Introduction
Gene therapy is a rapidly growing field of medicine in which genes are
introduced into the body to mitigate or cure a disease. Genes control heredity and
provide the basic biological code for determining cell's specific functions. Gene
therapy is not based on altering the human germline, but on aiding the human body
to fight a disease or the expected onset of a disease. Initially conceived as an approach
for treating inherited diseases, like cystic fibrosis and huntington's disease, the scope
of gene therapy has grown to include treatment for cancer, arthritis, and infectious
diseases. At onset, the primary goal of gene therapy was to swap a deficient gene in a
genetically inherited disease with a normal copy to restore production of functional
protein. Later this goal was broadened to include genetic defects beyond inherited
disorders, as numerous acquired diseases also involved alteration in the regulation of
gene expression. Therefore, gene therapy apart from replacing a defective gene could
also modulate gene expression and integrate functions into cells not originally present
but which could serve as a therapeutic purpose. Accordingly, in a modern concept,
gene therapy refers to the potential use of gene medicines such as nucleic acids,
antisense oligonucleotides or siRNA, to modulate the expression of genes in cells for
therapeutic purposes.
Most research and testing has been done by introducing a sequence that codes
for a required protein in order to counter a deficiency, induces a strong immune
response, or destroys tumor cells. The direct method of cure by replacing either a
deletion or mutation in genome includes treatment of hemophilia and cystic fibrosis
[1]. However, this method often may not work as many genetic diseases are polygenic
and do not easily lend themselves to such corrective methods.
The other approach of gene therapy involves altering the immune system.
Some individuals may be genetically predisposed to certain diseases due to an
immune deficiency, and therefore, may need a boosted immune response to
successfully combat diseases [1]. The immune response is, however, not always
helpful, especially when it is necessary to introduce foreign cells into the body. When
a transplant is performed, not only the body’s immune system may attack the needed
cells, but the transplanted T-cells could also attack some of the body’s vital organs
1
Introduction
such as liver, gut and skin. To defeat this disease called graft-versus-host disease
(GVHD), a gene therapy technique was developed involving a drug-activated
response. Genetically engineered with a self-destruct button, these “suicide” genes are
genetically altered to include a sequence that, when triggered by a drug, will make the
cell toxic. If a transplanted bone marrow’s T-cells begin to attack the host’s body, the
drug can be administered and the foreign cells would be destroyed before GVHD can
develop [2]. The ability to express an introduced gene at any time and for any
duration by simply swallowing a pill makes this type of gene therapy very practical.
This offers an attractive and controlled form of administering therapeutic proteins
such as monoclonal antibodies, interferons and even certain growth factors.
The significant achievement of clinical or therapeutic benefits with nucleic acid-
based gene medicines has, however, been challenged by several obstacles. The
journey of genes from needle to nucleus of the cell is fraught with barriers (Fig. 1). The
clinical applications of gene therapy remain limited today because the vectors (viral
and non-viral) for gene delivery encounter “cellular barriers” that affect the in vivo
expression levels. The major biological barriers to gene transfer are described in Table-
1 and suggest opportunities that can be employed to circumvent them.
Figure 1. Schematic representation of barriers to the expression of a transgene delivered by non-viral formulations. (1) Complexation of pDNA with delivery agent; (2) cellular internalization; (3) receptor-mediated endocytic pathway; (4) endosomal entrapment; (5) development of lysosomes; (6) endosomal escape; (7) nuclear translocation; (8) transcription; (9) transgene expression.
2
Introduction
Table 1: Biological barriers, challenges and opportunities
The in vivo barriers may broadly be classified into extracellular and
intracellular barriers:
Extracellular barriers
[i] Opsonins[ii] Phagocytic cells [iii] Degradative enzymes[iv] Extracellular matrix
Location Nature of barrier Challenges OpportunitiesBlood circulation
CapillaryEndothelium
Tissue interstitium
Cell surface
Endosome
Cytoplasm
Blood nucleases, Particle instability, Particle opsonization, Clearance by macrophages [in liver, spleen, etc.], Unwanted capillary blockade
Tissue specific characteristic:Continuous [muscle, skin, lung, etc.], Fenestrated [kidney, endocrine glands, etc.], Discontinuous [liver, spleen]
Extracellular nucleases,Poor distribution within tissues, High hydrostatic pressure in tumors
Poor cellular internalization
Trafficking to lysosome and consequent degradation
Inefficient cytoplasmic transport, Poor uncoupling of DNA and carrier
Protection of DNA from nucleases, Steric stabilization of particles, and Prevention of unwanted opsonization
Extravasationparticularly in organs with endothelia
Protection of DNA from nucleases, Prevention of unwanted binding, which prevents convective flow
Optimize physical properties for uptake, Understand uptake of naked DNA, Maximize rate of uptake by receptor mediated uptake
Incorporate endosomolytic agent
Optimize size of escape from the vesicular system
Systemic delivery after i.v. injection, Selectivity mediated by tissue-specific promoters
Delivery to hepatocytes via discontinuous endothelium,Delivery to tumors, Delivery to sites of angiogenesis, and Selective, receptor-mediated extravasation
Direct intramuscular injectionextended expression of genes for systemic effect, expression of DNA vaccines [also in skin], Direct injection into tumors
Selective uptake by receptor mediated endocytosis, Direct uptake into cytoplasm using membrane active peptides
Utilize viral peptides for escape of endosome or proton sponge properties of vector
Make use of microtubule transport system to deliver DNA to perinuclear region
3
Introduction
Opsonins are proteins that attach themselves to a gene or a delivery system
thereby making it visible to phagocytic cells. Phogocytes are cells that seek out, engulf
and actively digest the delivery systems. After opsonization, phagocytosis occurs,
which is the engulfing and eventual destruction or removal of foreign materials from
the bloodstream. Together these two processes form the main clearance mechanism
the blood. Without the presence of surface bound or adsorbed opsonin proteins, the
removal of undesirable components larger than the renal threshold limit from
phagocytes will typically not be possible. The bound opsonin proteins undergo
conformational changes from an inactive protein present in the blood serum to an
activated protein structure that can be recognized by phagocytes. Phagocytic cell
surfaces contain specialized receptors that interact with the modified conformation of
these various opsonins thus alerting them to the presence of a foreign material.
