Linear and branched chitosan oligomers as delivery systems for pDNA and siRNA in vitro and in vivo
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ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2006
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Pharmacy 46
Linear and Branched Chitosan
Oligomers as Delivery Systems
for pDNA and siRNA In Vitro
and In Vivo
MOHAMED MAHMOUD ISSA
ISSN 1651-6192
ISBN 91-554-6747-4
urn:nbn:se:uu:diva-7376
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“You are not given aught of knowledge but a little”
Holy Qur’an: 17:85.
To my family
Papers discussed
The thesis is based on the following papers, which will be referred to
by their Roman numerals:
I Köping-Höggård M, Vårum KM, Issa M, Danielsen S, Christen-
sen BE, Stokke BT, Artursson P. Improved chitosan-mediated
gene delivery based on easily dissociated chitosan polyplexes of
highly defined chitosan oligomers. Gene Ther. (2004)11(19):
1441-52.
II Issa M, Köping-Höggård M, Tømmeraas
K, Vårum KM, Chris-
tensen
BE, Strand SP, Artursson P. Targeted gene delivery with
trisaccharide-substituted chitosan oligomers in vitro and after
lung administration in vivo. J. Control. Rel. (2006) 115 (1): 103-
112.
III Issa M, Strand SP, Vårum KM, Artursson P. Chitosan oligomers
as siRNA delivery systems in vitro. In Manuscript.
IV Köping-Höggård M
*
, Issa MM
*
, Köhler T, Tronde A, Vårum
KM, Artursson P. A miniaturized nebulization catheter for im-
proved gene delivery to the mouse lung, J Gene Med. 7(9)
(2005): 1215-22.
*
shared first authorship.
Also published:
Mohamed M. Issa, Magnus Köping-Höggård and Per Artursson. Chitosan
and the mucosal delivery of biotechnology drugs. Drug Discovery Today:
Technologies (2005), 2 (1) 1-6.
Reprints were made with permission from the publishers.
Contents
Introduction 11
Nucleic acids and oligonucleotides as potential pharmaceutical
products 11
Gene delivery systems 14
Viral gene delivery systems 14
Non-viral gene delivery systems 14
Naked (unformulated) nucleic acids 15
Barriers to non-viral gene delivery 15
Pharmaceutical barriers 15
Extracellular barriers 16
Cell-surface and intracellular barriers 17
Bacterial gene delivery (bactofection) and alternative gene therapy
(AGT) 19
Physical methods of non-viral gene delivery 20
Chemical methods of non-viral gene delivery 20
Lipids 21
Polymers 22
Strategies to improve the in vitro/in vivo efficiency of non-viral gene
delivery: Structure-activity relationship 25
Molecular weight reduction 25
Positive charge shielding 26
Active targeting 26
In vivo toxicity of polyplexes 26
Chitosan chemical structure 27
Properties of chitosan 28
Physicochemical properties 28
Biological properties 29
General applications of chitosan in drug delivery 29
Chitosan as a delivery system for proteins and peptide drugs 29
Chitosan as non-viral gene delivery system 30
Aims 32
Materials and methods 33
Nucleic acids 33
Polycations 33
Cells 34
Formulation of complexes 34
Size and morphology of the complexes 35
Physical and enzyme stability 35
In vitro studies 35
Transfection experiments 35
Cellular uptake of chitosan complexes 36
Cellular toxicity (Intracellular dehydrogenase activity) 37
In vivo studies 37
Luciferase gene expression 37
Distribution pattern of gene expression in the mouse lung 37
Toxicological evaluations 37
Statistics 38
Results and discussion 39
Optimised linear chitosan oligomers as non-viral gene delivery systems
(Paper I) 39
Characterisation of DP
n
18 polyplexes in vitro and in vivo 39
Characterisation of polyplexes based on oligomer fractions isolated
from DP
n
18 in vitro and in vivo 41
Intracellular release of pDNA from chitosan oligomer-based
polyplexes 43
Trisaccharide-substituted chitosan oligomers as non-viral gene delivery
systems (Paper II) 44
Structure-activity relationship of polyplexes based on trisaccharide-
substituted chitosan oligomers (TCO). 45
Impact of trisaccharide substitution on chitosan oligomer-based
polyplexes 45
Chitosan oligomers as siRNA delivery systems in vitro (Paper III) 50
In vitro siRNA delivery under conditions previously optimised for
pDNA 50
Structure-activity relationship of chitosan oligomer-based siRNA
complexes 52
Improved aerosol gene delivery to the mouse lung in vivo (Paper IV) 56
Physical stability and in vitro transfection efficiency following
