure 50.

FT-IR spectrum of polymer II ......................................................................... 67
MEF treated with polymer I at various concentrations. .................................. 69
Series of confocal images. ............................................................................. 69
MEF cells treated with Cy5 labeled antisense oligonucleotides in
combination with a solution of backbone polymer. ........................................ 70
COS 1 cells before treatment, phase contrast image ....................................... 72
COS 1 cells transfected with plasmid encoding for green fluorescent
protein, transfection mediated by the polymer II at 24 hours after
treatment ......................................................................................................... 72
COS 1 cells — no treatment ............................................................................. 73
The upper panel represent the overlay of the phase contrast image and the

fluorescent image of cells treated with a 20:1 of polymer II:DNA ratio,
the bottom panel the corresponding phase contrast images of the upper
panel at 72 hours after treatment ............................. . ....................................... 73

C081 cells — (a) the phase contrast image and (b) the corresponding
fluorescent image, and (c) the overlay of the phase contrast image and the
fluorescent image at 72 hours after treatment with a polymer II:DNA
ratio of 20:2. ................................................................................................... 74

C081 cells — (a) the phase contrast image and (b) the corresponding
fluorescent image, and (c) the overlay of the phase contrast image and the
fluorescent image at 72 hours after treatment with a polymer II:DNA
ratio of 20:3 ..................................................................................................... 74

Comparison of the overlay of the phase contrast image of three treatment
conditions (a) 20:1 polymer II:DNA ratio, (b) 40:2 polymer II:DNA ratio,
and (0) 60:3 polymer II:DNA ratio. ............................................................... 75

C081 cells (a) the phase contrast image, (b) the corresponding
fluorescent image, and (c) the overlay of the phase contrast image and the
fluorescent image at 72 hours after treatment with Lipofectamine 2000
and 100 ng plasmid. ....................................................................................... 76

COSl cells (a) the phase contrast image, (b) the corresponding
fluorescent image, and (c) the overlay of the phase contrast image and the
fluorescent image at 72 hours after treatment with Lipofectamine 2000
and 200 ng plasmid. ....................................................................................... 76

Figure 51.

Figure 52.

Figure 53.

Figure 54.

Figure 55.

Figure 56.

Figure 57.

Figure 58.

Figure 59.

Figure 60.

Figure 61.

Figure 62.
Figure 63.
Figure 64.

Figure 65.

C081 cells (a) the phase contrast image, (b) the corresponding
fluorescent image, and (c) the overlay of the phase contrast image and the
fluorescent image at 72 hours after treatment with Lipofectamine 2000
and 300 ng plasmid. ....................................................................................... 77

C081 cells 72 hours after treatment with 20:1 ratio polymer II:DNA at a
lower magnification under the fluorescence mode. The distribution of
cells exhibiting fluorescence is even. ............................................................. 78

C081 cells 72 hours after treatment with Lipofectamine 2000 and 100 ng
plasmid at a lower magnification under the fluorescence mode, (a) many
cells exhibit fluorescence and (b) no cells are fluorescent. ........................... 79

C081 cells 72 hours after treatment with 20:1 polymer II:DNA ratio,
20:2 polymer II:DNA ratio, and 20:3 polymer II:DNA ratio at a lower
magnification under the fluorescence mode. ................................................. 80
Evaluation of GFP expression 72 hours after treatment. Comparison of
polymer H with Lipofectamine 2000 treatment. ............................................ 81
Evaluation of GFP expression 72 hours after treatment. Comparison of
different polymer II treatments. ..................................................................... 82
Phase contrast images of €081 cells with treatments of polymer H or
Lipofectamine 2000 at various concentrations ............................................... 85
Total number of cells grown under treatment with polymer H or
Lipofectamine 2000 and 100 ng plasmid ........................................................ 87
Total number of cells grown under treatment with polymer H or
Lipofectamine 2000 and 200 ng plasmid ........................................................ 88
Percentage of the number of cells that are lysed under Lipofectamine
2000 or polymer H treatment ......................................................................... 90
Percentage of the number of cells that are larger under Lipofectamine
2000 or polymer 11 treatment. ........................................................................ 91
Critical aspartic residue in the 0 subunit of the F0. ........................................ 93
FITC labeled polymer solution. ..................................................................... 94
C081 cells, no treatment. .............................................................................. 94

C081 cells treated with FIT C labeled polymer 11, immediately after
treatment. ....................................................................................................... 95

xi

Figure 66. C081 cells treated with FIT C labeled polymer II. ......................................... 95
Figure 67. C081 cells treated with FITC labeled polymer H in the presence of

DCCD inhibitor. Images recorded 10, 15 and 20 minutes after treatment
respectively. ................................................................................................... 96

xii

List of Abbreviations

CMV — cytomegalovirus

NLS — nuclear localization signal

RES — reticuloendothelial system

RISC — RNA-induced silencing complex

RT — reverse transcriptase

LTR - long terminal repeats

HSV - herpex simplex virus

AAV — adeno-associated viruses

DOPE — dioleoylphosphatidyl ethanolamine

DOPG — 1,2-dioleoyl—sn-glycerol-3-(phosphor-rac-(1-glycerol))
DOTMA — [2,3—bis(oleoyl)propyl]trimethyl] ammonium chloride
DOTAP — 1,2-diacyl-3-trimethylammonium propane

DOPC - dioleoylphosphatidyl choline

DMRIE -1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide

DOSPA - 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-
propanaminium trifluoroacetate

DOGS — dioctadecyl amido glycyl sperrnine
SUV -— small unilamellar vesicles

LUV — large unilamellar vesicles

GUV - giant unilamellar vesicles

MLV — multilamellar vesicles

PEG — polyethylene glycol

xiii

PTD - protein transduction domain

PEI — Polyethyleneimine

PAMAM - polyamidoamine

NLS — nuclear localization signal

EPR - enhanced permeability and retention effect
NMR - nuclear magnetic resonance

FI-IR - Fourier Transform infra red spectroscopy
EDC - 1—ethyl-3-dimethylaminopropylcarbodiimide
FITC -— fluorescein isothiocyanate

MEF — mouse embryonic fibroblasts

PBS - phosphate buffer saline

DMEM — Dulbecco's Modified Eagle Medium
DCCD - dicyclohexylcarbodiimide

MTT —3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

xiv

Chapter 1

Introduction

1.1 Drug delivery

Although the development of efficient drugs has recorded remarkable progress
during the last century, one of the most challenging issues in modern pharmacology
remains their efficient delivery to the target. The goal of developing drug delivery
systems is to simultaneously increase the therapeutic activity of the drugs and reduce their
toxicity and side effects. Drug delivery is an interdisciplinary research area, comprising
findings from biology, chemistry, biochemistry, physics, pharmacology, and physiology.
The development of effective drug delivery systems that can transport a drug safely to the

target is the “holy grail” of current pharmaceutical research‘.

1.2 Gene therapy

Solving the DNA structure (Waston, Crick and Franklin, 1953) opened a new era
in sciencez. The understanding of how the genetic material is stored, passed to new
generations and what is encoded offered a better understanding of function of the genes

and the effects of mutations. French Anderson3

, considered “the father of gene therapy”,
used the term to describe the delivery of a gene(s) to cure diseases. If one could replace

the bad copy of a gene with a good one, the cause of the disease would be treated, as

opposed to the effects of it as in traditional medicine. The completion of the human

genome project also has a great impact on identification of gene function and selection. In
particular the identification of genes that are involved in diseases has an immediate
application. This leads to development of nucleic acid based drugs which include
plasmids, antisense oligonucleotides, aptamers, ribozymes, DNAzymes and small
interfering RNA (siRNA). Oligonucleotide based therapies target either the DNA and in

this case is called anti gene therapy, or target the RNA and it is called antisense therapy.

The spectrum of diseases that could be treated using this approach is broad. Gene
therapy was initially introduced to treat genetic diseases like cystic fibrosis, severe
combined immunodeficiency (SCID/ADA), hemophilia, sickle cell anemia, and
Duchenne muscular dystrophy" to mention only a few. In 1990 the first protocol for gene
therapy that targeted the adenosine deaminase deficiency obtained the FDA approval. The
concept was later extrapolated to treat acquired diseases such as several types of cancer,
neurodegenerative diseases like Alzheimer’s and Parkinson’ss, cardiovascular diseases
(restenosis, arteriosclerosis) and infectious diseases (AIDS, hepatitis B). For cancer
treatment, gene therapy is applied as replacement of missing tumor suppressor gene
and/or inhibition of oncogenes6. The first gene therapy product was approved to the
market, in China, in 2003. It is a cancer drug, which using a viral method interferes with

the tumor suppressor gene p537, thus stimulating apoptosis.

Although very appealing, introducing foreign DNA into cells is a more difficult
task than it was first thought. To date, Achilles’ heel of gene therapy remains gene
deliverys. After the completion of the Human Genome Project, the challenges of gene
therapy are not identification of genes that need to be replaced, but the effective delivery

of nucleic acids into the nucleus of cells.

Gene therapy can interfere with gene expression either in vivo or ex vivo. The ex
vivo approach assumes that malfunctioning cells are collected from the patient, or a
compatible donor, to prevent rejection by the immune system if foreign cells are used,
and manipulate them outside the body. The genetic material is introduced into cells in
vitro, followed by insertion of cells back into the patient. This approach is suitable for
diseases that affect the blood system, since cells from blood or bone marrow can be
removed from the patient and grown outside the body in culture media. When the genetic
disease is localized to a tissue like lung in case of cystic fibrosis, or skeletal muscle in
case of Duchenne muscular dystrophy, the in vivo approach is more appropriate. This is a
better alternative when cells cannot grow in culture in sufficient quantities or when the re-
implantation process is difficult. The major challenge of in vivo gene therapy is that there

is no safe and efficient gene delivery systems.

1.2.1 Nucleic acid based therapeutics

Recent advances in molecular biology and molecular genetics have resulted in a
new concept in treating diseases. Intracellular delivery of genetic material is the key step
in gene therapy9. Plasmids are high molecular weight circular double stranded DNA
which contains the gene of interest (Figure 1). Besides the gene of interest, the promoter
and the enhancer sequence are compulsory for regulating gene expression. The promoter
is essential in initiation of transcription; it contains the recognition sites for the RNA
polymerase. A commonly used promoter is extracted from cytomegalovirus (CMV). The
enhancer is introduced into the plasmid construct for the same purpose that exists in

endogenous DNA, to enhance the production of the gene of interest. One hundred times
3

more protein can be obtained using a proper enhancer“). At a molecular level, plasmids
can be considered prodrugs since they require transport into the nucleus where the
transcription/translation machinery of the cell synthesizes the protein that the gene
encodes for. The problems with delivery of plasmid DNA to cells are related to some
properties of plasmid DNA. First, the size of plasmid DNA is 3-30 Kb, too big to cross
the cellular membranes. The charge of DNA is highly negative due to the phosphate
backbone. The nucleic acids are susceptible to enzymatic degradation both outside and
inside the cell. One of the major challenges for the plasmid molecule is to enter the
nucleus. In dividing cells nuclear entry takes advantage of the disruption of the nuclear
membrane. This is why it is much easier to transfect cells in logarithmic growth, in cell
culture. In non-dividing cells, the plasmid DNA should pass through the nuclear pore.
There are proteins responsible for transport through nuclear pores and they contain

nuclear localization signal (NLS), usually a sequence of basic amino acids.

Intron
Promoter

Enhancer it

  

cDNA
DNA

plasmid
3-30 kb

 
     
   

Bacterial
amplification Poly A

Figure 1. A schematic representation of plasmid with sequences needed for effective

expression and replication. (modified from Walther et al., 1996)

The discovery of non-conventional drugs like RNA interference (RNAi)11 or
antisense RN A”, where the oligonucleotides are the therapeutic agents, has expanded the
concept of gene therapy from introducing genetic material into cells to introduction of
molecules that have the ability to disrupt gene expression. Currently gene therapy means
not only the replacement of a dysfunctional gene, but use of nucleic acid transfer, either
RNA or DNA, to treat or prevent a disease. Novel drugs like RNA interference and
antisense RNA introduced new challenges in drug delivery. The cellular delivery of
nucleic acids raises the same problems as the delivery of other drugs: the danger of
clearance of the drug due to the reticuloendothelial system (RES) and difficulty to cross
the cellular membrane. In the absence of a carrier molecule, even small oligonucleotides
are not able to diffuse through the hydrophobic cellular membranes due to the high
negative charge of the phosphate backbone. Besides the fact that the nucleic acids should
avoid the immune system, the enzymatic degradation of the nucleic acids is a problem
both outside and inside the cells”. A vector to transport the nucleic acids to target is

required.

Oligonucleotides are short segments of single stranded DNA that are used for
disruption of one protein’s function. Antisense oligonucleotides are designed to interact
in a complementary fashion with a determined sequence of mRNA and inhibit its

translation into protein (Figure 2).