Alternatively, the non-specific adherence of phagocytes to surface adsorbed blood
serum proteins can result in the stimulation of phagocytosis as well [3]. Complement
activation also results in the binding and phagocytosis of the foreign particle by the
mononuclear phagocytes. The other barrier faced by the delivery vehicles is the
DNases present in the serum and extracellular fluid, which can rapidly digest
unprotected DNA. Finally, before entering the cell, the delivery vehicle has to traverse
through the extracellular matrix, which is a zone of polymerized proteins and
carbohydrates present between cells protecting the plasma membrane of the target
cell, and it can be difficult for a relatively large DNA carrier system to pass through
this barrier.
Intracellular barriers
[i] Plasma membrane[ii] Endosome[iii] Nuclear membrane
Once the gene delivery system reaches the target cell, it encounters the plasma
membrane, which must be traversed before the gene can be expressed. The uptake of
most of the macromolecules or particles into cells by passive diffusion across the
plasma membrane is limited due to their low solubility in lipid bilayers and therefore,
4
Introduction
occurs pinocytosis, adsorptive endocytosis, receptor mediated endocytosis or
phagocytosis [4, 5]. After crossing the membrane, it is endocytosed but then the
delivery system must have a mechanism to escape from the endosome lest it is
degraded in the lysosomal compartment. The implication is that a specific or generic
means of escape is required, or much of the internalized DNA will be lost by
degradation. Finally, the gene is able to cross these barriers and enters the cell
cytoplasm, it must still have a means of getting across the nuclear membrane. The
nuclear membrane is a barrier preventing uptake of most macromolecules greater
than 70 kDa into the nucleus, unless they are able to interact with the nuclear pore
active transport system [6]. Any gene delivery system that is intended to be viable for
in vivo applications or gene therapy must at the very least be equipped with the
capacity for rapid endosomal uptake followed by efficient endosomolysis, cytosolic
trafficking and nuclear entry. Obviously, efficient nuclear entry is only required for
DNA but not if RNA is involved. Conceivably, benefits could be had alternatively by
avoiding endocytosis altogether and harnessing alternative cellular uptake
mechanisms. However, this may well depend upon both vector characteristics and the
nature of cells in the organs of choice that have been selected for nucleic acid delivery.
In order to overcome these barriers, various gene delivery systems have been
developed that involve aspects of molecular biology, DNA condensation technology
and ligand conjugation chemistries. Therefore, for successful gene therapy, an
efficient, safe and selective vector, an appropriate technique and an appropriate gene
are the prerequisites.
The vectors for delivering genes into cells can broadly be classified into the
following categories:
[i] Physical methods,[ii] Viral vectors, and [iii] Non-viral vectors
Each of these classes of gene delivery vectors have been employed to
selectively deliver therapeutic gene to various cells.
Physical Methods
5
Introduction
A naked DNA injection, without any carrier, into local tissues or into the
systemic circulation is probably the simplest and safest ‘physical/mechanical’
approach. However, due to rapid degradation by nucleases and fast clearance by the
mononuclear phagocyte system, the expression level achieved by this approach is
severely limited [7, 8]. Consequently, attention has turned to a number of other so-
called ‘physical methods’ to improve the efficiency of gene delivery. In order to
augment the gene expression in vivo, numerous physical approaches have been
developed, such as the hydrodynamic method, gene gun, electroporation,
sonoporation, and laser irradiation.
Hydrodynamic method
The hydrodynamic method consists of the very rapid injection through the
mouse-tail vein of a large volume of pDNA solution (e.g. 5µg of DNA in 1.6 ml of
saline solution for a 20g mouse, which is almost equivalent to the total blood volume
of the animal) in order to induce efficient gene transfer in internal organs including
the lung, spleen, heart, kidney and liver, with the highest level of expression being
observed in the liver [9, 10]. Hydrodynamic method for gene delivery was used to
express proteins of therapeutic value such as hemophilia factors, alpha-1 antitrypsin,
cytokines, hepatic growth factors, and erythropoietin in mouse and rat models [11-18].
Gene gun method
The gene gun method employs use of heavy metal particles coated with pDNA
propelled into the target cell at a high velocity and acceleration is achieved by a high-
voltage electric spark, or a helium discharge. The technique was first used in 1987 to
overcome the inherent difficulty of transgene expression in plant cells [19]. The major
application of this technology is genetic immunization with the most obvious target
being the skin [20-22]. The approach has been used for genetic vaccination,
immunomodulation, and suicide gene therapy to treat cancer [23]. Encouraging
results have also been reported at other target sites, including the liver and the brain
[24-28].
Electroporation
6
Introduction
Electroporation technique causes transient and localized destabilization of the
cell membrane with high-intensity electrical pulses. As a result of this perturbation,
the cell membrane becomes highly permeable to exogenous molecules, such as DNA,
present in the surrounding medium. Ideal targets for electroporation are skin, muscle,
liver and solid tumors [29-38]. The efficiency of gene transfer by electroporation is
influenced by several physical (especially, pulse duration and electric field strength)
and biological (including DNA concentration and conformation, cell size) factors [39,
40].