nebulisation with the NCD 57
In vivo nebulisation with the NCD VS intratracheal instillation 58
Summary and conclusions 60
Acknowledgements 62
References 64
Abbreviations
A-A-M 2-acetamido-2-deoxy-D-glucopyranosyl-E-(1-4)-2-
acetamido-2-deoxy-
D-glucopyranosyl-E-(1-4)-2,5-
anhydro-
D-mannofuranose
AGT Alternative gene therapy
AUC Area under the curve
CMV Cytomegalovirus promoter
Da Dalton
D
A
Degree of acetylation
DNA Deoxynucleic acid
DP Degree of polymerisation
DP
n
Number-average degree of polymerisation
DS Degree of substitution
FA Fraction of acetylated units
GFP Green fluorescent protein
GlcNAc N-acetylglucosamine
GMP Good manufacturing practice
HSPG Heparan sulphate proteoglycans
Luc Luciferase reporter
mRNA Messenger ribonucleic acid
M
w
Molecular weight
NCD Nebulisation catheter device
NLS Nuclear localisation signal
NPC Nuclear pore complex
ODN Oligodeoxynucleic acid
PBS Phosphate-buffered saline
pDNA Plasmid DNA
PEI Polyethyleneimine
PLL Poly-L-lysine
RNA Ribonucleic acid
RNAi RNA interference
siRNA Small interfering RNA
TCO Trisaccharide-substituted chitosan oligomers
UPC Ultrapure chitosan
11
Introduction
Despite the increasing importance of biotechnology drugs (peptides, proteins
and nucleic acids) in drug treatment, these drugs remain difficult to deliver
by routes other than parenteral, using conventional formulation approaches.
Most biotechnology drugs are hydrophilic, and have much higher molecular
weights (M
w
) than conventional drugs. These characteristics limit their dis-
tribution across biological barriers and make them sensitive to degradation in
the body fluids by endogenous processes. Drug development scientists are
therefore searching for new approaches that improve the delivery of macro-
molecular drugs, especially across mucosal tissues [1]. In this thesis, the use
of the natural polysaccharide chitosan as a delivery vehicle for nucleic acids
is described as one of these newly developed approaches.
Nucleic acids and oligonucleotides as potential
pharmaceutical products
The concept of gene medicine is based on the use of nucleic acids as drugs
for gene therapy with the aim of restoring or shutting down a specific cellu-
lar function [2]. In contrast to conventional medicines that often focus on the
treatment of clinical symptoms, gene medicines provide treatment at the
molecular level of deoxyribonucleic acids (DNA) or ribonucleic acids
(RNA), i.e. at the intracellular gene expression level. The first successful
gene therapy-based clinical trial was reported in the early 1990s. In this
clinical trial, immune competence was restored to children suffering from X-
linked severe combined immunodeficiency (SCID), so that they were able to
live outside the sterile environment “bubbles” to which they have been con-
fined [3,4]. By September 2006, 1192 clinical gene therapy trials had been
approved and conducted worldwide; most of those involved devastating,
acquired or inherited diseases such as cancer, monogenic disorders
(e.g. cystic fibrosis) and infectious diseases (e.g. AIDS)
( In addition to gene therapy,
gene vaccination is another application of gene medicine [5]. The introduc-
tion of genes encoding for various pathogenic antigens into target cells can
result in the production of cellular and humoral (antibody) immune re-
sponses [6].
12
Two major approaches are used for the transfer of therapeutic nucleic acids
(genetic material) to the target area for gene therapy (Figure 1). 1) Ex vivo:
the genetic material is first inserted into cells grown in vitro (cell cultures).
The transfected cells are then selected, expanded, and introduced into the
patient. To avoid rejection by the host immune system, cells (e.g. bone mar-
row cells) are usually obtained from the same patient (autologous cells) prior
to the procedure. 2) In vivo: the genetic material is transferred directly into
the target cells in the patient. This process involves less manipulation than
the ex vivo approach. It may also be the only option in tissues where individ-
ual cells cannot be obtained or cultured in sufficient quantities or the cul-
tured cells cannot be re-implanted.
Figure 1. In vivo and ex vivo strategies for the transfer of genetic materials in
human gene therapy [7].
Nucleic acids that can be used as gene medicines can take a variety of forms.