RNA Initiation A Translation

 

—_—AAAAA —>4>

 

 

L .5
HM! ’_' HH! Notranslational
+ — a?) 5 5! complex formed
1“ Steric inhibition
+ _ AAAAA 3 —‘~" ”\AAAAA 3 E! of elongation

Figure 2. Mechanism of action of antisense oligonucleotides. Antisense oligonucleotides
(blue) form a hybrid with their RNA target (black). There are at least three ways that the
formation of the oligonucleotide: RNA duplex might prevent translation of a mRNA
resulting in protein production 1) The oligonucleotide: RNA duplex may form a
substrate for endogenous RNase H, leading to mRNA cleavage. 2) The oligonucleotide:
RNA duplex may prevent the productive assembly of the ribosomal complex (red)
preventing translation. 3) The oligonucleotide:RNA duplex may arrest a ribosomal
complex already engaged in translation leading to a truncated protein (green). (modified

from Dagle et al., 2001)

The prevention of the protein synthesis can occur at various levels, inducing
degradation of mRNA, inhibition of ribosome binding or arrest of translation. For
example, if the antisense sequence is designed to interact with the mRNA at the
spliceosome level the inhibition of protein expression will be accomplished”. Another
alternative is to degrade the message by activating the RNAse H, the ribonucleotide

6

responsible for mRNA degradation, thus altering the synthesis of the protein that that
specific mRNA encoded for15 . In designing the oligonucleotides, one important factor is
the length, which usually varies from 12 to 28 bases. If the sequence is too short, it may
lose in specificity. It may self-hybridize or achieve secondary and tertiary structure if too

long.

One advantage of using oligonucleotide based gene therapy is that the size of the
prodrug is diminished compared to the plasmid DNA. Another advantage is that,
depending what process is targeted to be inhibited, the requirement for nuclear
localization is not necessary. One of the major disadvantages consists of their high
instability; the single stranded oligonucleotides are very susceptible to enzymatic
degradation. To increase the oligonucleotides stability, several modifications have been
proposed. The most common are the replacement of the phosphodiester backbone with
phosphorothioate, peptide-nucleic acids (PNA)16 or 2-O’-methyl modifications to name
just a few. Replacing the oxygen atom with sulfur in phosphorothioate molecules confer
greater stability, since these molecules are not substrates for ribonuclease. The methyl
phosphonate modification is beneficial since it introduces hydrophobicity, thus enhanced

diffusion through cellular membranes.

Antigene oligonucleotides have a portion of a gene as a target, resulting in a
triplex formation”, and thus the inhibition of the transcription process (Figure 3). The

nuclear delivery of antigenes is compulsory.

S'Pu 33),:

3%
mfi
31%: 515;

Figure 3. Triplex forming oligonucleotides binding in the major groove of the duplex.

(modified from Dagle et al., 2001)

To achieve a greater stability of oligonucleotides inside the cellular milieu,
chimeric molecules have been synthesized. RNA/DNA systems consists of 25 nucleotides
targeted to the gene of interest, connected through a 5 bp GC clamp and two hairpin ends

of T loop. To increase the stability the RNA is 2’-O-methyl modified18 (Figure 4).

GC clamp mismatch 2'0 methyl-RNA

"‘ 'nlmfi)

 

 

...E'”ll”“l‘"H”

3' .5'|<-— Homologous region “I

I *

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T-loop
Figure 4. Diagrammatic structure of RNA/DNA chimera. (modified fiom Liang et al.,

2002)

Other versions of nucleic acid therapeutics include ribozymes, DNAzymes and
aptamers. Ribozymes are RNA molecules that have catalytic activity, cleaving the mRNA

molecules”. Two common types of ribozymes used for inhibiting the expression of

8

specific genes are hairpin ribozymes and hammerhead ribozymes. Similarly, the
DNAzymes are analogs of ribozymes but they have increased stability because the ssRNA
backbone is replaced by the dsDNA. Zang and collaborators injected DNAzymes directed

against VEGF into tumor to inhibit angiogenesiszo.

Aptamers are either single stranded or double stranded oligonucleotide molecules
that interact with specific molecular targets. Aptamers targeted against the coagulation
factor IXa21 and HIVl trans activation responsive element22 prove the successes of this

technology.

Small interfering RNAs (siRNA) are short fragments of oligonucleotides, 21-23
bp with 2 overhanging bases on the 3’ end. They degrade the mRNA that encodes for a
specific protein so that the protein cannot be synthesized. After administration, the siRNA
molecules integrate into the RNA-induced silencing complex (RISC), where the antisense
sequence binds to the complementary target mRN A (Figure 5). The mRNA is degraded
by a nuclease similar to RNase H. The RISC complex is still being studied intensively,
since its function and structure are not completely clear yet. The siRNAs can also be used
as primers for the generation of new dsRNA by RNA-dependent RNA polymerase
(RdRp). If longer sequences of dsRNA are delivered to the cells, those are cut by an
enzyme called DICER into the 21-23 oligonucleotide segments”. Among the advantages
of using siRNA instead of plasmid DNA to interfere with gene expression are the size of
siRNA and its site of action into the cells’. The relationship between the size of the drug
and the efficiency of the delivery is well known: the bigger the size the more difficult the
uptake“. SiRNA delivery to cytosol should to result in more efficient interference with

gene expression since translocation to the nucleus is an extremely inefficient process. In

9

this case, the integration into the host cell genome is not an issue. SiRNA, being dsRNA,

also has better resistance to ribonuclease.

dsRN
; W

l /.

TIIIIIEL 'IIIIEIIL 'EEEIIIIL siRNAs

RdRp / \ / RISC

\

 

 

 

 

 

 

|||||| * AAAAAAA mRNAmet
InIEmJIEE'ImImJI _ — - Immfi“

Figure 5. Overview of RNAi pathway. Intracellularly synthesized or exogenously
administered dsRNA is cleaved by the enzyme Dicer into 21—25 nucleotide siRNAs.
siRNAs become associated with the RNA-induced silencing complex (RISC), which uses
the antisense strand of the siRNA to bind to and cleave the target mRNA. The siRNAs
can also be used as primers for the generation of new dsRNA by RNA-dependent RNA
polymerase (RdRp). This newly formed dsRNA can then also serve as a target for the

Dicer enzyme. (modified from Shuey et al., 2002)

Several variants of generating the siRNA are known25 . Long dsRNA can be
delivered to cells and rely on the DICER enzyme to cut it into fragments of 21-25
oligonucleotides long (Figure 6). In vitro chemical synthesis, or in vitro transcription of
optimum fragments of siRNA are other alternatives. In vivo synthesis of short hairpin

RNA inserted into a plasmid and transfected to cells in culture is another option.
10

I II

m Chemical plasmid”

synthesis/ in vitro Retrovims
dSRNA transcription Adenovims

In vitro
transcription
Dicer JImIO

JEEEEIIF

siRNA

 

 

Figure 6. Gene silencing by RNA interference (RNAi). RNAi is triggered by siRNAs,
which can by generated in three ways. (I) Long double-stranded RNA molecules are
processed into siRNA by the Dicer enzyme; (11) chemically synthesized or in vitro
transcribed siRNA duplexes can be transfected into cells; (III) the siRNA molecules can
be generated in vivo from plasmids, retroviral vectors or adenoviruses. (modified from

Kurreck, 2003)

1.2.2 Nucleic acid delivery techniques

1.2.2.1 Naked DNA

It was shown that direct injection with naked DNA induces gene expression.
When naked DNA solution in 5 % sucrose was injected into skeletal muscle of mice, the

reporter gene that was encoded in the plasmid DNA was expressed for several days, even

11

weeksz". This type of expression is accessible only to a limited number of organs, and the
muscle is one of them. Recently, efficient delivery of naked DNA to kidney, after
intravenous administration, was reported 27 . The exogenous DNA was detected
extracellularly 10 minutes after administration at the target organ, and after 30 minutes,
intracytoplasmatic and intranuclear. The reporter gene B-galactosidase was detected for
up to 35 days. According to the official site of the Journal of Gene Medicine, as of

October 2005, 16 % of gene therapy clinical trials involve naked DNA”.

1.2.2.2 Physical methods

29.30 31.32

Physical methods like electroporation , microinjection, sonication , and
biolistic particle delivery require high equipment cost. Electroporation consists of
creating pores into cellular membranes under an electric field. The method is well known
for delivering nucleic acids in vitro, but the cell mortality is very high. Nucleofection33 is
a new technique based on electroporation. The genetic material is delivered directly to the

nucleus“.

Particle bombardment was first introduced in late 19808, and consists of gold
microparticles coated with DNA. An inert gas under high pressure is used to deliver the
DNA coated particles in vivo. The drawback of this method is that the gold particles
cannot be eliminated and remain in the body”. This technique could find immediate
application to deliver nucleic acid to skin, superficial tumors, and muscle or ex vivo gene

therapy. Electrical or mechanical techniques for nucleic acid delivery are in general

12

difficult to be setup in a clinical environment, require specific training of the personnel,

and are very invasive.

1.2.2.3 Viral vectors

The best systems that deliver genetic material into cells remain viruses”. Viral
vectors are biological systems derived from naturally evolved viruses capable of
transferring their genetic materials into the host cells. Viral vectors are modified viruses,
tailored for the designed purpose. In recombinant viruses 37, the genes responsible for
virus replication are substituted by the gene(s) of interest (i.e., therapeutic genes). This is

illustrated in Figure 7.

13

- Foreign DNA

A
v

 

Cut and insert
foreign DNA
Plasmid with
viral gene
___m____
Introduce into
cell with wild-
type virus
Recombination
___m
Wus with foreign
gene and inactivated
viral gene

Figure 7. Introduction of foreign DNA into the HSV-1 genome by recombination.

(modified from Latchman 2001)

Viruses have evolved genetically to infect cells. However, their use as drug
delivery agents has several drawbacks because during the same evolutionary time line,
higher organisms developed immune mechanisms for defense to combat the viral

8

infection3 . For example, even for viruses depleted of their genome, the viral capsids

include proteins that are toxic or highly immunogenic. Also, they have limited loading

14

capacity and may be a carcinogenic hazard due to insertional mutagenesis 39. Drug
delivery viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes

simplex viruses and lentiviruses‘o.

Retroviruses have their genome expressed as RNA. Their name “retro” comes
from using an enzyme, reverse transcriptase (RT) to convert their genetic information into
DNA in the infected cell. Three proteins are encoded in their genome, gag, pol and env,
with LTR (long terminal repeats) at both ends. The LTR are important in integration of
viral genome into host’s genome“. One severe disadvantage in using retroviruses for
gene therapy comes from their ability to infect cells during a specific phase of the cell
cycle — mitosis”. This may be a drawback in cancer therapy, since most tumor cells
contain cells that are in the resting phase — Go. By replacing the viral genes with the

gene(s) of interest, modified retroviruses can be used as gene carriers.

Lentiviruses are retroviral vectors that can infect both dividing and non-dividing
cells”. The best known lentivirus is the human immunodeficiency virus (HIV), which
contain coding sequences for six other proteins besides gag, pol and env. One of them,

the tat protein, is responsible for transport to the nucleus in non-dividing cells.

The advantage of using adenoviruses as gene carriers is that the adenovirus
genome consists of double stranded DNA“, and it can infect both dividing and non-
dividing cells“. Also, by depleting the coding sequence of the viral genome responsible
for integration of viral genome into the host genome, no integration of the genetic

material is acquired“.

15

Herpex simplex virus (HSV) is a double stranded linear DNA virus, which is
modified in order to be used for gene delivery. It has a large genome, approximately 152
kb, encoding 81 viral genes 47 , with 43 coding sequences being non essential for
replication in vitro. The possibility of insertion of multiple genes, about 30 kb of foreign
genes, makes HSV one of the largest viral vectors“. Another advantage of HSV is the

ability to infect non-dividing cells”.

Adeno-associated viruses (AAV) are ss DNA viruses which infects both dividing
and nondividing cells. They are named AAV because they require a helper virus for
replication, which can be either adeno-virus or herpes simplex virus. Their genome
encodes for two genes, cap and rep, which are flanked by inverted terminal repeats that
mark the end of the genome”. The cap gene encodes viral ‘capsid, the coating protein,
whereas the rep gene is responsible for proteins involved in replication and integration
into the host’s genome. If integration into the host’s genome occurs, the end result will be
stable transfection, which enables persistent gene expression. The integration into the
human genome occurs at a specific location in chromosome 19“. If the rep and cap genes
are replaced with therapeutic genes, AAVs can be used as viral vectors to transport DNA
at the desired location. One problem arises though: if AAV is rep negative, the
integration in the host genome occurs randomly, generating insertional mutagenesis.

Another drawback of AAV is that the capacity of the virus is small, only 4 kb.

Alternatively, non-viral methods for gene delivery have been developed. Non-
viral vectors can avoid many of the problems encountered with viral vectors. Among the
advantages of using non-viral vectors for gene delivery are the ability to be administered
repeatedly with little or no immune response, production at large scale with high

16

reproducibility at acceptable costs and generally good stability in time. The main

disadvantages are low transfection efficiency and toxicity.

1.2.2.4 Chemical methods

The association of DNA with a cationic carrier helps by compacting the DNA and
reducing the negative charge. Cationic carriers (polymers or lipids) can be used to ferry
nucleic acids to cells and also to counterbalance the negative charges that appear due to
the phosphate backbone. The interaction between the nucleic acids and cationic carriers

results in a condensed form of DNA that shows better stability against nucleasessz.

1.2.2.4.] Cationic lipids

Chemical methods to improve the DNA delivery into cells rely on development of
positively charged vehicles, the most popular ones being liposomes. Anionic liposomes
also have been used, but their efficiency is very low. Divalent metal ion solutions help to
bring together the anionic liposomes and plasmid DNA into a ternary complex53. One
example of a mixture of anionic lipid l,2-dioleoyl-sn-glycerol-3-(phosphor-rac-(1-
glycerol) DOPG, and neutral lipid dioleoylphosphatidyl ethanolamine DOPE“, was used
to deliver a plasmid encoding for green fluorescent protein into CHO-Kl cell line, in the

presence of Ca2+ ions.