Sonoporation
Sonoporation enhances cell permeability via the application of ultrasound (Fig.
2). Ultrasound covers a broad range of frequencies and wave-forms, but attention has
been principally focused on sonoporation using sinusoidal probes at megahertz
frequencies. Lower frequencies (e.g. 20 kHz) are mainly used for cell lysis and
disruption, while high intensity shock waves are employed for lithotripsy of kidney
and gall bladder stones. Reports on sonophoretic gene delivery were first published in
the mid-1990s [41-43]. Sonoporation has been broadly applied to different tissues,
including solid tumors, muscles, and vasculature [44-48].
Figure 2. Delivery of nucleic acids via sonoporation
Laser irradiation
Laser irradiation method of gene delivery requires a laser source (e.g.
neodymium–ytrium–aluminium garnet, argon ion, holmium–YAG, titanium
sapphire), the power of which is controlled by a pulse generator. The laser beam is
7
Introduction
commonly focused onto the target cell via a lens that leads to the perturbation in the
cell membrane at the site of the beam impact (probably) by a local thermal effect. This
perturbation is sufficient to allow a gene present in the surrounding medium to be
transferred into the cell. At present, gene delivery via laser irradiation is not widely
used and remains a relatively novel approach. The high cost and the physical size of
the laser sources required, as well as the need of an appropriate “know-how”, are the
minor limiting factors, although some reports on in vitro gene delivery via this
method have been published [49-53]. The in vivo gene transfer using a femtosecond
infrared laser has also been reported [54].
Viral Vectors
Viral vectors generally provide the most efficient gene transfer, which is a
result of the viruses evolving to develop efficient methods to introduce DNA into host
cells. The principle of viral gene delivery is to use recombinant viruses that are
genetically modified to make them replication deficient. Viruses are produced using
helper cell lines that create attenuated viruses that can efficiently deliver the
therapeutic gene but are incapable of replicating in vivo. They are the most competent
gene delivery systems owing to their ability to condense nucleic acids in a way to
provide protection against enzymatic degradation, together with their highly
specialized mechanisms for cell infection (cell binding and penetration, escape from
the intracellular compartments, active transport of the genetic material into the
nucleus, etc.) [55]. Class of viruses that have been used for gene delivery includes
retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses and herpes
simplex viruses [56-59].
Retroviruses
Retroviruses are the class of enveloped viruses having single stranded RNA as
its genome. The principle feature of this family is its replicative strategy, which
includes reverse transcription of the viral RNA into linear double-stranded DNA and
the subsequent integration of this dsDNA into the genome of the host cell [60]. A
requirement for retroviral integration and expression of viral genes is that the target
cells should be dividing. This limits gene therapy to proliferating cells in vivo or ex
8
Introduction
vivo, whereby cells are removed from the body, treated to stimulate replication and
then transduced with the retroviral vector, before being returned to the patient.
Lentivirus Vectors
Lentiviruses are a subclass of retroviruses which are able to infect both
proliferating and non-proliferating cells. Early results using marker genes have been
promising, showing prolonged in vivo expression in muscle, liver and neuronal tissue
[61-63]. The lentiviral vectors used are derived from the human immunodeficiency
virus (HIV) and are being evaluated for safety, with a view to removing some of the
non-essential regulatory genes.
Adenovirus Vectors
Adenoviruses are non-enveloped viruses containing a linear double stranded
DNA genome. Adenoviral vectors are very efficient at transducing target cells in vitro
and in vivo, and can be produced at high titres (>1011/ml). Following intravenous
injection, 90% of the administered vector is degraded in the liver by a non-immune
mediated mechanism [64]. Approaches to avoid the immune response involving
transient immunosuppressive therapies have been successful in prolonging transgene
expression and achieving secondary gene transfer [65].
Adeno-Associated Viruses
Adeno-associated viruses (AAV) are non-pathogenic human parvoviruses,
which depend on a helper virus to proliferate. They are capable of infecting both
dividing and non-dividing cells [66]. Interest in AAV vectors has been due to their
integration into the host genome allowing prolonged transgene expression. Gene
transfer into vascular epithelial cells, striated muscle and hepatic cells has been
reported, with prolonged expression [67-70].
Herpes Simplex Virus
Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus. The wild
type HSV-1 virus is able to infect neurons and either proceeds into a lytic life cycle or
persists as an intranuclear episome in a latent state. Although they are less pathogenic
and can direct transgene expression in brain tissue, they are toxic to neurons in
9
Introduction
culture [71]. There has been some success in using HSV-1 for gene therapy in
Parkinson’s disease by expressing tyrosine hydroxylase in striated cells [72, 73].
Because of their immunogenicity and potential oncogenicity, possibly resulting
from mutational insertion defects when the viral genome integrates into the host
genome, viral gene delivery vectors may pose serious problems in terms of safety [74-
76], thus jeopardizing their application in gene therapy. Yet another serious concern is
the long-term effect of the integrated transgene and of the virus in the host individual.
There are other problems also associated with viral delivery systems such as their
limited gene-carrying capacity, restricted cell-targeting and high large-scale
production cost. Consequently, many non-viral vectors are currently being tested for
the delivery of transgenes that, unlike their viral counterparts, are safe and also
amenable to large-scale production.