One such is plasmid DNA (pDNA), a macromolecule that carries a specific
gene sequence (transgene) encoding the desired protein(s). Upon delivery to
the target cells (the transfection process), the gene sequences are eventually
translated as the desired therapeutic functional or structural protein(s) (Fig-
ure 2). In contrast, gene-silencing techniques (antigenes) such as antisense
13
oligodeoxynucleotides (ODN), ribozymes, DNAzymes and the more re-
cently introduced small interfering, double-stranded RNA (siRNA) are nu-
cleic acids that can bind to specific target sequences of intracellular mRNA
and subsequently block its translation (Figure 2) [8-10]. This process can
lead to specific knock down (silencing) of the targeted cellular proteins or
functions. More specifically, the process of RNA interference (RNAi) by
siRNA involves incorporation of the short double-stranded RNA into a pro-
tein structure to produce an RNA-induced silencing complex (RISC), which
recognizes and binds to the target mRNA sequence, resulting in cleavage
[11]. In this thesis, pDNA and, to a lesser extent, siRNA are used.
Figure 2. The central dogma presented by Crick, showing the flow of genetic
information from DNA to messenger RNA (mRNA) to protein [12]. In addition, a
general model of gene silencing is presented. ODN and siRNA recognize mRNA
sequences and subsequently cleave the mRNA or block its translation.
One antisense drug, for the treatment of cytomegalovirus (CMV) rhinitis in
AIDS patients, has been approved by the FDA for clinical use [9]. Several
more drug products that are based on gene-silencing techniques, especially
siRNA are being investigated in clinical trials at various phases, which sug-
gests that more marketed nucleic acid-based drug products may be available
in the coming few years [13,14].
The achievement of significant clinical or therapeutic benefits with nucleic
acid-based drugs has, however, been challenged by several obstacles. The
low efficiency and toxicity of various systems (viral or non-viral) or methods
(physical or chemical) used for the cellular delivery of nucleic acids have
caused various problems for successful clinical gene therapy [15,16].
14
Gene delivery systems
Viral gene delivery systems
Viral gene delivery systems are based on recombinant viruses that are ge-
netically modified to be replication-defective and unable to cause diseases
when introduced into the human body. Viruses that have been used in gene
therapy protocols include retroviruses, adenoviruses, adeno-associated vi-
ruses, vaccinia viruses and herpes simplex viruses [17-22]. The first clinical
gene therapy protocol approved for the treatment of SCID, in 1990, involved
a retroviral vector. Since then, almost 70% of clinical protocols for gene
therapy have used viral delivery systems
( The ability of viruses to
condense nucleic acids and provide protection against enzymatic degrada-
tion, together with their highly specialised mechanisms for cell infection
(cell binding and penetration, escape from the intracellular compartments,
active transport of the genetic material into the nucleus, etc.) has resulted in
their current position as the most effective gene delivery systems [23]. How-
ever, significant limitations are inherent to their use in humans. Viral vectors
(e.g. retroviruses) may provoke insertional mutagenesis, in which the ran-
dom integration of the viral DNA with the host cell genome may result in
disruption of the expression of a tumor-supressor gene or activation of an
oncogene leading to the malignant transformation. Recent reports on the
development of leukaemia in children treated with viral vectors for SCID
provided evidence for the toxicity problems associated with viral delivery
systems [24]. Repeated administration of viral vectors (e.g. adenoviruses)
may induce an immune response, which can abolish transgene expression
[18]. Other problems associated with viral delivery systems include their
limited gene-carrying capacity, restricted cell-targeting and high large-scale
production cost.
Non-viral gene delivery systems
The increased risk of using viral delivery systems for gene therapy of genetic
or acquired human diseases has motivated the search for, and utilisation of,
safer carrier molecules. Non-viral delivery systems such as naked or formu-
lated nucleic acids have important advantages over viral approaches, includ-
ing their reduced propensity for insertional mutagenesis and pathogenicity,
as well as their relatively low cost and ease of production [25].