Liposomes are colloidal vesicular structures formed from (phospho) lipid bilayers,

and can be used as drug delivery agents since they provide a cavity where the drug is

17

protected. Felgner"s and collaborators, in 1987, introduced liposomes as DNA delivery
vectors. They showed that the DNA interacts spontaneously with [2,3-
bis(oleoyl)propyl]trimethyl] ammonium chloride (DOTMA) and a neutral lipid
dioleoylphosphatidyl ethanolamine (DOPE). Practical experience demonstrated the
importance of the neutral lipids into efficiency of transport. They observed that DNA
loaded liposomes fuse with COS7 cells in culture and that the complex mediates the
nucleic acid delivery into cells. In Figure 8 are presented the chemical structures of some
molecules that are able to form cationic liposomes, the molecules known as cationic

lipids”, along with some neutral lipids57 (Figure 9).

18

Cationic lipids

DOBAB \L
Br/

Cl’ C —
DO mafia __

DOTAP ATko

 

 

 

 

DC—Chol

I.

NH
/ + \/\N
H

 

Figure 8. Structures of cationic lipids used as materials for gene therapy. DOTMA: [2,3-
bis(oleoyl)propyl]trimethyl] ammonium chloride; DODAB: dioctadecyldimethyl
ammonium bromide; DOTAP: 1,2-diacyl-3-trimethylammonium propane; DC-Chol: 3[N-

(N‘N-dimethylaminoethane)-carbamoyl] cholesterol.

l9

O
3 H 0W
DOPE HaN wo‘g-O‘XLWW
O _
e

O
a I H ‘ WWN
DOPC /T\/‘o—g—o ‘\
O —
e

Chol

 

Figure 9. Structures of neutral lipids used as materials for gene therapy. DOPE:
dioleoylphosphatidyl ethanolamine; DOPC: dioleoylphosphatidyl choline; Chol:

cholesterol; (modified from Segura et al., 2001)

The commercially available transfection reagent Lipofectin from Invitrogen
contains a 1:1 mixture of DOTMA and DOPE”. Other commercially available products
for this purpose are Lipofect-AMINE reagent, a 3:1 (w/w) liposome formulation of the
polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarbor.amido)ethyl]-N,N-dimethy‘.~1.
propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl

phosphatidylethanolamine (130%)” (Figure 10).

20

Lipofectin

 

r
DOTMA 3 I" —
30
6 H 0
WWW
DOPE Hano‘g‘o‘Xf‘Ln/VMW
% O ‘—

' Lipofectamine
0 W
+
OW

NH;
DOSPA
5 craco; A

NH 3+

0
a H OWVVW
DOPE H3N \Wo-SELOW
O ——
6

0
Figure 10. Structures and compositions of some commercially available cationic lipids
formulations for nucleic acid transfer Lipofectin and Lipofectamine. (adapted from

Schuber et al., 1998)

If we analyze the structures of the most commonly used lipids we notice that all
possess the same elements: a head group connected by a linker to a hydrOphobic anchor60

(Figure 11).

21

Oleoyl —— O I +
N—
Oleoyl — o |
¢ b 4——-> 4 >

Hydrophobic Linker I Head group I
anchor .

 

 

 

 

 

 

 

Figure 11. Graphic representation of cationic lipids. (modified from Chesnoy et al., 2000)

The choice of lipid in cationic lipids is between double chain hydrocarbons and
cholesterol“. Single chain hydrocarbons form micelles in solution and are known as
surfactants. Their application in gene delivery is limited by their toxicityéz. Cholesterol
was used successfully as a hydrophobic anchor and it was found to confer rigidity to the
lipid bilayer. Two examples of cationic lipids containing cholesterol residues as a

hydrophobic anchor are DC-cholesterol and GL-67.

Double chain hydrocarbons are the most popular hydrophobic residues used in
cationic lipid formulations. Saturated acyl chain lengths vary from 10 carbons (lauryl), 12
(myristoyl), 14 (palmitoyl) and 16 (stearoyl). Oleoyl residue (C18:1) is the most
commonly used unsaturated hydrocarbon chain, although two double bonds (C1812
lynoleyl) and three double bonds (C18:3 lynolenyl) are also common. The unsaturated
acyl chain is thought to enhance membrane fluidity. One of the drawbacks of using
molecules that contain double bonds is their limited shelf life, since the double bonds can

be oxidized easily.

22

The linker is the part of the molecule that connects the hydrophobic anchor with
the head group. The linker group is an important determinant of the (bio) degradability of
the cationic lipid; depending on the chemical bond that connects the linker, the catalytic
activity of various hydrolytic enzymes like esterases or peptidases can degrade the lipid.
For the cationic lipids that contain two hydrocarbon chains, the most commonly used
linker molecule consists of a glycerol residue (e. g., DOTMA, DOTAP). When
cholesterol derivatives are responsible for hydrophobicity, a spacer of 3-6 carbon atoms is

inserted between the linker and the head group”.

A lot of variation in structure and properties can be introduced using various
cationic head groups. Quaternary ammonium groups, primary, secondary or tertiary amine
groups introduce the positive charge that is responsible for electrostatic interactions with
the highly negatively charged phosphate backbone of the DNA molecule. For example,
monocationic molecules like N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-
hydroxyethyl) ammonium (DMRIE) and N-[l-(2,3 dioleyloxy)propyl]-N,N,N-
trimethylammonium chloride (DOTMA), as well as polycationic molecules like
dioctadecyl amido glycyl spermine’ (DOGS), and 2,3-dioleyloxy-N-[ 2
(sperminecarboxarnido) ethyl ] - N , N, -dimethyl-1 - propanaminiumtrifluoroacetate
(DOSPA) are used extensively. The multication groups, like “T-shaped” spermine, are

more efficient than the monocation ones.

As a summary, cationic lipids are amphiphilic molecules, with double
hydrocarbon chains or cholesterol derivatives responsible for the hydrophobic part, while
the hydrophilic part can carry various charged groups. Various combinations are possible
and some examples are presented in Table 164. Some of the acronyms in the table are

23

mentioned in the previous paragraph while DC-chol stands for 3[N-(N‘,N'-
dimethylaminoethane)-carbamoyl] cholesterol and Lipid 67 is cationic lipid amphiphile

(GL-67) consisting of a cholesterol anchor linked to a spermine head group“.

Table 1. Cationic lipids classified according to structural properties

 

 

 

 

, , Hydrophobic part
Hydrophllrc part
double chained lipids cholesterol derived lipids
monocationic DMRIE, DOTMA DC-chol
polycationic DOGS, DOSPA Lipid 67

 

 

 

 

 

Liposomes are colloidal vesicular structures formed from (phospho) lipid bilayers,
and can be used to some extent as drug delivery since they provide a cavity where the
drug is protected. Depending on size, liposomes are known as small unilamellar vesicles
(SUV, <100 nm), large unilamellar vesicles (LUV, 100-500 nm) and giant unilamellar
vesicles (GUV, >1 am)“. Considering lamellarity, liposomes can be unilamellar or
multilamellar vesicles (MLV, 01-10 am)“. Liposomes have been used as carrier systems
since they confer some advantages of delivery, compared to naked drugs. Hydrophobic
drugs are difficult to be administered intravenously, but they can be solubilized into the
liposome’s phospholipids bilayer. Also, hydrophilic drugs can be encapsulated into

vesicle’s cavity (Figure 12).

24

Phospholipid vesicle

Hydrophilic Mam}
drug N.S\?;§~iw‘/h’%water

g” f g Hydrophobic

Figure 12. Liposome structure. The phospholipid bilayer can transport hydrophobic drugs

while hydrophilic drugs can be encapsulated inside the aqueous core.

It was shown that liposomal drug formulation is beneficial for increasing
circulation time, since the vesicle protects the drug fiom enzymatic degradation. In a slow
release mode, the concentration of the therapeutic agent is constant over a longer period
of time. The active dose that the tissue is exposed to is also smaller, diminishing the toxic
effect of certain drugs. For example, it is well known that, the anticancer drug
doxorubicin has side effects on heart muscle. By encapsulating the doxorubicin into a
liposome the direct exposure of heart to the cytotoxic drug is diminished. DOXIL is a
liposomal formulation of doxorubicin that is manufactured by Alza Corporation. Doxil
was approved in June 1999 for treatment of Kaposi’s sarcoma. Another advantage of .

liposomal drug delivery is the ability to direct the drug to a specific target.

Four types of liposomes are known: conventional liposomes, cationic liposomes,
targeted liposomes and stealth liposomes (Figure 13). Conventional liposomes, also

known as “naked liposomes”, consist only of a phospholipid bilayer and are either neutral

25

or negatively charged. They are not chemically modified, so nothing protects them from
phagocytes. These formulations usually carry drugs that target macrophage cells (Kupffer
cells) in the liver or spleen. For example, delivery of antimicrobial agents loaded into
conventional liposomes to macrophages is a potential application in case of an
infection“. To control the circulation time of liposomes, they were chemically modified
with polyethyleneglycol (PEG)69’ 70. The PEG chains become hydrated and thus provide
steric protection, blocking the enzymes and nucleophiles, and also limiting diffusion of
encapsulated drug into liposome, thus creating a “stealth liposome’m. A half-life of
approximately 48 hours in terms of circulation time was achieved in humans for PEG-

ylated liposomes.

Cationic liposomes have both interior and exterior surfaces positively charged.
They were designed for delivery of genetic material. The positive charges from the
cationic lipid interact with negatively charged nucleic acid molecules, thus condensing
the DNA into a more compact structure. The complex ensures DNA protection and
promotes cellular internalization. Cationic liposomes can successfully deliver their load
but they are not stable, have low loading capacity, are subjected to leaking and have a

limited storage life”.

26

Conventional

 

Figure 13. Schematic representation of four major liposome types. Conventional
liposomes are either neutral or negatively charged. Sterically stabilized (‘stealth’)
liposomes carry polymer coatings to obtain prolonged circulation times.
Irnmunoliposomes (‘antibody targeted’) may be either conventional or sterically
stabilized. For cationic liposomes, several ways to impose a positive charge are shown

(mono-, di- or multivalent interactions). (modified from Storm, 1998)

Targeting liposomes to different organs or tissues was achieved by using specific
antibodies or antibody fragments (like Fab’ or single chain antibodies). These liposomes
are thus also known as immunoliposomes. These fragments are attached to liposomes’
surface to promote target site binding. The primary application of targeted liposomes is
the delivery of cytotoxic anticancer drugs. A variation of immunoliposomes coated with

PEG, to increase their circulation time, is also possible. The antibody fragments can be

27

attached directly to the liposome surface (which may provide steric hindrance to antigen
binding), or to PEG molecules (which do not give a steric hindrance problem)73 (Figure
14). Targeted liposomal gene delivery can be achieved also by attaching different

fragments that bind to extracellular receptors, facilitating receptor-mediated endocytosis.

74,75,

It is known that cancer cells can be targeted using folate receptor transferrin receptor

76’77'78and LDL receptor. By chemically attaching these kinds of ligands to liposomes,

preferential tissue or tumor targeting can be achieved.

 

Figure 14. Immobilization of antibody on PEG-liposomes by (a) direct coupling to the
liposome surface and (b) coupling to the terminal ends of the PEG chains. (modified from

Klibanov et al., 1992)

1.2.2.4.2 Cationic polymers

Another fairly successful way to deliver cargo into cells are the so called “trojan

” 79' 80. Natural polypeptides like HIV-1 TAT protein, Antennapedia

peptides
homeodomain and the herpes simplex virus 1 DNA binding protein VP22 have been
shown to enter cells when added to culture media. These proteins contain a motif, the

protein transduction domain (PTD) that is rich in basic amino acids lysine and arginine
28

and is responsible for internalization. Highly cationic peptide sequences like
polyarginine81 and polylysine 82 (Figure 15) have been synthesized after this model and
were successful in nucleic acid delivery but have limitations including iterative synthesis,
and small size associated with low capacity of protection the genetic material from

nucleases.

Polyethyleneimine (PEI) (Figure 15) was shown to successfully transfer nucleic
acids into COSl cells, but one of its major limitations is the cytotoxicity 83. Other
chemical methods use specifically designed activated dendrimers that possess precisely
defined size and shapem’ss. Dendrimers have a tridimensional spherical architecture, with
branches radiating from a central core and terminating with charged amino groups. One
of the advantages of synthesizing dendrimers is their monodiSpersity. An example of a
widely used dendrimer is polyamidoamine (PAMAM) 8" (Figure 16). Their tedious
synthesis and low protection capacity doesn’t make them the best candidates for gene
delivery. Examples of commercially available sixth generation dendrimeric structures are

Superfect and Polyfect (Quiagen, Valencia, California) used for in vitro gene delivery.

29

Polyamino acids "“2

Polylysine
R o
H
N
o
11
o R H
N
H2N OH
O n
Polyethylene irrrine
H
«M5
n
H2N

Figure 15. Cationic polymers used for gene delivery (polylysine, polyethyleneimine).

(modified from Segura et al., 2001)

30

Polyanridoamine (PAMAM)

Figure 16. Cationic polymers - polyamidoarnine structures. (modified from Segura et al.,

2001)

Natural polymers have been used also for gene delivery because they are
biodegradable and ensure better compatibility with cells. One example is chitosan87 ,
obtained from deacetylation of chitin which is found in crab’s exoskeleton (Figure 17). At
an appropriate ratio, the positively charged chitosan interacts electrostatically with

negatively charged DNA and generates a positively charged complex that mediates the

nucleic acid uptake into cells”. One of the limitations in using chitosan for gene therapy

31

in humans is that chitosan was reported to cause hypocholesterolemiasg. In addition its
structural complexity makes its purification and functionalization difficult to accomplish.