Non- Viral Vectors
The increased peril of using viral delivery systems for gene therapy of genetic
or acquired human diseases has triggered the search for, and utilization of, safer non-
viral gene delivery vectors. Compared to viral vectors, the non-viral vectors have
certain advantages that make them more attractive candidate for gene delivery such
as their reduced propensity for insertional mutagenesis and pathogenicity as well as
their relatively low cost and ease of production. Additionally, in contrast to viral
vectors they are not limited to the delivery of coding nucleic acids but can
accommodate a greater variety of cargo, including antisense ODNs, siRNAs and
entire genes. Finally, non-viral vectors are better amenable to chemical modifications
for the purpose of effectuating therapeutic applications. One of the widely used non-
viral gene delivery system comprises of an expression repository, inserted into a
plasmid which is ionically complexed to cationic lipid (lipoplex), cationic polymer
(polyplex), or a mixture cationic polymer and lipid (lipopolyplex). These complexes
carry an overall positive charge, which is responsible for interaction with the cellular
membrane. The complexes are subsequently endocytosed and the DNA complex or
DNA alone is transferred to the nucleus.
Lipid-based Non-viral vectors
10
Introduction
A variety of non-viral vectors have been explored for gene delivery in vitro and
in vivo till date. With the introduction of the transfection reagent LipofectinTM, there
has been an increase in the number
of cationic lipids that have been
developed
(Fig. 3).
Figure 3: A representation of the various steps involved in gene transfection by cationic lipids
Quaternary ammonium salt lipids
The family of quaternary ammonium salt lipids comprises of quaternary
ammonium salts covalently attached to a lipid moiety. Examples of quaternary
ammonium salt lipids include biodegradable 1,2-bis(oleoyloxy)-3-
trimethylammoniumpropane (DOTAP), 1,2-dimyristyloxy-propyl-3-dimethyl
hydroxyethyl (DMRIE), 1,2-dioleoyloxy-propyl-3-dimethyl hydroxyethyl ammonium
bromide (DORI), N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-
propylammonium bromide (GAP-DLRIE) [77-83]. The principle underlying these
systems is the formation of electrostatic complexes between the negatively charged
phosphate backbone of DNA and the cationic head groups of the lipids resulting in
the release of low-molecular weight counter-ions associated with the charged lipids
into the external media, which is accompanied by a substantial entropy gain [77, 84,
85]. The common feature of the quaternary ammonium salt lipids is the co-
11
1. Formation of vector/DNA complexes [lipoplexes]2. Cellular uptake via endocytosis3. Endosomal escape to avoid DNA degradation in lysosomes4. Trafficking through the cytoplasm and nuclear entry.5. Transgene expression in the nucleus
Introduction
formulation with a non-charged lipid such as (dioleylphosphatidylethanolamine)
DOPE or cholesterol for efficient transfection [86].
Lipopolyamines
A second family of cationic lipids, lipopolyamines, was designed to take
advantage of the DNA condensing property of a naturally occurring oligoamine such
as spermine [87]. Compared to the quaternary ammonium salt lipids, dioctadecyl-
glycyl-carboxyspermine (DOGS), a carboxy-spermine lipopolyamine yielded
increased transfection efficiency. DOGS efficiently condenses DNA and unlike
quaternary ammonium salts, does not need the help of co-lipids such as DOPE or
dioleoylphosphatidylchloine (DOPC) for efficient transgene expression [88]. The
fluorinated derivatives of DOGS exhibited higher transfection efficiency than that of
DOGS in lung epithelial A549 cells and less cytotoxicity [89, 90].
In another approach, Gao and coworkers [91] employed cholesterol instead of
the double fatty acid chains as lipid entity. They designed 3-{N-(N’,N’-
Dimethylaminoethane)-carbamoyl} cholesterol (DC-Chol) harboring a tertiary amine
group linked via a spacer to cholesterol. The novelty of this approach was the
introduction of a tertiary amine as a cationic entity instead of the quaternary
ammonium salt. Compared to quaternary ammonium salts, tertiary amines are
weaker protein kinase C inhibitors and thus, they display potentially fewer side
effects and toxicities. Although, cholesterol lipids are not capable of forming bilayers,
they, however, can intercalate into bilayers formed by at least 20 mol% of DOPE. DC-
Chol was the first cationic lipid used in clinical trials [92, 93]. Cholesteryl derivatives
are mixed with a neutral co-lipid DOPE or DOPC to form liposomes for efficient
transfection, in a way similar to quaternary ammonium salt lipids.
Amidinium salts cationic lipids
The family of amidinium salt cationic lipid consists of members with a double
lipid chain linked to an amidinium headgroup [94]. The leading reagent of this family
is Clonfectin composed of two C14 lipids linked to the two arms of amino-ethyl
amidine. Other derivatives include lipids based on positively charged guanidinium
headgroups [95, 96].
12
Introduction
Targeted cationic lipids
Cationic facial amphiphiles are a good scaffold to support on one face a
targeting ligand such as glycoside, while the other hydrophobic face remains
fusogenic for cell penetration [97]. Another approach for targeting is by the addition
of targeting ligand to cationic lipid/DNA complexes after appropriate formulation
[98].
Biodegradable cationic lipids
Cytotoxicity of cationic lipids is a major concern limiting their uses for in vivo
purposes [99]. One of the approaches to reduce the cytotoxicity is the use of
biodegradable cationic lipids. Carboxylic esters were the first biodegradable
functionality to be used [82, 83]. Another approach for synthesis of biodegradable
cationic lipids is based on guanidinium head groups [100]. These lipids contain acetal
functionalities, which undergo successive hydrolysis in acidic conditions. More
advanced promising structures contain a sensitive disulfide bridge between the polar
and aliphatic domains in the cationic lipids [101, 102]. The early release of DNA
during or just after penetration into the cell, probably promoted by the reduction of a
disulfide bridge located between the polyamine and lipid, as in the case of
RPR128522, resulted in a total loss of transfection efficiency. On the other hand,
proper modulation of DNA release by the insertion of the disulfide bridge between
one lipid chain and the rest of the molecule (RPR132688) yielded increased
transfection efficiency when compared to the analog (RPR1205353). These unique
cationic lipids do not require DOPE or other co-lipid(s) for optimal transfection
efficiency [102].