15
Naked (unformulated) nucleic acids
In non-viral gene delivery, the desired therapeutic gene(s) is usually incorpo-
rated into pDNA, which is a circular double-stranded DNA molecule of bac-
terial origin. Besides the therapeutic gene(s), pDNA contains other important
gene sequences such as promoter/enhancer elements, which are responsible
for controlling the transcription and expression levels of the encoded protein
once it is introduced into the target cells [26]. As a result of the inclusion of
these elements, a typical plasmid comprises 5,000-10,000 base (nucleotide)
pairs and has a molecular mass of more than 1 million Daltons (Da) [23].
pDNA is a strong polyanion because of the phosphate groups in its nucleo-
tide backbone. It can exist in three different conformations (supercoiled,
open circular and linear), of which the supercoiled form is preferred because
of its more compact structure. In physiological salt solutions, the phosphate
groups of the pDNA bind counter ions, which may further contribute to the
compactness of the plasmid structure [27]. pDNA can elicit immune re-
sponses as a result of the unmethylated CpG motifs [28]. While this may be
an advantage in development gene vaccines, this effect can be masked by the
methylation or exclusion of these motifs from the pDNA sequence [29].
Barriers to non-viral gene delivery
Although non-viral gene delivery systems largely lack the adverse effects of
viral delivery systems, their clinical application has been limited as a result
of their poor gene transfection efficiency. A better understanding of the vari-
ous barriers encountered by non-viral gene delivery will significantly con-
tribute to the development of nucleic acid-based formulations into therapeu-
tic products for human gene therapy. These barriers can generally be classi-
fied into three major categories: pharmaceutical, extracellular and cell-
surface and intercellular barriers (Figure 3) [25,30,31]
Pharmaceutical barriers
One of the main pharmaceutical barriers involves the manufacture of suit-
able, non-toxic delivery systems that possess backbone structures amenable
to chemical modification. These delivery systems should be able to compact
various nucleic acids into physically stable particles. The ability to prepare
well-defined particles that are stable upon storage is crucial for the develop-
ment of any pharmaceutical product, and nucleic-acid formulations should
possess such a property to provide a practical “bedside” medicine [32].
16
Figure 3. General schematic representation of cell-surface and intracellular barri-
ers to the expression of a transgene delivered by non-viral plasmid-based formula-
tions. These include (1) complexation of pDNA with the delivery system; interaction
of DNA-based complexes with the cell membrane; cellular internalisation via (2)
nonspecific or (3) receptor-mediated endocytic pathways; (4) endosomal entrap-
ment followed by (5) the development of lysosomes or (6) the rupture and cyto-
plasmic release of the complexes or pDNA alone [cytoplasm is the site of action for
antisense oligonucleotides (ODN and siRNA), ribozymes, DNAzymes and aptam-
ers]; (7) nuclear translocation [the nucleus is the site of action for transgenes in
plasmids for gene therapy and siRNA-generating plasmids]; and (8) transcription
followed by (9) the expression of the transgene into the desired therapeutic product.
Extracellular barriers
On introduction into physiological fluids, either cell culture media in vitro
or, the body fluids such as blood in vivo, naked or formulated nucleic acids
will encounter a hostile environment. In vitro, particle size stability is one of
the most critical issues that can affect the transfection process [33]. Non-
stabilised nucleic acid formulations tend to aggregate in physiological media
and sediment onto the surface of cells as a result of the reduced colloidal
stability. While this could be an advantage in transfecting adherent cells,
uncontrolled aggregations could impair cellular uptake because of size con-
straints associated with cellular uptake mechanisms, mainly endocytosis
17
(vesicular uptake of extracellular molecules) [34,35]. Various uptake path-
ways involved in non-viral gene delivery are shown in Figure 4.
Following in vivo systemic administration of formulated nucleic acids, inter-
action with the plasma proteins and blood cells can result in aggregation, fast
clearance and a reduced circulation time in the human body [36]. Further,
the abundance of nucleic acid-degrading enzymes (nucleases) in the blood or
in the mucosal sites can lead to a substantial loss of the expected therapeutic
effect [37]. However, besides the poor serum stability and unfavourable
pharmacokinetics in vivo, one of the most critical toxic effects preventing
repeated administration of these formulations is stimulation of the immune
response [38].
Figure 4. Portals of entry into the
mammalian cell. The endocytic
pathways differ with regard to the
size of the endocytic vesicle, the
nature of the cargo and the mecha-
nism of vesicle formation [34,35].
Cell-surface and intracellular barriers
Binding and uptake
Once the naked nucleic acids are at the cell surface, the negatively charged
pDNA and oligonucleic acids, in general, exhibit little interaction with the
negatively charged components of the cell membrane (glycoproteins, glycol-
ipids and proteoglycans). Further, size-restrictions can hamper cellular up-
take via endocytosis [34,35]. Therefore, an optimal delivery system should
physically condense nucleic acids into small particles (e.g. through ionic
complexation) and should facilitate their cellular binding and uptake. This
can be achieved, either non-specifically by providing excess positive surface
charges on the particles or specifically via receptor-mediated endocytosis by
actively targeting specific receptors on the cell surface [39-42].