Significant “batch-to-batch” variations depend on the source.

OH OH OH
0 0/
H0

32. it. it.
cu,

EH3 CH3
Chitin
0H 0H 0H
/
mommo
NH2 ""2 "'12
Cllitosan

Figure 17. Structures of chitin and chitosan.

(modified from http://dalwoo.com/chitosan/structure.htm )

1.3 Mechanism of uptake

The association by electrostatic interactions between nucleic acid therapeutics and
the cationic carriers results in complexes that ensure a tight compaction and protection of
the DNA. Subsequently, these positively charged complexes bind to the cell surface and
are taken up by endocytosis. Once the endosome is formed and the cargo crosses the
cellular membrane, the hydrophobic site of lipids acts synergistically with polycationic
residues to release the complex from the endosome. The efficient escape from the

endosome results in release of the nucleic acids into the cytosol, followed by nuclear
32

uptake. The interaction between drug and carrier should be strong enough to allow a
complex to be formed yet weak enough to enable the drug to dissociate from the
complex, once it has reached its target. The final product, the protein expressed after the
reporter gene allows the evaluation of this multistep transfection process. Yokoyarna90
proposed a model for how a cationic carrier accomplishes the task to deliver plasmid
DNA to cells. The cationic carrier-DNA complex is able to adhere to the cell surface
through electrostatic interactions, followed by endocytosis of the complex. Once the
complex or the DNA released from the complex escapes from the endosome, it

translocates to the nucleus and allows transcription to initiate. A graphic representation of

this model is presented in Figure 18.

Plasmid JW cationic carrier,
DNA | l
complex formation

loose orl . I
dissociated “8‘“

BAD for uptake and for evasion good for uptake and for evasion

 

 

 

from enzymatic degradation from enzymatic degradation
good for EAD fl.” ti
transcription 31159“? 011

Figure 18. The dilemma of the DNA — polymer complex: how tightly should DNA be
complexed? For the cationic DNA-polymer complex a tight complex will be favorable for
cellular uptake and provide protection against enzymatic degradation, and a loose

complex will allow release of DNA from the complex in order to be transcribed.

(Modified from Yokoyama, 2002)

33

1.3.1 External barriers

In order to reach the target, the active component of a drug must overcome several
biological barriers. The immune system has developed evolutionarily to guard against
foreign material spreading throughout the body. One of the most common ways of
systemic administration is intravenous injection. The interactions between the therapeutic
agent and blood compartments, serum proteins and other cellular elements like
erythrocytes cannot be disregarded. If the DNA carrier complex interacts with serum
proteins (opsonins) that mark the complex for clearance by the reticuloendothelial system,
it is easy to realize that once destroyed, the DNA will not get to the target and obtain the
desired therapeutic effect. The liver and the kidney are the organs responsible for
detoxification of the organism, and after degradation of the complex, these are the places

where the therapeutic DNA ends up.

1.3.2 Internal barriers

An efficient gene delivery system should protect the nucleic acid from

degradation, both outside and inside the cell.

For both viral and non-viral vectors, the intracellular barriers consist mainly of
cellular membranes, intracellular trafficking, and the nuclear membrane. Cellular entry,
internalization, is considered to be achieved mainly through endocytosis, though other

alternatives like membrane fusion have also been considered.

In case of plasmid DNA complexed with either cationic polymers or cationic

lipids, the endocytotic mechanism is considered responsible for cellular uptake”. For
34

cationic lipid complexes, the lipoplexes, an alternative mechanism, membrane fusion,

was also proposed92 (Figure 19).

cationic liposome ...-

 

 

Figure 19. Cell entry mechanisms for cationic liposome/DMA complex. Endocytosis

versus membrane fusion”. (modified from Smyth Templeton, .2002)

Once the free DNA or DNA complex enters the cell, in order to be efficient it has
to reach its final target, the cell nucleus. For some eukaryotic cells, the distance from
cellular membrane to the nucleus can range from a few um, up to 100 pm in larger cells.
The cytoplasm is not a bag that contains the cellular organelles and metabolic products
but is a complex system, with a highly branched network of rnicrofilaments, microtubules
and intermediate filaments. The cytoplasm’s consistency resembles a gel-sol system more
than an aqueous solution. It was reported that protein concentration inside the cytosol is
up to 100 mg/mL 94. Lucaks and collaborators examined DNA mobility through the
cytoplasm, and they found that large DNA fragments are not able to passively diffuse to
the nucleus. When DNA fragments of about 2000 bp and macromolecules larger than
2000 kDa were microinjected in HeLa cells and fibroblasts, their movement through

cytoplasm was not detectable. This result suggests that plasmid DNA movement through
35

cytoplasm by physical diffusion is very limited”. It was not determined if the efficient
transport of free DNA to the cell nucleus takes place using the cytoskeleton network and

molecular motors96.

After internalization of the DNA complex via endocytosis, several alternatives are
possible for the fate of the DNA. One is that the DNA complex is entrapped into the
endosome, and is never released. Another alternative is that the early endosomes mature
into late endosomes, the DNA escapes from the endosome by membrane destabilization
and enters the cytoplasm. Once in the cytoplasm the plasmid DNA has to dissociate from
the complex and find its way to the nucleus97 before being degraded by cytoplasmic
nucleases. It was suggested that the plasmid DNA is released from the cationic polymer
complex 98 , but not from cationic lipid complex” , through transfer reactions. The
cytoplasm contains DNA-binding proteins and RNA-binding proteins that interact with
free DNA/RNA. This interaction could promote stabilization of the DNA and protection
from nucleases. It is also possible that this DNA protein interaction should mediate
interaction of nucleic acids with microtubules or actin-based motors’oo. It is known that
free DNA does not bind motor proteinslm. When entry to the nucleus is facilitated by
disruption of the nuclear membrane the timing is important also. The DNA should be able

to survive enzymatic degradation until the appropriate phase of the cell cycle, mitosis.

Zabner and collaborators found by gold electron microscopy that in the case of
cationic lipid mediated DNA delivery the lipid/DNA complex accumulates in the
perinuclear regionloz. Huang and collaborators tried to prove the hypothesis that the
lipid/DNA complex should escape from the endosome before mixing with the lysosome.
When lipid/DNA complex was microinjected into the nucleus, the gene expression failed

36

to occur, since the DNA was not accessible to the transcription complex. The third
alternative for the fate of DNA/carrier complex, if the complex is entrapped into the
lysosome the DNA will be degraded by the lysosomal enzymes, and the expected

therapeutic effect will fail to happen.

1.3.2.1 Endosomal escape

Most of the cationic lipid based methods for nucleic acid delivery consist of
mixture of lipids. The lipid complex contains also a neutral amine, a hexagonal phase
forming lipid, dioleoylphosphatidylethanolamine (DOPE), which is expected to facilitate
the escape from the endosome by membrane destabilization at acidic pH103 . It was shown
that at lower pH a phase transition from lamellar to inverted hexagonal phase resulted in
destabilization of endosome membrane with release of the nucleic acid cargo into the
cytoplasm. When the neutral lipid DOPE was replaced by DOPC the effect of
destabilization of endosomal membrane was not seen. The structure of DOPE with a
small polar head and a large hydrophobic tail allows it to form micelle like structures,
inverted hexagonal phase104 (Figure 20). Cholesterol had also been used as a co-lipid or
neutral lipid, but the complexes formed with cholesterol were less efficient compared to

DOPE containing carriers’os.

37

 

WW _., 85
I! 38158 ggg [fill 959!!!” % é
Figure 20. Phase change of lipid structure from lamellar phase to hexagonal phase.

(modified from Monkkonen etal., 1998)

Safinya and collaborators studied the structure of complexes formed between
cationic lipids and nucleic acids'06. X-ray diffiaction studies had shown that one possible
structure is similar to a sandwich, with nucleic acids in between sheets of cationic lipid
bilayers. A model is presented in Figure 21. Under cellular conditions, due to differences
in intracellular compartments, some lipids undergo phase transitions. One common
structure that is adopted by the neutral lipid DOPE at low pH is the inverted hexagonal

phase. This phase transition may play a role in DNA release from the complex.

   

Figure 21. Condensed multilamellar lipids incorporating DNA and hexagonal
arrangement of lipids with DNA. An inverted hexagonal phase of cationic liposome-DNA

complexes related to DNA release and delivery. (modified from Koltover et al., 1998)

38

Xu and Szoka proposed a model for destabilization of the endosomal membrane
in which the anionic lipids that face the cytoplasm from the endosome, through a flip-flop
mechanism will pair with cationic lipids from the cationic liposome/DNA complex
(Figure 22). This pairing results in displacement of the DNA from the cationic lipid and
release of the DNA into the cytoplasm. Displacement of the cationic lipid from the DNA

prior to its entering the nucleus is critical for gene expressionm.

X Cationic lipid
. V Plasmid DNA

 

Figure 22. Mechanism of uptake and release of plasmid DNA from the complex. (Step 1)
After electrostatic interaction with the cell membrane, cationic liposome/DNA complexes
are endocytosed. (Step 2) In the early endosome, membrane destabilization results in
anionic phospholipid flip—flop. (Step 3) The anionic lipids diffuse into the complex and
form a charge neutral ion pair with cationic lipids. (Step 4) The DNA dissociates from the

complex and is released into the cytoplasm. (modified from Xu et al., 1996)

39

In case of cationic polymers, carriers that contain many amine groups like
polyethylene imine (PEI) or other polyamines, the proton sponge mechanism was

proposed to be responsible for escaping the endosome108 (Figure 23).

Emknmons
H+ CI-
“mun

pH=13

“mu!

 

pH=45-sn

Figure 23. The PEI proton sponge hypothesis. (modified from Remy et al., 1998)

The cationic polymers have the capacity to buffer the influx in protons that
acidifies the endosome from the pH 7.4 — 7.5 to 4.5 — 5.0. The influx of protons brought
by endosomal ATPase is coupled with an influx of chloride ions. The influx of water is
also increased and the result is an increase in osmotic pressure inside the endosome. The
endosomal compartment swells and bursts, releasing its content into the cytoplasmlog.
Interestingly, if for cationic lipids the dissociation of the complex prior to entry to the
nucleus is required, in the case of cationic polymers, it was shown that gene expression

was obtained even when complexes cationic of polymer/DNA were injected into the

nucleusl 10.

40

1.3.2.2 Nuclear entry

Nuclear entry represents the major barrier for nucleic acid therapeutics. In case of
small molecules, oligonucleotides can passively diffuse through the nuclear membrane. In
case of plasmid DNA there are two alternatives to reach the nucleus, one is to pass
through nuclear pores and the other is to take advantage of the nuclear membrane
disruption during mitosis. Since 1980, it is known from microinjection experiments, that
nuclear membrane represents a major limitation to gene expression. Plasmids
microinjected into the nucleus were able to express the gene whereas the plasmids
microinjected into the cytoplasm did not express the gene'”, and not more than 0.1 —
0.001 % of cytosolic microinjected plasmid DNA was transcribed. In another
microinjection experiment, with the same number of plasmid copies injected in the
cytoplasm and the nucleus, the gene expression for plasmid microinjected into cytoplasm
was about 3 % of what was reported for plasmids microinjected directly into nucleusm.
These experiments suggest that passing the nuclear membrane is the rate-limiting step in
gene expression. It is well known that the nuclear membrane had been developed
evolutionarily to enclose the genetic material and keep it safe from foreign material that
could result in mutations. Recently Ludtke’s group reported that the plasmid DNA is
localized into cytoplasm in divided cells, even after microinjection into the nucleus’”.
Thus, the exclusion of foreign genetic material from the nucleus is another limiting factor
in achieving the desired gene expression, and should be considered in addition to
enzymatic degradation. One example of expressing plasmid in dividing cells is the
expression of reporter gene in cells that look like twins (Figure 24), a clear indication that

the plasmid origin is from the mother cell that had divided into two daughter cells1 ’4.

41

 

Figure 24. BNL.C1-2 hepatocytes transfected with nuclearLacZ/PEI appear as twins.

(modified from Zuber et al., 2001)

The transfer of plasmid DNA to the nucleus is regulated by the nuclear pore
complexes, which restrict passive diffusion to molecules no larger than 40 — 50 kDa. The
passage of larger molecules through nuclear pores is an active process, requires energy,
ran-GTP gets hydrolyzed to Ran-GDP, a protein complex is formed from importin a/B,
proteins that contain nuclear localization signal (NLS) bound to the plasmid DNAHS. A
schematic representation of a possible nuclear import mechanism is presented in Figure

25.

42

"Ar—,A\

5565143443“ ......
Nuclear pore - NLS-oligopeptide

Cytosol “a” A complex - Ran-GTP
= Ran-GDP
\A/ \ D m DNA
«Kw

Figure 25. Hypothetical mechanism of nuclear import of plasmid DNA by importin
transport receptors. DNA is covalently attached to a NLS or bound to a NLS containing
protein, such as transcription factor. The complex binds to importin in the cytoplasm and
translocates into the nucleosol. Following nuclear entry, the importin-Ran GTP complex
is recycled back to the cytoplasm where Ran-GTP is displacedfrom the complex upon the

hydrolysis of GTP. (modified from Lechardeur et al., 2002)

43

Chapter 2

Design of novel carriers for nucleic acid delivery

The percentage of clinical trials for gene therapy involving viruses to deliver the
nucleic acids decreased fiom 72.4 % in October 2000 to 50 % in October 2005. Charts
presenting vectors used in clinical trials in 2000 and 2005 are presented in Figure 26 and

Figure 27.