A major driving force for the lipid/DNA complex formation is the release of
low-molecular weight counter-ions that makes a large entropic contribution to the free
energy of binding [103]. The lipid’s hydrophobic segments are the key determinant in
the macroscopic characteristics of the ensuing liposomes, particularly their size,
shape, and stability in the dispersed state, as well as interactions with other lipids, cell
membranes, and DNA. This, in turn, affects the transfection efficiency of the resulting
lipoplexes [104, 105]. The ability to control these parameters is rather limited, leading
13
Introduction
to the instability of their macroscopic properties over time [106] and thus restricting
their pharmaceutical potential. Furthermore, liposomal formulations often require an
adjuvant, such as di-oleylphosphatidylethanolamine, for efficient delivery [107].
Cationic liposomes, especially those composed of monovalent cationic lipids, cannot
condense DNA efficiently, resulting in a very heterogeneous size distribution of the
complex.
In contrast, self-assembly of polyplexes does not entail interaction of the
polycation molecules with each other, resulting in greater control of their macroscopic
properties, and is quite efficient without adjuvants. Additionally, since polycations
are ensembles of a certain repeating structural unit, they can be easily manipulated by
chemical modification to achieve higher efficiency or cell targeting without the loss of
activity [108]. Many of the polyplexes have superior transfection efficiency and serum
sensitivity compared to lipoplexes [109]. These considerations make polycations a
compelling target for future exploration in non-viral gene delivery.
Polycationic-based Gene delivery vectors
Polycationic polymers have been widely used as an alternative to lipid
formulations for efficient gene delivery. Cationic polymers interact with DNA to form
a relatively small sized particulate complex, polyplex, capable of gene transfer into the
targeted cells [110]. This can be crucial for gene transfer, as small particle size may be
favorable for improving transfection efficiency. The cationic polymers commonly
used as gene carrier backbones are chitosan, poly (2-(dimethylamino) ethyl)
methacrylate (pDMAEMA), polyamidoamine (PAMAM) dendrimers, poly (L-lysine)
(PLL) and polyethylenimine (PEI) (Fig. 4).
Chitosan
Chitosan is a biodegradable amino-polysaccharide composed of two subunits,
D-glucosamine and N-acetyl-D-glucosamine linked together by β-(1,4) glycosidic
bonds. The amino groups of chitosan present in the N-deacetylated confer positive
charge to chitosan. These amino groups exhibit intrinsic pKa values of 6.5 and thus
chitosan behaves as a polycation at acidic and neutral pH [111]. The cationic charge of
chitosan enables it to interact with negatively charged polymers, macromolecules and
14
Introduction
certain polyanions in an aqueous environment. The low toxicity, biocompatibility and
biodegradability of chitosan make it attractive for gene delivery purposes [112]. The
important
chitosan
derivatives and
their potential
applications are
described below:
Figure 4: Commonly used cationic vectors
(i) Deoxycholic acid modified chitosan
Liu et al. and Lee et al. [113, 114] synthesized hydrophobic chitosan by
conjugating deoxycholic acid to chitosan in methanol/water mixture using EDAC as
coupling agent with the degree of substitution as 5.1 (5.1 deoxycholic acid groups
15
Introduction
substituted per 100 anhydroglucose units). DNA complexes of hydrophobic chitosan
exhibited relatively higher transfection efficiency in comparison to naked DNA but
significantly lower than the LipofectamineTM/DNA formulation.
(ii) Quaternized chitosan
The poor solubility of native chitosan at physiological pH limits its application
as gene delivery carriers. The solubility of chitosan was enhanced by forming
ammonium salts of free amine groups of chitosan with inorganic and organic acids.
Reaction of chitosan with excess of methyl iodide in alkaline conditions resulted in N-
trimethyl-chitosan derivative. M. Thanou et al. [115] synthesized trimethyl-chitosan
oligomers (TMO) of 40% (TMO-40) and 50% (TMO-50) degrees of quaternization and
tested these for transfection efficiency. TMO-50 markedly increased the transfection
efficiency from 5-folds to 52-folds in COS-1 cells, whereas, TMO-40 displayed even
higher transfection efficiency ranging from 26-folds to 131-folds in COS-1 cells.
However, none of the TMO-based vectors were able to increase the transfection
efficiency in differentiated cells such as Caco-2.
(iii) Chitosan modified with hydrophilic polymers
Numerous methods have been developed for the grafting of amphiphilic
polymers onto chitin or chitosan to improve affinity to water or organic solvents [116,
117]. PEG-chitosan derivatives with various molecular weights of polyethyleneglycol
(PEG) and degrees of substitution were synthesized [118]. Higher molecular weight
PEG was found to enhance water-solubility of chitosan. PEG modification minimized
aggregation and prolonged the transfection potency for at least one month in storage.
Intravenous injection of chitosan-DNA nanoparticles and PEGylated chitosan-DNA
nanoparticles resulted in majority of nanoparticles to localize in kidney and liver
within the first 15 minutes. The clearance of the PEGylated nanoparticles was slightly
slower in comparison to non-PEGylated nanoparticles [119].
(iv) Galactosylated chitosan
Park et al. prepared and examined galactosylated chitosan-graft-dextran-DNA
complexes [120, 121]. This system efficiently transfected Chang liver cells expressing
asialoglycoprotein receptors (ASGR), which specifically recognize the galactose
16
Introduction
ligands on modified chitosan. In parallel work, galactosylated chitosan-graft-PEG
(GCP) [122] was also developed for gene delivery. In another protocol, galactosylated
chitosan-graft-poly (vinyl pyrrolidone) (GCPVP) was synthesized [123], which
showed improved physicochemical properties over the unmodified chitosan.