18
Release from endosomes
Following uptake and internalisation, the formulated nucleic acids are
trapped in intracellular compartments called endosomes. The endosomes
mature from early to late stages with a simultaneous drop in pH from 6.0 to
5.0. They then fuse with lysosomes, and a significant fraction of the trapped
nucleic acids is eventually degraded by the lysosomal hydrolytic enzymes,
leading to a substantial reduction in the transfection efficiency [43]. It would
thus be preferable if the delivery system could facilitate early escape of the
nucleic acids from the endosomes. This can be achieved either by disrupting
the endosomal membrane or by inducing endosomal swelling and rupturing
[44-46]. Membrane active peptides, either synthetic or derived from viruses
have been shown to enhance the endosomal release of non-viral gene deliv-
ery systems [47-49].
Unpacking
In general, the disassembly of nucleic acids from the delivery system is es-
sential for the transcription and expression of the desired function [50]. This
process can take place during endosomal release, where anionic cell mole-
cules such as lipids can displace the nucleic acids [44]. However, unpacking
of the formulated nucleic acids can occur after endosomal release in the cy-
tosol or even later in the nucleus [51-53]. The chemical nature of the deliv-
ery system can influence the release by controlling the strength (tightness) of
the interaction with the nucleic acids. In other words, the physical stability of
the nucleic acid formulation can be a limiting factor in the disassembly of
the nucleic acids [54].
Diffusion in the cytoplasm
For oligonucleic acids that exert their mechanism of action in the cytoplasm,
such as siRNA, the critical intracellular steps include efficient cellular bind-
ing and uptake, early escape from the endocytic vesicles and stability against
intracellular enzymatic degradation [55]. However, larger pDNA has to
overcome additional hurdles to access the nucleus for the transcription of the
encoded therapeutic genes into mRNA. After endosomal escape, pDNA has
to diffuse through the dense microfilaments and microtubule (cytoskeleton)
network, as well as through a variety of subcellular organelles bathing in the
cytosol [56]. In the highly packed cytosol, the movement of pDNA is size-
dependent, and DNA larger than 3,000 base pairs appears to be practically
immobile [57]. The restricted mobility of pDNA in the cytosol necessitates
its active transport to the nucleus [58].
Nuclear translocation and expression
The nuclear pore complex (NPC), which is located on the nuclear mem-
brane, is the ultimate obstacle to the entry of pDNA into the nucleus [59]. As
19
with diffusion in the cytoplasm, passage through the NPC is size-dependent
[60,61]. The diameter of the NPC is 9 nm, but this can be extended to 20-25
nm during active translocation [62,63]. This pore size range implies that it is
almost impossible for pDNA to passively diffuse to the nucleus through the
NPC [58,64]. However, coupling of a nuclear localising signal (NLS) to the
pDNA or to the delivery system can provoke the enlargement of the NPC
through conformational changes [65-67]. Alternatively, pDNA can access
the nucleus during cell division (mitosis) when the nuclear envelope is disas-
sembled [68]. However, this method does not significantly contribute to the
efficiency of nuclear translocation in vivo since most human cells are non-
dividing [69].
Nuclear entry does not guarantee long-term expression of the encoded trans-
gene. In most cases, only transient gene expression can be obtained as a re-
sult of transcriptional silencing of the episomal pDNA (extrachromosomal;
non-integrating pDNA into the cellular genome) in the nucleus [70,71].
Recent advances suggest that efficient, long-term expression could be attain-
able through site-specific integration of pDNA into “safe sites” on the cellu-
lar genome, i.e. those that are not associated with cell proliferation or tumour
suppression [72]. In addition, the introduced transgene may be maintained in
the nucleus in a stable manner by the design of DNA molecules that can be
replicated and transferred to daughter cells in an extrachromosomal form
[73,74].