 

Gene therapy protocols by vector Viral vectors

Retrovirus 35.8% (n=212)
Adenovirus 27.7% (n=164)

,.
. V “31 “cm“ Poxvirus 6.2% (n=3 7)
. . Adeno-assoc virus 2.2% (n=13)

O Lipofectron Herpex simplex virus 0.5% (n=3)
0 Naked DNA ,

Non-Viral vectors
0 Gene gun Lipofection 13% (n=77)

Naked DNA 9.3% (n=55)
. Others Gene gun 0.8% (n=5)

Rna transfer 0.8% (n=5)

 

 

 

 

Figure 26. Overview of vectors used in clinical trials in October 2000. (adapted from http:

/ /www.wiley.co.uk/genetherapy / clinical 0116.

 

Vectors Used in Gene Therapy Clinical Trials

Relrovirus 25‘; (n- 72)
Adenovrrus 25“. (ne27l)
Naked/Plasmid DNA I61. (n=l74)
Lipolechon 8 o (r1493)

Pox virus 716- (n—'76l

Vacrima vrrus o 3‘ . (rt—67)
Herpes srmplex Virus 3 3'3. (rt-36)
Adena-associated vurus 3 l"-. ln=33l
RNA transfer I 2‘1. (n43)

Others II 5c (n—2l)

N/C 3 9". (n'—42)

 

 

 

 

Figure 27. Overview of vectors used in clinical trials in October 2005. (adapted from

www.wiley.co.uk/genmed/clinical)1 17

Because of irnmunogenic responses, toxicity, and the threat of insertional
mutagenesis, the trend has been to switch to develop safer, non-viral methods. Non-viral
vectors can avert many of the problems encountered with viral vectors. Among the
advantages of using non-viral vectors for gene delivery are the ability to be administered
repeatedly with little or no immune response, the production at large scale with high
reproducibility at acceptable costs and generally good stability in time. The main

disadvantages are low transfection efficiency and toxicity.

As described above, there is a plethora of drug delivery systems; however, each is
limited by significant shortcomings. Cationic liposomes can successfully deliver their

load but they are not stable, have low loading capacity, leakage is not uncommon and

45

118

limited storage life A commercially available dendrimer product, SuperFect (Qiagen,

CA), is commonly used for transfections, but it can cause cell lysis (Figure 28).

 

Figure 28. Transmitted images of MEF cells (a) control, (b) treated with SuperFect. The

cells are destroyed by SuperFect treatment.

The limitations of using short peptides for gene delivery come from the iterative
synthesis, small size associated with low capacity of protection the genetic material from
nucleases. The use of natural polymers is limited by the fact that their structural
complexity makes their purification and functionalization difficult to accomplish. They
have significant “batch-to-batch” variations, depending on the origin. The current non-
viral methods used gene delivery show low transfection efficiency and are not suitable for
therapeutic use. Development of new delivery agents that have better compatibility with
cells, exhibiting lower toxicity and also better efficiency is necessary for turning the gene

therapy into a successful therapeutic method.

46

There are a few features that were found to be important in delivering nucleic
acids. The molecular weight is one of them. Drugs that have molecular weight less than
40 Kda are susceptible to clearance from the systemic circulation due to renal filtration. If
gene therapy targets a tumor, the enhanced permeability and retention effect (EPR) can be
used to achieve higher drug concentrations at the tumor site. The advantage of coupling
the drugs with high molecular weight polymers consists of accumulation of the

”9. This effect is characteristic of tumor

complexes at the tumor site due to the EPR effect
tissues, is also known as passive targeting. There are two aspects related to this effect.
First, the tumor vasculature allows the macromolecules with a MW of 50 kDa or even
higher to enter the tumor. Second, the lymphatic system, that is responsible for the
drainage of macromolecules from normal tissues, is not as efficient in tumors. Not only
can macromolecules reach the tumor site, but also they remain there for a longer period of
time (more than 100 hours), since the removal of macromolecules from the tumor site is

120. Low molecular weight compounds are able to reenter systemic

not as efficient
circulation, since they can diffuse through the biological barriers. A schematic

representation of the EPR principle is presented in Figure 29.

47

 

 

Normal tissue
anflpuufl Tissue cells

...
Blood . .

capillary
5&3
$22)”

0
Lymphatic
W U capillary

iii r
l

it???

 

 

 

Tumor tissue

 

Cancer cells

004
r——\
-
L.._J

' -
\_)
F‘fi
-

Blood
capillary

 

 

an;
i a

 

 

 

[11

i

@— Large molecules (proteins, polymers) 0 Small molecules

2%?

Figure 29. Schematic illustration of the EPR effect principle. Angiogenesis and enhanced
vascular permeability of tumor capillaries and impaired or missing lymphatic clearance of

macromolecules result in accumulation of macromolecules (polymers) in tumor tissue

(modified from Ulbrich et al., 2004)

Another important characteristic of molecules that mediate nucleic acid delivery is

charge; positive charge seems to be important. Cationic polymers like polyethyleneimine,

48

polyarginine, polylysine, cationic liposomes of various compositions and natural
polymers like chitosan and DEAE dextran, all have in common the positive charge. It was
reported that anionic liposomes were also used to deliver nucleic acids to cells, but they

were successful only when they formed a ternary complex with calcium ions.

Hydrophobicity is another characteristic that some of the non-viral vectors have
in common. Cationic lipids contain a cationic head and a lipid tail connected by a

linker122

(Figure 30). It is believed that the hydrophobic tail mediates membrane fusion.
The ratio between the charged part and the non-polar part is about ranges between (30-

40):(70-60).

Etch—«— Hydrophobic part

Figure 30. General representation of a cationic lipid. (modified from Trenchant et al.,

2004)

If we analyze the structures of the most commonly molecules used as transfection
reagents, we see that there is a lot of diversity among the structures of efficient delivery
systems, various functional groups seems to play a role in efficient delivery of genetic
material to cells. Three physico-chemical features are important for delivery: charge
(cationic polymers and cationic lipids have positive charge), hydrophobicity (cationic
lipids have non-polar functionalities) and size. Size of the efficient drug delivery systems
can vary from 1 kDa (small peptides like polyarginine, polylysine, and cationic lipids) to
25 kDa (Polyethyleneimine) and up to 500 kDa (DEAE-dextran, chitosan). The goal of

gene therapy is to develop a vector that can be used to deliver nucleic acids to treat
49

human diseases. Biocompatibility of the carriers and their metabolic products plays a

major role in developing safe solutions to this issue.

Preliminary studies in our lab resulted in development of a platform that permits
incorporation into a single molecule of different functional groups. The properties that
were shown to be important for nucleic acid delivery are charge (positive charge) and
hydrophobicity. A schematic representation of a molecular scaffold that allows
incorporation of various side chains is presented in Figure 31. Our laboratory developed a
synthetic scheme for a biodegradable platform that can be functionalized via amidic
bonds with molecules that contain positive charges and molecules that have a
hydrophobic side chain. The platform consists of a polymeric backbone that that contains
free carboxylic groups. The relative proportion in which these specific moieties are
introduced is easy to control. By controlling the reaction conditions (temperature, reaction
time) the molecular weight of polymers can be tailored at will. Membrane fractionation
can be used to obtain fractions of molecules that fall in a particular range. The
biocompatibility issue is addressed by having biodegradable linkages — the amide bond is
a substrate for many proteases. This mechanism may be involved in releasing the nucleic

acids from the carrier, by degradation of the polymer.

50

.. .__a_ a

X16 gatively
charged gr cup

 

“U »
hydrophobic positively
chains charged groups

Figure 31. Model for the general platform that allows incorporation of hydrophobic chain,

and charged groups in the same molecule.

The elements of design of a novel non-viral nucleic acid delivery platform are
positive charge and biocompatibility, and

molecular weight, hydrophobicity,
biodegradable linkages. A fine balance of all these elements would ensure the success in

delivering plasmids to cells.

51

Chapter 3

Materials and methods

All chemical materials were obtained from Sigma-Aldrich Company and were
analytical grade, unless otherwise noted. NMR measurements were made on a Varian
VXR 300 MHz Spectrometer with deuterated water as a solvent. The FT-IR spectra were
collected with a Nicolet 710-FT-IR spectrometer. The images for the evaluation of the
uptake of antisense oligonucleotides mediated by polymer I and the characterization of
the uptake mechanism of polymer II were collected using a Zeiss LSM 5 Pascal laser
confocal microscope. For the evaluation of efficiency of transfection of plasmid DNA
mediated by polymer II and the cytotoxicity of polymer II a Nikon inverted microscope
was used. The plasmid encoding for green fluorescence protein, pEGFP-C2 was a gift

from Dr. John Lapres’ lab.

3.1 Synthesis of polymer I

A polymeric material that is positively charged has been synthesized. The
synthesis takes place in two steps. First a polymeric backbone is synthesized according to
the reaction scheme presented in Figure 33. After purification of the product, the second
step consists of functionalization of the polymeric backbone. The reaction scheme of

backbone functionalization is underlined in Figure 36.

52

Cysteine (0.01 moles, 1.21 g) and sodium hydroxide (.012 moles, 0.48 g) were
dissolved in warm Mili-Q water (2 ml) in a 50 ml round bottom flask. The reaction
mixture was kept on ice. After adding the 2(5H) furanone (0.01 moles, 0.84 g) cooled on
ice, the solvent was removed with a rotary evaporator at 70°C for three hours. It is
important that the water is removed for good yield of the Michael addition reaction. The
reaction mixture was heated on an oil bath at 100°C overnight, followed by 7 hours at
140°C. The reaction was monitored by 1H NMR. After the polymeric backbone was

purified, the second step of the reaction was done as follows.

The high molecular weight fraction (described below) of polymeric material
(1x10'4 moles, 0.02 g) was dissolved in 1ml of Milli-Q water and 1-ethyl-3-
dimethylaminopropylcarbodiirnide (EDC) (2 x104 moles, 0.0041 g) was added. The pH
was adjusted (final pH=3) using hydrochloric acid. A corresponding 1 x10'4 equivalents
of N,N-dimethylethylenediamine (0.0113 g) was added to the mixture. The reaction
mixture was incubated at room temperature for 24 hours. The reaction was stopped by
adding sodium carbonate (1 x104 moles, 0.0135 g). The polymeric material was further

purified.

Purification of the backbone polymer was done using biogel p10 exclusion
chromatography. The polymeric material from the first reaction step (0.2 g) was dissolved
in Milli-Q water (1 ml), and then loaded into the biogel p10 column (3x80 cm) with water
as eluant. Five ml of polymeric fractions were collected and 0.2 ml samples were loaded
into a 96 well plate. Absorbances were measured using a Quant Universal Microplate
Spectrometer at a wavelength of 220 nm. High, medium and low molecular weight

polymer fractions were identified and collected together, following the absorbance

53

reading. The solvent was removed with a rotary evaporator and the polymeric fractions
were lyophilized for 24 hours. Another technique used for polymeric materials
purification was selective precipitation using acetone/water solvent systems. The
backbone polymer (0.2 g) was dissolved in minimum amount of Milli-Q water (0.5 ml),
followed by addition of acetone in excess (2 ml). The mixture was left in the vial for two
hours. The higher molecular weight polymeric material, the precipitate, was collected and

the procedure was repeated.

3.2 Synthesis of polymer [1

A polymeric material that is positively charged but also contains hydrophobic side
chains has been synthesized. The synthesis takes place in two steps. First a polymeric
backbone is synthesized according to the reaction scheme presented in Figure 33. After
purification of the product, the second step consists of functionalization of the polymeric

backbone. The reaction scheme of backbone functionalization is underlined in Figure 37.

The polymeric backbone was obtained using equimolar amounts of cysteine
methyl-ester treated with furanone and sodium bicarbonate dissolved in minimum amount
of water. The use of cysteine methyl-ester is beneficial because the methyl ester has a
better water solubility than cysteine. The water was removed using a rotor evaporator,
heating the reaction mixture at 70°C for one hour. The reaction mixture was further
heated at 70°C for 24 hours. The reaction progress was monitored by NMR. The ester was
hydrolyzed further using N aOH until the solution achieved the pH=11. After one hour the

reaction mixture was treated with acetic acid, until the solution achieved pH=4.

54

In order to obtain molecules that fall into a nominal molecular weight range,
membranes are used to fractionate the compounds by size; the polymeric backbone was
fractionated using a membrane with a 12-14 kDa cut-off. The high molecular weight
fraction was used in the second step of the reaction - the coupling of carboxylic groups
with amines. The second step of the reaction consists of functionalization of the
polymeric backbone by coupling the carboxylic groups with either octylamine or N, N
dimethylethylenediamine. In either case the reaction take place in the presence of the
coupling reagent 1-ethyl-3-dimethylarninopropylcarbodiimide (EDC), at pH=5. For one
equivalent of polymeric backbone were used 0.66 equivalents of octylamine and 0.33
equivalents of N,N-dimethylethylenediamine, in the presence of 2 equivalents of coupling
reagent. The reaction was stopped after 24 hours by adding sodium bicarbonate. The
excess reagents are removed by equilibrium membrane size fractionation (membrane cut-
off 3500 Da). The resulting product (polymer H) has a molecular weight higher than 20

kDa.