Erbacher et al. [124] synthesized lactosylated-modified chitosan derivatives and tested
their transfection efficiencies in many cell lines.
(v) Transferrin/KNOB/endosomyltic proteins conjugated chitosan
Two strategies were explored by Mao et al. [119] to bind transferrin onto the
surface of chitosan-DNA complex. In the first strategy, aldehydic groups were
introduced after oxidation with periodate, and thereafter, allowed to react with
chitosan amine groups via the formation of Schiff’s base linkages. The transfection
efficiency of transferrin-modified chitosan carriers were examined in HEK293 cell line
and were found to exhibit a 2-folds higher transgene expression compared to
unmodified chitosan. In the second strategy, transferrin was attached to the
nanoparticle surface through a disulfide bond, which resulted in a 4-folds increase in
transfection efficiency in HEK293 cells and only 50% increase in HeLa cells. To further
enhance the transfection efficiency, KNOB (C-terminal globular domain on the fiber
protein) was conjugated to chitosan by the disulfide linkages, which improved gene
expression level in HeLa cells by ~130-folds [125].
Dextran
Dextran is a polysaccharide made of glucose molecules joined through a -1,6-
glycosidic linkages into chains of varying lengths (from 10 to 150 kDa). Azzam et al.
[126] synthesized dextran-oligoamine based conjugates, wherein the side chain
oligoamines were attached to either a linear or branch hydrophilic dextran backbone.
The oligoamine, spermine, conjugated to dextran efficiently transfected cells in culture
[127]. DEAE-Dextran, a polycationic derivative of dextran, was one of the first
chemical reagents used for transfer of the foreign genes into cultured mammalian cells
[128]. It is obtained by reacting diethylaminoethyl chloride with dextran in basic
17
Introduction
aqueous medium [129]. Although DEAE-Dextran was found to allow superior
transfection compared to other techniques in the transfer of DNA to human
macrophages [128], its efficiency to deliver genes to a wide range of cell lines is still
very low in comparison to “modern” cationic vectors such as PEI, dendrimers, etc.
Polyaminomethacrylates
In recent years, researchers have developed polymethacrylates as gene delivery
vectors. Poly (2-(dimethylamino)ethyl methacrylate) (PDMAEM), the leading polymer
of this family, was synthesized by free radical polymerization of 2-
(dimethylamino)ethyl methacrylate in water using ammonium peroxydisulphate as
an initiator. The resulting high molecular weight linear polymer was able to condense
DNA and provided a physical barrier to nuclease digestion [130]. Cherng et al. [131]
showed that PDMAEM promoted cellular uptake and expression of exogenous DNA.
Wolfert et al. [132] examined the transfection and cytotoxicity of poly (N-2-
hydroxypropyl methacrylamide)-b-poly (trimethylaminoethyl methacrylate)
(PHPMA-b-PTMAEM), an amino methacrylate polymer with quaternary amine
groups connected to uncharged hydrophilic polymer of similar structure. It has been
proposed that this neutral hydrophilic block will coat the surface of the complex and
shield the surface charge to reduce non-specific interactions with fluid components in
vivo. Chloroquine was added as a lysosomotropic agent to enhance transfection
efficiency and it was found that while cytotoxicity of the copolymer did not differ
much from the homopolymer, the transfection efficiency was enhanced by the
addition of the PHPMA block.
Cationic Dendrimers
Dendrimers are spherical, highly branched polymers prepared either by
divergent or convergent strategies. The degree of branching is expressed in the
generation of the dendrimer. Most commonly used dendrimers for non-viral gene
delivery are synthesized via the divergent strategy [133] and represent sixth
generation StarburstTM polyamidoamines (PAMAM) dendrimers either in intact
(PolyfectTM) or fractured (SuperfectTM) form. Intact dendrimers bear two new polymer
18
Introduction
arms at each point of branching, whereas in fractured polymers either one or two
arms originate or the polymer is terminated at this point.
The terminal amino groups of polyamidoamine dendrimer are known to bind
DNA electrostatically to form positively charged complexes, which are taken up
during endocytosis. The star shape of the polymer is advantageous as DNA appears
to interact with the surface primary amines only, leaving the internal tertiary amines
available to assist endosomal escape of the dendrimer-gene complex. Unfortunately,
dendrimers have also been reported to be toxic to cells, which is the major limitation
for its application in human patients. In addition, only polyamidoamine dendrimers
with high generation showed practicable gene transfection efficiency, but the cost of
preparing these polymers is very high.
Imidazole-containing polymers
Polymers containing the heterocycle, imidazole, have shown promising
transfection capabilities. Modification of ε-NH2 groups of poly (L-lysine) using
histidine or other imidazole containing structures showed a significant enhancement
of reporter gene expression compared to un-modified poly (L-lysine) [134-136].
Swami et al. [137] synthesized imidazolyl-PEI nanoparticles, which delivered pDNA
efficiently in mammalian cells in vitro. The incorporation of imidazolyl group led to
the increased buffering capacity and also masked the excess cationic charge in PEI.
The imidazole ring displays a pKa of ~6 thus possessing a buffering capacity in the
endo/lysosomal pH range, and possibly mediating vesicular escape by a ‘proton
sponge’ mechanism.