Bacterial gene delivery (bactofection) and alternative
gene therapy (AGT)
Bacteria-mediated transfer of pDNA (bactofection) is a technique, in which
genetically modified (nonpathogenic) bacteria are used to transfer genes
directly into the target organ or tissue [75]. The basic idea of bactofection
was introduced in 1980; transformed bacteria were used to deliver genes
located on plasmids into cultured mammalian cells [76]. The main advan-
tages of bactofection are the simplicity of application and the possibility of
selective gene transfer. Another interesting approach; alternative gene ther-
apy (AGT) was also proposed to be useful for gene therapy application. In
AGT, instead of using the transferred bacteria for pDNA delivery, they are
exploited as a factory for the production of therapeutic peptides or proteins
in situ [77]. AGT allows the levels of the expressed therapeutic product to be
controlled or even stopped by using specific antibiotics or activation of sui-
cidal genes in the bacteria. Several studies have reported successful applica-
tion of bactofection and AGT in genetic vaccination and gene therapy of
several tumors, and monogenic and infectious diseases in vivo [78-80]. How-
ever, both bactofection and AGT share several serious adverse effects that
20
are mainly related to host-bacteria interactions. Stimulation of the host im-
mune system by the introduced bacteria or the products of their lysis can
lead to rapid clearance of the bacteria or even autoimmune reactions.
Physical methods of non-viral gene delivery
Several strategies have been developed with the aim of enhancing the effi-
ciency of gene transfection using pDNA in vitro and in vivo. One strategy
involves the use of physical methods of delivery such as nuclear microinjec-
tion, particle bombardment (ballistic delivery via gene gun), and electropora-
tion (short, controlled electric pulses) [81-84]. These methods avoid the
problems associated with endocytosis and facilitate the introduction of
pDNA into the intracellular environment either directly (injection) or
through the disrupted cell membrane. Examples of physical methods other
than nuclear microinjection were shown to be efficient in the local in vivo
delivery of naked pDNA to tissues such as the skin and skeletal muscles
[85,86]. One important application of such methods is gene vaccination
[86,87].
An alternative physical method for in vivo targeting involves hydrodynamic
injection, where the naked nucleic acids are injected in large volume under
high pressure into the tail vein of animal models. This technique has proved
to be efficient for pDNA and siRNA targeted delivery (passive targeting)
mainly to the liver and, to a lesser extent to the skeletal muscles [88,89].
However, as for nuclear microinjection, hydrodynamic injection is of minor
application in clinical practice [90].
Chemical methods of non-viral gene delivery
Chemical methods of gene delivery involve the formulation of negatively
charged nucleic acids with various polycations. e.g. cationic lipids and cati-
onic polymers, into micro- or nanoparticulate structures (complexes) through
electrostatic, ionic interactions (Figure 5) [7,91]. The driving force behind
the complex formation is the release of the counter ions associated with the
polycations and the accompanying substantial gain in entropy [92]. The
physicochemical (size, morphology, surface charge and stability) and bio-
logical properties of the complexes are dependent on factors such as the
chemical structure of the polycation, the polycation/nucleic acid stoichiome-
try (+/- charge ratio), the pH and the order of mixing. To distinguish the
origins of the complexing molecule, lipid-based formulations are referred to
as lipoplexes and polymer-based formulations as polyplexes [93].
21
Figure 5. Schematic illustration of polyplex (pDNA/cationic polymer) formation
and pDNA compaction as visualised by atomic force microscopy.
Lipids
The early attempts to formulate pDNA with neutral or anionic lipids were
not successful because the resultant liposomes (spherical lipid-bilayer vesi-
cles that can encapsulate nucleic acids) had poor physical properties (large
particle size) as well as poor transfection efficiency [94]. In 1987, the suc-
cessful use of a cationic lipid (DOTMA; N-(1-(2,3-diolyloxy)-propyl)-N,N-
trimethylammonium bromide) as a non-viral gene delivery system was re-
ported for the first time [95]. Following this report, a variety of cationic lip-
ids were introduced to the field of gene delivery. These lipids can be either
monovalent (DOTMA, DOTAP) or multivalent (DOSPA: diolyloxy sper-
minecarboxamidoethyl diethylpropanaminium trifluoroacetate; DOGS: dioc-
tadecylamido-glycylspermine, Transfectam™) [96,97]. Other neutral, helper
lipids (co-lipids) were also introduced to improve the transfection efficiency
of cationic lipids especially those of the monovalent type. Such helper lipids
include DOPE and cholesterol [98-100]. Commercially available lipid com-
binations for gene delivery include Lipofectin™ (DOTMA/DOPE) and Li-
pofectamin™ (DOSPA/DOPE).