3.3 Labeling of polymer II with Fluorescein isothiocyanate (FITC)

One fraction of the polymer H was labeled with fluorescein isothiocyanate (FIT C).
One mg of polymer was lyophilized and after the water was removed it was mixed with
0.1 mg FITC in 200 [1L pyridine. The coupling of the fluorescent label took place at room
temperature for 4 hours. The pyridine was removed under a nitrogen flow and the sample
was dissolved in 1 mL water. The removal of the free FITC was performed using a
reverse phase (C18) column, SEP-PAK cartridge. The elution of the sample was

performed using mixtures of waterzmethanol in various ratios (4:1, 3: 1, 2:1 and 1:1). The
55

FIT C labeled polymer II was collected in the fraction eluted with 4:1 water:methanol

mixture.

3.4 Cy5 labeled antisense oligonucleotide treatment

The ability of polymer I to deliver Cy5 labeled oligonucleotides to MEF cells was
tested by laser multi channel fluorescence scanning confocal microscopy. Mouse
embryonic fibroblasts (MEF), passage 8-12, were grown in DMEM media supplemented
with non-essential amino acids, heat inactivated fetal bovine calf serum (10 %) and Hepes
at 37°C, 5 % C02. MEF cells were plated at low density (5x 105 cells) on cover slips in 6
well plates. After 24 hours, one cover slip was flipped over a microscope slide which had
5 uL of the following mixture: 4 uL of polymeric solution (1 mg/mL) and 1 uL of Cy5
labeled antisense oligonucleotide solution (0.1 mg/mL) dissolved in PBS. Control cells
with no treatment and positive control cells were treated with 1 uL of antisense
oligonucleotide solution (0.1 mg/mL dissolved in phosphate buffer saline - PBS) diluted
with 4 uL PBS. Confocal microscopy images of cells treated with solutions containing
polymer I in various concentrations in combination with Cy 5 labeled antisense
oligonucleotide were collected. The microscope settings were as follows: laser 633 nm,
the first dichroic mirror (HFI‘) 488/543/633nm, the second dichroic mirror (NFI‘) 635
nm, long pass filter (LP) 650 nm for collecting the Cy5 signal. Images collected from this

experiment are presented in color.

56

3.5 Evaluation of plasmid DNA transfection mediated by polymer 11

C081 cells (African green monkey kidney cells) were grown in Dulbecco's
Modified Eagle Medium (DMEM) media that was supplemented with non-essential
amino acids (1 %), heat inactivated fetal bovine calf serum (10 %),
penicillin/streptomycin (l %), and glutamine (1 %). Cells were grown for 24 hours at

37°C and 5 %C02.

COSl cells were grown in 96 well plates at initial seeding density of 2000
cells/well followed by treatment with polymer 11 DNA complexes using the commercially
available plasmid encoding for green fluorescent protein gene, pEGFP-C2 (Clontech,
CA). Figure 32 presents the map of pEGFP-C2 plasmid123 , containing the gene encoding
for GFP along with promoter region pCMV, origin of replication and

kanamycin/neomycin resistance gene. Various sites for restriction enzymes are specified.

polyA
E000 109 I

  
 

Kan/Neo

SV40 ori

Figure 32. pEGFP-C2 encodes a red-shifted variant of wild-type GF P.

57

The plasmid DNA was diluted in serum free media at a concentration of
0.01ag/uL, and either 100 ng, 200 ng or 300 ng were distributed in a 96 well PCR plate.
Various amounts of polymer 11 solution containing 1, 2, 3, 4, 5 or 6 pg polymer H were
added to each well. Control wells with no carrier and positive control wells with
commercially available transfection reagent Lipofectamine 2000 (Invitrogen, CA) were
set up. The manufacturer’s protocol suggested that 0.5 ug of Lipofectamine 2000 be used
for each well of a 96 well plate. The Lipofectamine 2000 was diluted in 25 aL serum free
media 20 minutes prior to each treatment. The homogeneity of each mixture was ensured
by pipetting up and down with a multichannel pipettor, followed by capping the 96 well
plate and vortexing at high speed for 30 seconds. The mixtures were allowed to incubate
at room temperature for 20 minutes. Then the samples were diluted with serum free
media, to a total volume of 200 uL. The media was removed from the 96 well plate
containing the cells, followed by the distribution of the mixtures from the PCR plate. The
cells were incubated at 37°C for 6 hours followed by changing of media with normal
growth media. The efficiency of transfection was evaluated by capturing images with an
inverted microscope at 72 hours after treatment. Phase and fluorescence images of cells

under various treatment conditions were collected with a Nikon inverted microscope.

3.6 Effects of polymer 11 treatment on cells viability and proliferation

COSl cells were grown in 96 well plates at initial seeding density of 2000
cells/well followed by treatment with polymer H DNA in concentrations varying from 0,
0.5 ug/mL, 1 ug/mL, 2.5 uglmL, 5 ag/mL, 7.5 ug/mL, 10 ug/mL, and 100 ug/mL. Two

parallel sets of experiments were set up, one using 100 ng of plasmid DNA and the other
58

using 200 ng of plasmid DNA. The effects of polymer H on cell growth and proliferation
were compared with the commercially available reagent Lipofectarrrine 2000 treated cells.
COS 1 cells were treated with complexes of Lipofectamine 2000 (0, 0.5 ug/mL, l ug/mL,
2.5 ,ug/mL, 5 ug/mL, 7.5 ug/mL, 10 ug/mL, and 100 ug/mL) and either 100 ng or 200 ng
plasmid. The complexes were allowed to incubate for 20 minutes and the cells were
treated in a similar manner that was previously described in Section 3.5. Phase contrast
images of cells under various treatments were collected with a Nikon inverted microscope

72 hours after treatment.

3.7 Investigation of the uptake mechanism of FITC labeled polymer II

The uptake mechanism of FITC labeled polymer H to COS 1 cells was
investigated. COSl cells were seeded at 10000 cells/well 24 hours prior treatment, in 35
mm tissue culture dish, on cover slips. They were incubated at 37°C, with 5 % C02. After
24 hours, one cover slip was flipped over a microscope slide which had a droplet of 10
liL of stock solution of FITC labeled polymer H (0.1 mg/mL). To inhibit the ATP
synthesis, the cells were incubated with 100 ,uM solution of DCCD (stock solution in
ethanol) for 15 minutes. After the incubation period, the cover slip was flipped over a
microscope slide which had a droplet of 10 uL of stock solution of FIT C labeled polymer
(0.1 mg/mL). The uptake of fluorescently labeled polymer was analyzed in the two
situations by confocal laser fluorescence microscopy within the next 30 nrinutes. The
slides were analyzed by confocal microscopy immediately after treatment using an 100x
oil objective. The microscope settings for collecting the FITC signal were as follows:

laser 488 nm, the first dichroic mirror (HFT) 488 nm, the second dichroic mirror (NET)
59

__.__ .
M --‘
M

F

545 nm, and band pass filter (BP) 505-530 nm. Images in this thesis are presented in

color.

60

Chapter 4

Results and discussion

4.1 Principle

Our laboratory developed a synthetic method that allows incorporation of different
moieties into a molecular scaffold. Since our goal is to develop molecules that could be
used to deliver nucleic acids into cells, we are interested in functionalizing the scaffold

with groups that carry positive charges, as well as hydrophobic groups.

The synthesis of macromolecular compounds consists mainly of two steps. These
are illustrated in scheme presented in Figure 33. First, a backbone that carries free
carboxylic groups is synthesized. The thiol group of the amino acid cysteine reacts with
the double bond of the lactone 2(5H)-furanone via a Michael addition mechanism. The
head to tail polymerization reaction occurs via opening the resulting y lactone, resulting
in a linear polymer backbone with alternating thio-ether and amide linkages. The free
carboxylic group from cysteine provides the negative charge and allows functionalization

using one or more reagents in order to achieve the desired properties.

61

cysteine 2(5H) furanone

do
S

OH

monomer unit

gSl

Figure 33. Polymerization of cysteine with furanone via a Michael addition mechanism

leading to synthesis of backbone polymer.

The reaction was monitored by NMR. In Figure 34 is presented the spectra of
starting material consisting of cysteine and furanone. The signal from C2 of furanone is
around 6 ppm, C3 gives a signal at 7.7 ppm while C4 gives a signal at 5 ppm. Analyzing
cysteine, the CH2 gives a signal at 3.0 ppm, while the CH has a signal at 3.7 ppm. After
the reaction was completed, the signals from furanone beyond 5.0 ppm disappeared
(Figure 35), consistent with the opening of the lactone ring. The signals from the amino
acid are labeled (c) for CH at 4.5 ppm and (d) for CH2 at 3.0 ppm. The CH2 next to

hydroxyl group from the former lactone ring is labeled (b) and gives a signal at 3.5 ppm,

62

while the CH2 labeled (a) gives a signal at 2.2 ppm. Also, the peaks are broader indicating

that the product is polymeric.

 

 

 

 

 

 

 

 

2 U NH2
3 l 0 Sll\/l\“/Oll
4 a
b o
3 4
1‘ D I
i b
'
. a
1
..JW HL . ..-..._._.../ ; M. LJJE'I “use
"’“*"*"Tr"rrvr"r'rrnrvn'rvr. ...]..—.—.A,.a..,.-—f—r—4~-r——i——.--r—.—T—.-—.~~.-—
6.0 5.5 5.0 4.5 4.0 3.5 3.0 ppm

7.5 7.0 6.5

Figure 34. The 1H NMR spectra of the cysteine and furanone mixture before

polymerization

63

_ *-

II
a No d I
fl rs h
o W
rm b n on] o
Ill) (

i
) (II I
I I0

 

 

 

 

 

 

 

Figure 35. The 1H NMR spectra of the polymeric backbone. The signals from the starting

material disappeared.

In the second step, a pendant group is added to the carboxylic groups. The best
candidates are amines to form amidic bonds. If N,N-dimethylethylenediamine is used to
functionalize all the free carboxylic groups the final product is a polymer that is positively
charged. In Figure 36 is presented the reaction scheme that leads to this product. In

further discussion, this product will be referred as polymer 1.

“flxw 523w ”If???” 3:127er1;

EDC/(CH3)2NCH2CH2NH2 “0 H
”OJ/T :JSWIE IW Us ifs n1?“ Him fl”

/\

Figure 36. Functionalization of the free carboxylic groups with N,N-

dimethylethylenediamine resulting polymer 1.

If a mixture of amines is used to couple the free carboxylic groups, , the final
product can be engineered to contain moieties at a desired proportion by controlling the
composition of the mixture. Besides the positive charges introduced by the N,N-
dimethylethylenediamine, amines that carry a non-polar residue will introduce
hydrophobicity in the final product. If octylamine is used besides the N,N-
dimethylethylenediamine for coupling the free carboxylic groups, the ratio in which the
two amines are added is important. Cationic lipids are very common non-viral nucleic
avid delivery vectors. This class of molecules has a positive to hydrophobic chain ratio of
about 35:65. This ratio was mimicked at a macromolecular level, and a polymeric
material that contains 66 % hydrophobic chains introduced by octylamine and 33 %

positive charges introduced by N,N-dimethylethylenediamine was synthesized. The

65

a-

F

synthetic scheme underlying the coupling reaction is presented in Figure 37. The polymer

with this composition will be referred as polymer H.

fir “if?“ “thud £wa

OHOj J\OH
' EDC/ CH3(CH2)6 CHzNHz and (CH3hNCH2CH2NH2

flgflrlrflf

N
\
,N

ouo fl:;\

/
\

Figure 37. Functionalization of the polymeric backbone by coupling amines to the free
carboxylic groups. Polymer 11 contains 33 % positive charges and 66 % hydrophobic side

chains.

In the FT-IR spectrum of the polymer II presented in Figure 38 the broad signal at
3300 cm'1 indicates the presence of -OH and -NH, signals at 2972 cm", at 2798 cm'1 and
2720 cm'1 indicate the presence of -CH2, and -CH stretches, while the signals at 1637 cm'

1 were attributed to carbonyl group.

66

80~
76 j
72 -
68 q
64 i
60 1 2720.93

%T

56 - . .
52 - 2972.86

48 7 1566.10
44 . 1637.77

 

 

‘ I

I l I l
4000 3200 2400 1600 1200 cm-1

Figure 38. FT—IR spectrum of polymer II. The carbonyl group gives signal at 1637 cm",
the -OH and -NH at 3300 cm", -CH2, and -CH stretches are present at 2720 cm'1 and

2972 cm".

4.2 Evaluation of the performance of polymers in vitro

4.2.1 Evaluation of the ability of polymer I to deliver antisense oligonucleotide to

MEF cells

The membranes of mammalian cells typically have a net negative charge. Because
of this, due to the highly negative charge of the phosphate backbone, introducing RNA
into cells is a challenging task. To test the efficacy of the new molecular systems at the
transfer of oligonucleotides of the sizes used for interference of gene function by the

antisense strategy, transfer of the antisense oligonucleotide 5’-GCG CGG GGA GCA
67

AAA GCA C-3’ from the hTR subunit of human telomerase RNA ‘24 into mouse
embryonic fibroblast cells was attempted. To aid visualization, the 5’ end was labeled
with the fluorophore CyS, which emits at 670 nm when it is excited with a laser operating

at 633 nm.