Poly (L-lysine)
Poly (L-lysine) polymers, liner polypeptides with lysine as the repeat unit,
were one of the first cationic polymers employed for gene transfer [138]. Poly (L-
lysine)-based polymers, pioneered in 1987, was used for gene delivery by employing a
targeting ligand, e.g. asialoorosomucoid and folate to facilitate receptor-mediated
uptake [138-140]. The gene transfer efficiency of PLL was also improved by
employing lysosomotropic agents (such as chloroquine) or inactivated adenovirus, or
peptide derived from haemophilus influenza envelope proteins to facilitate PLL/DNA
19
Introduction
complex release from the endosomes [141, 142]. These studies implied that without
the use of either targeting ligands or endosome lytic reagents, gene transfer is poor
with PLL polyplexes alone because PLL is composed only of primary amine.
Increasing the molecular weight of PLL rendered the PLL-DNA complex toxic to the
cells.
Degradable cationic polymers
Lim et al. [143] reported successful transfection of mammalian cells with
decreased cytotoxicity employing degradable cationic polymers as delivery vehicles.
A biodegradable cationic polymer was synthesized using diacrylates as linker
molecule between cationic compounds resulting in formation of linear polymers with
low cationic density [144]. The low cationic charge density was insufficient to
effectively condense DNA. Moreover, synthesis of these polymers required days to
complete and the amount of effective product to be used in gene delivery, was low.
These factors made the preparation of high molecular weight polymers by this
method difficult to achieve. The transfection efficiency of the degradable cationic
polymers was observed to be lower compared to non-degradable polymeric
backbones, which may be due to the rapid degradation of these polymers in aqueous
solution resulting in rapidly lost gene transfer efficiency. The difficulty of controlling
degradation rate and synthesis limit the applications of degradable cationic polymers
in vivo and in clinical patients.
Polyethylenimine (PEI)
PEI (b 25 kDa) has become the gold standard of non-viral gene delivery owing
to its high transfection efficiency [145]. Highly branched PEIs (25 kDa and 800 kDa)
have been used for gene delivery both in vitro and in vivo [146]. Polyethylenimine
effectively complexes DNA molecules [147, 148], leading to the formation of
homogeneous spherical particles of size ~100 nm or less, which efficiently transfect
cells. PEI offers significant protection to the complexed nucleic acids against
enzymatic degradation, possibly due to their higher charge density and more efficient
complexation. PEI mainly exists in either linear or branched form. The high density of
primary, secondary and tertiary amino groups in branched PEI confers significant
20
Introduction
buffering capacity to the polymers over a wide pH range. This property, known as
‘proton sponge effect’, is likely one of the crucial factors for the high transfection
efficiency obtained with PEI [149]. The factors affecting the efficiency/cytotoxicity
profile of PEI polyplexes include molecular weight, degree of branching, zeta
potential and particle size [146, 148]. With increase in molecular weight, branched PEI
exhibits high transfection efficiency, however, cytotoxicity has also been found to
increase concurrently [150]. Although associated with high transfection efficiency,
bPEI (25 kDa) exhibits cellular toxicity at higher doses possibly due to its high cationic
charge [146, 151], while, linear PEI (25 kDa)/DNA complexes display excellent
transfection efficiency with relatively low toxicity [152-154].
Although low molecular weight PEI (800 Da) displays minimal toxicity, its
transfection efficiency was found to be very low. As the transfection efficiency of PEI
depends on its molecular weight, Gosselin et al. [155] synthesized high molecular
weight PEI from low molecular weight PEI by using disulfide-containing linkers,
dithiobis (succinimidylpropionate) NHS ester (DSP) and dimethyl-3,3'-
dithiobispropionimidate-2HCl (DTBP). The resulting polymers showed high
transfection efficiency and low cytotoxicity. The disulfide bonds introduced via cross-
linking reagents was reduced in the cytoplasm, resulting in the breakdown of PEI
conjugates before genes were delivered into nucleus. Tang et al. [156] developed and
tested a new PEI polymer synthesized by linking low molecular weight PEIs with
beta-cyclodextrin. The polymer displayed improved biocompatibility over non-
degradable branched PEI (25 kDa) and high transfection efficiency in cultured
neurons and in the central nervous system of mice.
Kim et al. [157] designed a class of degradable PEIs with acid-labile imine
linkers. The acid-labile PEI may be rapidly degraded into low molecular weight PEI in
acidic endosomes. The acid labile PEI was less toxic than PEI (25 kDa) due to the
degradation of acid-labile linkage. Therefore, the use of acid labile PEIs may be
helpful for gene delivery. Kircheis et al. [158] linked PEGylated PEI polyplexes to
tumor-specific ligand transferrin, an asialoglycoprotein and then applied
intravenously, resulting in 5-folds increase in the transfection efficiency with lower
21
Introduction
toxicity in comparison with PEGylated (transferrin free) PEI polyplexes. The addition
of PEG as a crosslinker to form nanoparticles of PEI [159] markedly decreased the
toxicity and resulted in enhanced transfection efficiency.
To combine the advantages of PEI and liposome, water soluble PEI-Chol
lipopolymer was synthesized by Han et al. [160] for gene delivery. PEI-Chol
lipopolymer is amphiphilic in nature because PEI is hydrophilic and water soluble,
while cholesterol is hydrophobic. With the increase in concentration, PEI-Chol may
form multimolecular micelles or micellar aggregates in water, depending on the
hydrophilic–hydrophobic balance between the cationic headgroup and the lipid tail.
PEI-Chol, administered into jurkat cells, was found to be less toxic and showed high
level of green fluorescent protein expression [161].