Upon lipoplex formation, multilamellar structures of a size range of 0.2-1.0
µm are produced with the nucleic acid monolayers sandwiched between the
lipid bilayers. In general, lipoplexes formulated with monovalent lipids are
physically unstable; they aggregate to give rise to heterogeneous shapes that
have been described as “spaghetti and meatballs” [101,102]. In order to pre-
pare more defined lipid-based complexes, cationic thiol-detergents have
been used to compact individual pDNA molecules into particles of around
32 nm. The lipoplexes packed with pDNA molecules are physically stabi-
lised by an oxidation-induced dimerisation of the detergent into a disulphide
22
lipid on the template pDNA [103]. Recently, these lipoplexes were also
shown to be stable following intravenous injection in vivo [104].
The improved transfection efficiency of lipoplexes over naked pDNA is
attributed to their resistance to enzymatic degradation, improved cellular
uptake and efficient endosomal release. Besides their efficiency as pDNA
delivery systems, lipid-based formulations were reported as efficient deliv-
ery systems for siRNA in vitro and in vivo [105-107].
Although by 2005 8.3% of the gene therapy clinical trials were based on
lipoplexes ( several problems
continue to limit their application as drug products for human use. For in-
stance, the formation of lipoplexes involves complex interactions between
the lipid molecules, in addition to those with nucleic acids. Additionally, the
ability to control the size and morphology (colloidal satiability) of lipoplexes
is rather limited, with resultant instability problems over time [108].
Furthermore, toxicity associated with the use of lipoplexes in terms of im-
mune stimulation of the host can also limit their in vivo application. The
toxicity of lipoplexes is closely associated with the administered dose and
may, in part, result from their large size and the high positive surface charge
[109,110]. However, the resulting inflammatory responses of lipoplexes
could be advantageous in specific applications such as vaccination and anti-
tumour immunotherapies [111].
Polymers
In contrast to the large number of clinical trials investigating lipoplex-based
gene therapy, gene therapy based on cationic polymers (polyplexes) is still in
its infancy. This seems “paradoxical” since polycations were being used for
the insertion of DNA into cells long before lipid formulations [112]. How-
ever, progress was marginal until the introduction of polyethyleneimine
(PEI) in 1995. This was primarily due to the low transfection efficiency of
the used systems such as poly-L-lysine and protamine sulphate [46]. Cati-
onic polymers are interesting alternatives to cationic lipids in many respects.
The self assembly of polyplexes does not involve interactions of the polyca-
tion molecules with each other, which results in better control of their physi-
cal properties compared with lipoplexes. In addition, the chemical structure
of various polycations comprises repeated units that can be easily manipu-
lated by chemical modification to improve the physical and biological prop-
erties of the resultant polyplexes, and consequently enhance their transfec-
tion efficiency [108,113].
Several naturally occurring proteins, such as histones, cationised human
serum albumin and gelatin, have been employed as non-viral gene delivery
systems [114-116]. However, the low transfection efficiency of these pro-
23
teins compared with that of the recently introduced synthetic cationic poly-
mers has limited their further application. Examples of commonly used syn-
thetic cationic polymers as non-viral delivery systems for nucleic acids in-
clude PEI, polyamidoamine (PAMAM) dendrimers, poly-L-lysine (PLL) and
chitosan (Figure 6).
Figure 6. Cationic polymers most commonly used for nucleic acid delivery [117].
Of the various cationic polymers, PEI has displayed several properties that
placed it as the gold standard and one of the most efficient non-viral systems
for nucleic acid delivery. Therefore, PEI has been selected as a reference
delivery system in this thesis, and the properties of PEI-based polyplexes
will be discussed below in more detail.
Polyethyleneimine (PEI)
Since the introduction of branched and linear PEI, the properties of PEI-
based polyplexes have been extensively studied with the aim of devising
safer, target-specific PEI derivatives [118-122]. Various PEI derivatives
have been used to deliver oligonucleotides, ribozymes, RNA and pDNA in
vitro and in vivo. The transfection efficiency and the toxicity of PEI depend
to a great extent on material characteristics such as the M
w
, degree of
24
branching and cationic charge density. While high M
w
PEI (800 kDa) was
associated with increased cellular toxicity in some studies, lower M
w
coun-
terparts (5-48 kDa) formulated at higher charge ratios were better tolerated
in cell cultures [123,124].
PEI is able to physically condense nucleic acids, especially pDNA, into
small nanoparticles (less than 100 nm) that are suitable for cellular uptake.