The antisense delivering capacity of a polymer I having all the carboxylic groups
coupled with N, N dimethylethylenediamine was tested in MEFs. Control experiments
with MEF cells (Figure 39 (A)), and cells treated only with antisense oligonucleotide
solution in PBS were set up. The cells were treated with polymer I at various
concentrations (0.1 g/mL, 0.01 g/mL, 0.001 g/mL, or 0.0001 g/mL) and CyS labeled
oligonucleotides. The amount of antisense oligonucleotide was constant through the
experiment (1 uL of stock solution (0.1 mg/mL) freshly prepared in PBS). The ability of
the highly positively charged polymeric materials to deliver their cargo to the nucleus was
successful in each case Figure 39 (C, D, E, and F). Antisense oligonucleotides in the
absence of a carrier are excluded from the cells, demonstrating that naked antisense
oligonucleotide constructs are unable to enter the cells Figure 39 (B). Series of images
(36 um apart) of MEF cells treated with 1mg/mL polymer I and 1 1.1L of stock solution
(0.1 mg/mL) of Cy 5 labeled antisense oligonucleotides confirmed that t polymer I and

the fluorescent label are inside the cells Figure 40.

68

10 pm

20 um 20 pm 20 um
I ~.. ‘-'.- .' ...

 

Figure 39. MEF treated with polymer I at various concentrations (A). Control — MEF no
treatment. (B). MEF with no carrier (CyS antisense oligonucleotide in PBS). (C, D, E, F)
MEF and antisense oligonucleotide with various concentrations of polymer 1 solution: C

(0.1 g/mL), D (0.01 g/mL), E (0.001 g/mL), F (0.000 lg/mL).

 

Figure 40. Series of confocal images through a group of MEF cells treated with polymer I

(1 mg/mL). The Z series consists of 4 images; the distance between images is 36 pm. The

scale bar is the same in all images, 20 um.

69

The unmodified parent polymer which is highly negatively charged due to the
carboxylic groups, was tested for the ability to deliver the antisense oligonucleotide to
MEF cells Figure 41. As expected, the fluorescently labeled antisense oligonucleotides

were excluded fi'om the cells.

A

207m 20_pm

 

Figure 41. MEF cells treated with Cy5 labeled antisense oligonucleotides in combination
with a solution of backbone polymer. A. Bright field image showing the MEF cells. B.
The fluorescent image. C. An overlay image of the transmitted and fluorescence image.

The drug was not delivered inside MEF cells.

The ability of polymer 1 to deliver fluorescently labeled oligonucleotides to MEF
cells demonstrated successful delivery of the antisense oligonucleotides to the cells.
There is no indication about the stability of the complex between polymer and antisense
oligonucleotide; in order to disrupt gene expression the nucleic acids should dissociate
from the complex formed between the antisense oligonucleotides and the positively

charged carrier.

70

4. 2. 2 Evaluation of plasmid DNA transfection mediated by polymer II

The polymer II, which contains 66 % hydrophobic side chains and 33 % positively
charged amine groups, was tested for the ability to transfect COSl cells with plasmid
encoding for GFP, pEGFP-CZ. Several combinations varying the amounts of polymer II
and the amounts of plasmid DNA were tested. Examples of ratio polymer Ilzplasmid
DNA that were tested are presented in table 2. The amount of plasmid DNA varied from
100 ng, 200 ng to 300 ng. The amounts of polymer 11 used for transfection varied from 1

to 6 ug. Combinations of these quantities were tested.

Table 2. Examples of combinations of polymer Hzplasmid ratios

 

 

 

10:1 20:1 30:1 40:1 50:1 60:1
10:2 20:2 30:2 40:2 50:2 60:2
10:3 20:3 30:3 40:3 50:3 60:3

 

 

 

 

 

 

 

 

The commercially available transfection reagent Lipofectamine 2000 was used as
a positive control. The commercially available transfection reagent was also tested with
100 ng, 200 ng or 300 ng plasmid DNA. That translates into Lipofectamine 2000:plasmid

DNA ratios of 5:1, 2.5:lor 5:3.

The image of €081 cells before treatment is presented in Figure 42.

71

 

Figure 42. COS 1 cells before treatment, phase contrast image.

As soon as 24 hours after treatment some cells exhibited green fluorescent protein
expression. One specific combination, the one with 20:1 polymer H DNA ratio was
identified Figure 43.

 

Figure 43. COS 1 cells transfected with plasmid encoding for green fluorescent protein,

transfection mediated by the polymer II at 24 hours after treatment. (a) the phase contrast
image and (b) the corresponding fluorescent image, (c) the overlay of the phase contrast

image and the fluorescent image.

The efficiency of transfection was tested by visualizing, under the fluorescence
mode, the cells that were able to express the green fluorescent protein, 72 hours after
treatment. Control cells with no treatment are presented in Figure 44. As expected, the
COS 1 cells do not exhibit fluorescence, since the cells do not contain the gene encoding

for green fluorescent protein.
72

 

Figure 44. COS 1 cells — no treatment, (a) the phase contrast image and (b) the

corresponding fluorescent image, at 72 hours after treatment.

At 72 hours after treatment, the C081 cells treated with carrier solution at a
polymer II:DNA ratio of 20:1 exhibited the highest efficiency of transfection. A few
examples are presented in Figure 45. Some cells were able to express the green

fluorescent protein.

 

Figure 45. The upper panel represent the overlay of the phase contrast image and the

fluorescent image of cells treated with a 20:1 of polymer II:DNA ratio, the bottom panel

the corresponding phase contrast images of the upper panel at 72 hours after treatment.
73

Other conditions that exhibited cells fluorescing green were the 20:2 polymer
II:DNA ratio, presented in Figure 46, and 20:3 polymer II:DNA ratio, presented in Figure

47. The efficiency under these conditions is lower when compared to the 20:1 polymer

II:DNA ratio condition. Other conditions did not exhibit any fluorescence.

 

Figure 46. C081 cells - (a) the phase contrast image (b) the corresponding fluorescent
image, and (c) the overlay of the phase contrast image and the fluorescent image at 72

hours after treatment with a polymer II:DNA ratio of 20:2.

 

Figure 47. C081 cells — (a) the phase contrast image and (b) the corresponding

fluorescent image, and (c) the overlay of the phase contrast image and the fluorescent

image at 72 hours after treatment with a polymer II:DNA ratio of 20:3.

74

Analyzing Table 2, where the condition treatments are presented, one would
expect that 20:1 polymer II:DNA ratio, 40:2 polymer II:DNA ratio and 60:3 polymer
II:DNA ratio would give similar results. When these conditions where compared (Figure
48) only the 20:1 ratio had a positive outcome, while the other two did not have any

fluorescent cells, thus no efficient transfection of the plasmid encoding for GFP.

 

Figure 48. Comparison of the overlay of the phase contrast image of three treatment

conditions (a) 20:1 polymer II:DNA ratio, (b) 40:2 polymer II:DNA ratio, and (c) 60:3

polymer II:DNA ratio.

The images of cells transfected with Lipofectamine 2000 are presented in Figure
49, 50, and 51 respectively, where the same amount of Lipofectamine 2000 was used in
combination with 100 ng, 200 ng and 300 ng plasmid DNA respectively. It seems that in
all conditions, the efficiency was similar and not affected by the amount of plasmid DNA

used.

75

 

Figure 49. C081 cells (a) the phase contrast image, (b) the corresponding fluorescent
image, and (c) the overlay of the phase contrast image and the fluorescent image at 72

hours after treatment with Lipofectamine 2000 and 100 ng plasmid.

 

Figure 50. C081 cells (a) the phase contrast image, (b) the corresponding fluorescent

image, and (c) the overlay of the phase contrast image and the fluorescent image at 72

hours after treatment with Lipofectamine 2000 and 200 ng plasmid.

76

 

Figure 51. C081 cells (a) the phase contrast image, (b) the corresponding fluorescent

image, and (c) the overlay of the phase contrast image and the fluorescent image at 72

hours after treatment with Lipofectamine 2000 and 300 ng plasmid.

When the cells were examined under the fluorescence mode at a lower
magnification, the area that can be visualized at once is increased. For the condition that
was identified that exhibit the highest transfection efficiency, 20:1 ratio polymer H2DNA,
several fields are presented in Figure 52. The distribution of cells that exhibit

fluorescence in any field is homogenous.

77

Figure 52. C081 cells 72 hours after treatment with 20:1 ratio polymer II:DNA at a lower

 

magnification under the fluorescence mode. The distribution of cells exhibiting

fluorescence is even.

When the C081 cells treated with Lipofectamine 2000 and 100 ng plasmid DNA
were examined at lower magnification, some fields showed very high fluorescence, while
other fields were completely dark, since no cells were fluorescing on these areas (Figure
53). This result may seem unexpected, but a very reasonable explanation exists. It is well
known that the actual non viral transfection reagents are toxic, causing cell lysis. If a cell
is lysed, its content is released outside the cell and the cell dies — it is not able to grow
and proliferate. The reason that no cells are exhibiting fluorescence on the right side of

figure 51 is that there are no cells there.

 

Figure 53. C081 cells 72 hours after treatment with Lipofectamine 2000 and 100 ng

plasmid at a lower magnification under the fluorescence mode, (a) many cells exhibit

fluorescence and (b) no cells are fluorescent.

The efficiency of fluorescence can be qualitatively identified using the lower
magnification of the microscope under the fluorescence mode. The conditions that were
identified to exhibit fluorescence 20:1 polymer II:DNA ratio, 20:2 polymer II:DNA ratio,
and 20:3 polymer II:DNA ratio were examined and the images are presented in Figure 54.

Again, the highest efficiency is achieved by the 20:1 polymer II:DNA ratio condition.

79

 

Figure 54. C081 cells 72 hours after treatment with 20:1 polymer II:DNA ratio, 20:2
polymer II:DNA ratio, and 20:3 polymer II:DNA ratio at a lower magnification under the

fluorescence mode.

The efficiency of transfection was expressed as the percentage of the ratio of the
number of cells that express GFP over the total number of cells in one field. The mean of
several fields was taken into account for each treatment condition. The highest efficiency
for the polymer II is observed when the ratio polymer IIzplasmid was 20:1 and is 6.5 %.
In comparison, the treatment using Lipofectamine 2000 in a 5:1 ratio exhibited 52.14 %

transfection efficiency (Figure 55).

80

 

Transfection Efficiencies

 

% fluorescent cells
8

 

 

 

 

30 +
20 _
10 - l
0 _ ‘ .
Lipofectamine 2000:DNA rau'o 5:1 ' PolynerzDNA ratio 20:1
Treatrrent conditions

 

 

 

Figure 55. Evaluation of GFP expression 72 hours after treatment. Comparison of

polymer II with Lipofectamine 2000 treatment.

When different polymer II:plasmid DNA combinations were compared, the results
are consistent with qualitative examinations of images in figures 43, 44, 45 and 52. The
highest transfection efficiency was obtained when the polymer II:plasmid DNA ratio was

20:1, followed by a 20:3 ratio and a 20:2 ratio. The quantitative results are plotted in

Figure 56.

81

 

Transfection efficiencies

 

 

% fluorescent cells
0‘

 

 

 

0- , , —_
PolymerDNA ratio 20:1 PolymerDNA ratio 20:2 PolymerzDNA ratio 20:3
Treatment conditions

 

 

 

 

Figure 56. Evaluation of GFP expression 72 hours after treatment. Comparison of

different polymer II treatments.

4.2.3 Effects of polymer 11 treatment on cell viability and proliferation

The impact of the carriers on the physiology and structural integrity of the cells
are important aspects of the evaluation of their performance in transfection. This includes
cell viability and ability to proliferate. To evaluate the cytotoxic effect of the newly
synthesized carrier on COS 1 cells, various concentrations of carrier were tested. The
C081 cells were grown in a 96 well plate and treated with polymer in concentrations
varying from 0, 0.5 ug/mL, 1 ug/mL, 2.5 ug/mL, 5 ug/mL, 7.5 ug/mL, 10 ug/mL, and
100 ug/mL. Two parallel sets of experiments were set up, one using 100 ng of plasmid

82

DNA and the other using 200 ng of plasmid DNA. Please recall that from the efficiency
of transfection experiment, the combination that exhibited the highest transfection
efficiency was when 100 ng plasmid DNA were delivered by 2 pig polymer. This
translates into a final polymer II concentration of 10 ug/mL in a 200 ML volume per well
in a 96 well plate. A concentration 10 times higher was tested for the toxic effect on cells.
Lipofectamine 2000 was again chosen as a positive control. It was used in the same
concentrations as the polymer, with two parallel sets using either 100 ng or 200 ng of
plasmid DNA. The manufacturer recommended a 2.5 ug/mL concentration to be used for

a 96 well plate set up.

A very common assay to evaluate the cytotoxicity that some treatments might
have on cells is the MTT assay. This assay is controversial, since it does not involve a
direct measure of the number of cells, but rather a measurement of the mitochondrial
dehydrogenase activity. The dehydrogenase enzyme cleaves the tetrazolium rings of 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) resulting in dark blue
crystals. Those crystals are able to accumulate only in intact cells. To evaluate the activity
of the enzyme, the cells are lysed and the forrnazan crystals are dissolved resulting in a
colored solution. The absorbance of the solution can be correlated with the number of

healthy cells.

We decided to use a more direct way to describe the effect of different treatments
on the ability of cells to grow and proliferate. At a time point of 72 hours after treatment
we collected phase contrast images of the cells using an inverted microscope. We
performed a qualitative analysis, looking at cells morphologies under different treatment

conditions when compared with controls. When analyzing the phase contrast image of the

83

control cells (Figure 57 a), which had no treatment, we observed that they reached
confluence and proliferated covering the entire well surface. The same phenotype was
observed for the cells that were treated only with plasmid DNA (Figure 57 b). The cells
treated with polymer II of various concentrations (Figures 57 c to 57 0) also proliferated,
the cells under this treatment being no different in morphology from the control cells. In
contrast, cells treated with Lipofectamine 2000 of the same concentrations as polymer II
(Figures 57 d to 57 p) exhibited a large reduction in growth. In some areas, only a few
cells were seen to grow, leaving a lot of the available area bare. Lipofectamine 2000
treatment had a highly negative impact on the viability of the cells interfering with their

capacity to grow and proliferate.

84

Figure 57. Phase contrast images of €081 cells with treatments of polymer II or
Lipofectamine 2000 at various concentrations. (a) control, no treatment, (b) treated with
100 ng plasmid DNA no carrier, (c) treated with 0.5 rig/ml polymer 11, ((1) treated with
0.5 ug/ml Lipofectamine 2000, (e) treated with 1 ug/ml polymer 11, (f) treated with 1
pig/ml Lipofectamine 2000, (g) treated with 2.5 rig/ml polymer 11, (h) treated with 2.5
pig/ml Lipofectamine 2000, (i) treated with 5 rig/ml polymer 11, (j) treated with S ug/ml
Lipofectamine 2000, (k) treated with 7.5 rig/ml polymer H, (1) treated with 7.5 ug/ml
Lipofectamine 2000, (m) treated with 10 ug/ml polymer II, (n) treated with 10 ug/ml
Lipofectamine 2000, (0) treated with 100 ug/ml polymer H, (p) treated with 100 ug/ml

Lipofectamine 2000.

 

 

A quantitative analysis was performed by counting the total number of cells
present in each image. The means of the total number of cells from 9 images collected for
each treatment condition are plotted in Figures 58, and 59. The influence of polymer II on
the ability of cells to proliferate was evaluated. A concentration 10 times higher than the
one that was identified to mediate transfection was tested. The averages of total number
of cells in 9 images that were collected for the same treatment are plotted in Figure 58. In
contrast to the results obtained from the Lipofectamine 2000 treatment, even at highest
concentration tested, the number of cells that are present in the images is comparable to
controls, indicating little or no deleterious impact on growth. The total number of cells is
steady, throughout the experiment, under various polymer 11 concentrations. The same
trend is noticed when Figures 58 and 59 are compared. These two figures differ only by
the amount of plasmid DNA that was transported. Again, the amount of plasmid DNA

tested to be transported does not influence the ability of cells to grow and proliferate.

86

 

Transfection using 100 ng plasmid

 

Total number of cells
8
O

 

 

 

_50 nnnrrnl L 1L5 l 7 5 5 7 fl III In!)

Transfection reagent concentration ug/ml

 

 

 

I Polymer 11 I Lipofectamine 2000‘

 

Figure 58. Total number of cells grown under treatment with polymer II or Lipofectamine

2000 and 100 ng plasmid.

87

 

Transfection using 200 ng plasmid

 

Total number of cells

 

 

Control 0 0.5 1 2.5 5 7.5 10 100

Transfection reagent concentration ug/ml

 

 

 

I Polymer II I Lipofectamine 2000}

 

Figure 59. Total number of cells grown under treatment with polymer II or Lipofectamine

2000 and 200 ng plasmid.

Figure 58 represents also the total number of cells that were counted for the
treatment with Lipofectamine 2000 at different concentrations and 100 ng of plasmid. At
a concentration of 2.5 pg/ml of Lipofectamine 2000 the total number of cells was
significantly smaller compared to the control condition. As the concentration of
Lipofectarrrine 2000 increases, the number of cells that are able to grow and proliferate
decreases. At the highest concentration tested (100 ug/mL) no intact cells were observed;
all of them had been lysed. The same trend is seen when 200 ng of plasmid DNA was

used and various concentrations of Lipofectamine 2000 (Figure 59), leading to the
88

conclusion that the amount of plasmid DNA that is to be transported does not influence
the ability of cells to grow and proliferate, the toxic effect that is seen is due to the

Lipofectamine 2000.

From Figure 57, the morphology of the cells treated with Lipofectamine 2000 is
seen to be very different when compared to non-treated cells. Three different phenotypes
were identified. One class of cells exhibits a difference in size: they are smaller,
condensed, shrunk and darker. The number of cells that exhibit this type of behavior were
counted and reported as percentage of the number of cells that are shrunk from the total
number of cells in a field. Averages from 9 different images describing the same
condition treatment were plotted in Figure 60. At a Lipofectamine 2000 concentration of
2.5 ug/mL about 30 % of cells belong to this category. When the Lipofectamine 2000
concentration was increased to 7.5 ug/mL and 10 ug/mL 100 % of the total number of
cells were shrunk. For the polymer 11 treated cells, the number of cells that fall into this

category is very small, under 2 %, for all the concentrations tested (Figure 60).

89

 

Cytotoxicity

 

120

100 ‘

00
O
r

 

O\
O
l

 

% lysed cells
‘5

 

 

 

0.5 l 2. ' 5 7.5 10 100

 

 

 

Transfection reagent concentration

 

 

 

I Polymer II I Lipofectamine 2000

 

 

 

 

Figure 60. Percentage of the number of cells that are lysed under Lipofectamine 2000 or

polymer II treatment.

Another class of cells is represented by cells that are larger than the control cells.
The number of cells that exhibit this type of behavior were counted and reported as
percentage of the number of cells that are large from the total number of cells from a
field. Averages from 9 different images describing the same condition treatment were
plotted in Figure 61. The percentage of cells that fall into this category is very small,

under 1 %, and is present only at low Lipofectamine 2000 concentrations.

90

 

 

 

 

 

 

 

 

Cytotoxicity of Lipofectamine 2000 and 100 ng
plasmid
2.5
2 ~ .
g 1.5 -
8
d)
be 1 ~
33
6° 0.5 .
0 d I I r
2.5 5 7.5 10
.05
Lipofectamine concentration ug/ml

 

 

 

Figure 61. Percentage of the number of cells that are larger under Lipofectamine 2000

treatment.

The third phenotype consists of cells that are totally lysed. This situation was
encountered only for the Lipofectamine 2000 treatment at a concentration of 100 ug/mL
(Figure 57 p). In comparison with Lipofectamine 2000 treatment at this concentration, the
cells treated with polymer H were able to grow and proliferate at a rate comparable to
control cells (Figure 57 o). The amount of plasmid DNA does not influence the cell

growth.

91

The results presented show that the polymer that we designed and tested has
potential to be used as a nucleic acid carrier. Under the treatment with polymer H of
various concentrations the cells are still growing and proliferating, covering the entire
surface available, up to confluence. Even at a concentration 10 times higher that the one
that was identified to mediate transfection, the ability of cells to grow and proliferate is

not influenced.

4.3 Characterization of the mechanism of uptake

Characterization of the mechanism of uptake of polymer—complexed nucleic acids
into cells is possible by analyzing confocal images of cells treated with FITC-labeled
polymer. Two types of transporters are known: passive transporters that do not require
energy (e.g., glucose transport), and active transporters that rely on ATP hydrolysis to
drive the transport (e.g., sodium/potassium pump). It is generally accepted that
macromolecules are taken up by endocytosislzs’ 126. In this process, the cellular membrane

pinches-off enclosing the molecule.

To determine if the uptake is an active or a passive phenomenon ATP synthesis
was inhibited using a classic ATP synthesis inhibitor, the dicyclohexylcarbodiimide
(DCCD). This molecule reacts with a critical aspartic acid residue in the F0 subunit of the
ATP synthase molecule (Figure 62), forming a covalent adduct, thus impeding the proton
flux necessary for ATP synthesis. The c subunit of F0, also known as proteolipid, because
of its hydrophobicity subunit, has a hairpin structure with two transmembrane a-helices

and a connecting loop. One a-helix includes the Asp residue whose carboxyl reacts with

92

DCCD. Mutation studies have shown that this DCCD-reactive carboxyl, in the middle of

the bilayer, is essential for H+ transport through F0 127.

PDB 1.-\.91

F0 subunit c

Figure 62. Critical aspartic residue in the c subunit of the F0 128.

N, N'-dicyclohexylcarbodiimide (DCCD) inhibits proton transport in endocytotic
vesicles. The half—maximum inhibition of proton transport is observed at 10 uM DCCD
afier 30 minm. Sabolic and Burckhardt demonstrated that a 0.1 mM DCCD solution
inhibits the proton transport for a 30 minute period of time and that ATP synthesis is

lowered to 3 % compared to control cells with no treatmentm.

COSl cells were treated with N, N'-dicyclohexylcarbodiimide (DCCD) as
described in Section 3.7. The confocal microscope settings were established using a
solution of FITC labeled polymer Figure 63. The control COSl cells, with no treatment,

are presented in Figure 64.

93

 

Figure 63. FITC labeled polymer solution.

10 till]

 

Figure 64. C081 cells, no treatment.

The images recorded for the FITC labeled polymer treated cells are presented in
Figure 65 and 66. Immediately afier treatment, rounded vesicles including the fluorescent
dye are visible at the periphery of the cells (Figure 65). Ten minutes afier the FITC
labeled polymer H treatment the polymer had already begun to accumulate in the nuclei of

the cells, which are more brightly stained than the corresponding cytosol (Figure 66).

94

1011111 ‘ 101”“ 1011111

 

Figure 65. C081 cells treated with FITC labeled polymer H, immediately after treatment.

1
1
1
1

1011111 1 1011111 1011111

 

Figure 66. C081 cells treated with FITC labeled polymer H. A confocal slice of several

COS 1 cells 10 minutes afier treatment.

To determine if the uptake mechanism requires energy, the ATP synthesis was
blocked by using DCCD treatment. The cells were pre-incubated for 15 minutes with 100
11M DCCD solution, followed by the treatment with FITC labeled polymer H. Images of
the COS 1cells under this treatment condition are presented in Figure 67. The images are

recorded 10, 15 and 20 minutes afier treatment respectively.
95

1011111 . - _. ”:. - 101.111

1011111

10.1““ ' i 1011111 ‘ . 1011111

 

Figure 67. C081 cells treated with FITC labeled polymer H in the presence of DCCD

inhibitor. Images recorded 10, 15 and 20 minutes after treatment respectively.

In Figure 65, rounded vesicles are present at the periphery of the cellular
membrane. In this particular situation, the C081 cells were treated with FITC labeled
polymer H. The vesicles present at the cellular membrane encapsulate the labeled polymer
H suggesting that endocytosis is responsible for the transport of fluorescently labeled
polymer H inside the cells. When the cells were pre-incubated for 15 minutes with the

inhibitor of proton transport the fluorescently labeled polymer H accumulated in the
96

nucleus of some cells 10 minutes after treatment. That means that either the attempt of
inhibition of the ATP synthesis with DCCD was not successful or the transport of FIT C
labeled polymer H does not rely on ATP resources. This result suggests several
explanations. Maybe an alternate pathway besides endocytosis is responsible for the
transport of the fluorescently labeled polymer H inside the cell. Some temporary
disruption of the cellular membrane or fusion of the hydrophobic side of polymer H with
the cellular membrane might be responsible for the efficient transport. Polymer H was
designed to have 66 % hydrophobic side chains introduced by octylamine. Possibly a
spontaneous phase transition at the cellular membrane favorized the formation of
vesicles. Another explanation may be that the inhibition of the proton transport with
DCCD was not effective, allowing the COS 1 cells to further synthesize ATP. It is
possible that this particular cell line, COS 1 cells, does not have that critical aspartic acid
that is important for proton transport, so that the inhibitor does not have the substrate to
bind. Another alternative might be that the endogenous energetic resources of the cells,
the ATP that was present in cells at the moment of incubation with DCCD were sufficient
to allow cell survival and endocytosis of fluorescently labeled polymer H. Further
studies, using different endocytosis inhibitors, or investigation of the efficiency of the

uptake at 4°C should shed more light on the mechanism of uptake.

97

4.4 Conclusions

A macromolecular platform that can incorporate functional groups at a desired
proportion was developed. The synthetic strategy consists only of two steps: the synthesis
of a polymeric backbone and its further functionalization. The functionalization of the
backbone relies on amide bond formation. This has two advantages. First, the chemistry
behind this reaction is well known, as is used at a large scale in any laboratory that
synthesizes peptides. Second, the peptide bond is a substrate for proteases, thus ensuring
polymer degradation. Two significant representatives that can mediate the transport of
nucleic based therapeutics were synthesized, characterized, and their in vitro performance

was evaluated.

Polymer I, which is positively charged, was effective in delivering CyS labeled
antisense oligonucleotides into the nuclei of MEF cells. The positive control, MEF cells
treated with Cy 5 labeled antisense oligonucleotide solution with no carrier, showed the

fluorescent dye excluded from the cells.

Polymer H, which contains hydrophobic side chains along with positive charges,
was tested for the ability to transport pEGFP, a plasmid that encodes for green fluorescent
protein to the nuclei of COSl cells. This carrier not only efficiently mediates transfection,

but also does not affect the structure of the cells.

After performing the tests that were presented we have an in depth
characterization of materials that are able to deliver nucleic acids into mammalian cells.
The characterization of the material covers the physico—chemical characterization, the

optimization of conditions for transfection of plasmid encoding for GFP into mammalian

98

cells, comparison of cytotoxicity of this material to other commercially available

transfection reagents, and preliminary characterization of the uptake mechanism.

99

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