The particle size and shape of PEI-based polyplexes depend on the structure
and M
w
of PEI, the pH and ionic strength of the surrounding environment,
the method of preparation and the charge ratio used for polyplex formula-
tion. An excess of PEI (i.e. higher charge ratios than 1:1 +/-) is required to
produce smaller, positively surface-charged polyplexes that display im-
proved colloidal stability [125].
In addition to enhanced pDNA packing and physical stability, the presence
of excess positive charges on the surface of the polyplexes facilitates interac-
tion with the negatively charged cell membrane components and conse-
quently enhances the internalisation of the polyplexes in vitro [126]. As for
cationic lipids, it has been proposed that extensive damage of the cell mem-
brane due to high positive charge density contributes to the cytotoxicity of
PEI polyplexes [109,127]. Fluid-phase and adsorptive endocytosis have been
reported as possible cellular uptake mechanisms of PEI polyplexes in vitro
[128,129].
It has been postulated that the high transfection efficiency of PEI may be due
to the ability of the polymer to capture protons in the slightly acidic en-
dosomal compartments. This buffering capacity makes PEI act as a “proton
sponge”, allowing the cell to pump more protons together with chloride ions
and water into the endosomes, leading to the swelling and eventual rupture
of the endosomal membrane [46]. Although this mechanism has been criti-
cised, several recent reports support the role of the buffering capacity of PEI
in its superior transfection efficiency in vitro [130,131].
Upon cytoplasmic release, PEI can protect the complexed nucleic acids from
enzymatic degradation. Moreover, PEI polyplexes undergo intracellular traf-
ficking and nuclear translocation more efficiently than lipoplexes or naked
pDNA [61]. Interestingly, PEI polyplexes were shown to be transported in
the cytoplasm by more than one mechanism: diffusive transport, restricted
transport (by altering the structure of the cytoskeleton) and active transport
through the microtubules [132]. Unlike lipids, intact polyplexes formulated
with branched PEI were found inside the nucleus [52,128]. Accordingly, cell
division is not a prerequisite for the nuclear translocation and expression of
nucleic acids formulated with PEI in vitro [133]. However, the presence of
25
free or complexed PEI in the nucleus may, in part, explain its increased in-
tracellular toxicity [134,135].
In contrast to studies of cationic lipids, initial investigations indicated that
cationic polymers (e.g. PEI) were unsuitable for the delivery of oligonucleic
acids such as ODN and siRNA [136]. However, several recent studies have
reported contradictory findings [137,138]. One possible reason for this dis-
crepancy is poor characterisation of the critical formulation parameters re-
quired for the efficient delivery of oligonucleic acids. Therefore, the struc-
ture-activity relationship of siRNA delivery will be addressed in this thesis.
Polyamidoamine (PAMAM) dendrimers
PAMAM dendrimers (e.g. Superfect™) are highly branched cationic poly-
mers that are of comparable efficiency to PEI [139].
Poly-L-lysine (PLL)
PLL is a linear biodegradable polymer composed of repeated lysine units.
Polyplexes based on PLL are generally less efficient than those of PEI and
PAMAM dendrimers. The most important reason for the poor transfection
efficiency of PLL-based polyplexes is their minimal ability to escape from
the endosomes [140]. Thus, PLL-mediated gene delivery requires co-
administration of endosomolytic agents such as chloroquine for an efficient
escape from the endosomal compartments [141]. However, this approach is
not practical for in vivo application because of the dose-dependent toxicity of
chloroquine. Further, it is hard to target chloroquine to the specific cells
transfected by PLL polyplexes [142]. The introduction of chemical residues
such as histidine, which can be protonated at the slightly acidic pH of the
endosomal compartments, to PLL has significantly improved the transfection
efficiency by facilitating endosomal escape of the polyplexes [142]. The
greater efficiency of the histidylated PLL compared to unmodified PLL pro-
vides further evidence for the “proton sponge” theory of the mechanism of
action of PEI.
Strategies to improve the in vitro/in vivo efficiency of
non-viral gene delivery: Structure-activity relationship
Molecular weight reduction
Several recent studies have reported higher transfection efficiency and lower
cellular toxicity of low M
w
PEI (less than 25 kDa) than for the higher M
w
counterpart [123,137,143-145]. In one of these studies, the biocompatibility
of low M
w
modified PEI was superior to that of non-degradable PEI (25