Nanoparticle aided gene delivery
For enhancing the bioavailability of the entrapped biomolecules and also to
achieve the desired therapeutic response of the biomolecules, the nanoparticulate size
of polymeric matrix is desired. Nanoparticulate systems are attractive methods of
DNA delivery owing to the versatility, ease of preparation, and protection conferred
to encapsulated plasmid DNA [162]. These carrier systems can efficiently encapsulate
various sizes of plasmids and provide protection during transit in the systemic
circulation. Nanoparticles usually have a high surface area to volume ratio and thus,
are able to efficiently encapsulate DNA even without pre-condensing step.
Nanoparticles can be made to reach a target site by virtue of their size and charge
[163]. By attaching cell-specific ligands, nanoparticle-based gene delivery vectors can
be targeted to reach specific tissues and cells in the body. To avoid uptake by the
mononuclear phagocytic system after systemic administration, polyethylene glycol
chains are attached to the nanoparticles [164-166]. For the delivery to solid tumors in
vivo, long-circulating PEG-modified nanoparticles were found to be preferentially
distributed in the vasculature due to the enhanced permeability and retention (EPR)
effect [167, 168].
Nanoparticles of pharmaceutical importance were initially prepared by
Birrenbach and Speiser in 1976 [169]. They polymerized acrylamide and cross-linked
22
Introduction
it with N,N’-methylenebisacrylamide in an inverse microemulsion (water-in-oil)
reaction to form nanoparticles. Kreuter and Speiser [170] used dispersion
polymerization methodology for the preparation of poly (methyl methacrylate)
(PMMA) nanoparticles. Till date, several methods have been reported for the
preparation of nanoparticles from poly (lactic acid), poly (l-glycolide), poly (lactide-
co-glycolide) and poly (cyanoacrylate) by using the preformed polymers [171].
Various crosslinkers have been designed to crosslink the polymers in the form of
nanoparticles. PEGylated gelatin nanoparticles efficiently transfected LLC cells and
the in vivo expression of β-galactosidase in tumor mass showed that PEGylated gelatin
nanoparticles could transfect with 61% efficiency after i.v. administration relative to
non-PEGylated nanoparticles [172]. Ichikawa et al. [173] synthesized biocompatible
nanoparticles by self-assembly of chitosan and carboxymethyl cellulose hydrolysates.
Polyethylenimine (PEI-PEG) nanoparticles [159] have shown remarkable
improvement in transfection efficiency compared to native PEI in mammalian cells.
Administration of polycation/DNA complexes to an organism in vivo (locally or
systematically) involves overcoming of barriers such as anatomical size constraints,
interactions with biological fluids and extra-cellular matrix, and binding to a variety
of non-target cell types for efficient and specific gene expression [174]. Optimal design
of site-specific gene delivery systems ought to take into account the physicochemical
parameters of the system with respect to biodistribution and transgene expression.
This could be achieved by establishing a relationship between a cationic polymer and
its gene delivery performance, followed by bringing about further modifications in
the structure to improve the activity in a predictable manner.
23
Introduction
Scope and objectives of the present work
Gene therapy continues to hold promise in treating a variety of inherited and
acquired diseases. Majority of gene therapy trials rely on viral vectors for gene
transduction because of their high efficiency. Viruses remain the vectors of choice in
achieving high efficiency of gene transfer in vivo. These vectors, however, pose safety
concerns unlikely to abate in the near future [74-76]. Issues of immunogenicity and
toxicity remain a challenge. Limitations of cell mitosis for retrovirus, contamination of
adenovirus, and packaging constraints of adeno-associated virus (AAV) also lessen
their appeal. Non-viral vectors, although achieving only transient and lower gene
expression level, may be able to compete on potential advantages of ease of synthesis,
low immune response, and ability to condense unrestricted plasmid size. They have
the potential to be administered repeatedly with minimal host immune response.
They can also satisfy many of the pharmaceutical issues better than the viral vectors,
such as scale-up, storage stability, and quality control. Development of safe and
effective non-viral gene carriers is still critical to the ultimate success of gene therapy.
The most frequently studied strategy for non-viral gene delivery is the
formulation of DNA into condensed particles by using cationic polymers. One of the
most extensively studied cationic polymers is polyethylenimine. Of the various
cationic polymers, PEI displays several properties that make it the gold standard and
one of the most effective non-viral vectors for gene delivery. Polyethylenimine is a
cationic polymer with a high density of a variety of amines with every third atom
being nitrogen that can be protonated. Various PEI derivatives have been used to
24
Introduction
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 the
molecular weight, degree of branching and cationic charge density of the polymer.
Unfortunately, the polycationic nature of PEI also appears to be the main origin of its
marked toxicity, a property it shares with many other polycations (e.g. polylysine)
limiting its use as a gene delivery vector in vivo. Toxicity of PEI is strongly influenced
by its molecular weight, polydispersity, structure, and concentration [146, 175]. To
circumvent the cytotoxicity of the polymer, various modifications like, PEGylation,
acylation and attachment of pendant groups such as dextran, have been carried out.
Though these modifications have led to an improved transfection efficacy, further
improvements are mandatory to make PEI-based gene delivery vectors more versatile
in terms of efficiency and cell viability.
Nanoparticles mediated gene delivery has attracted the attention of researchers
both in academia and industry. Nanoparticles owing to their small size easily traverse
across the cellular membrane. As the nanoparticles are easy to prepare and also confer
protection to the complexed DNA, they have become attractive gene delivery vehicles.
In view of great diversity within the field of cationic polymer-based non-viral gene
delivery and the promising results obtained with PEI-based transfection reagents, the
present investigation was, particularly, focused on to the development of strategies
for the synthesis of PEI-based nanoparticles for safe and efficient gene delivery.
Therefore, the main intention, to undertake the proposed study, was to develop
efficient and safe cargo systems that can effectively transfer nucleic acids to a wide
spectrum of cell lines with significantly high cell viability and gene expression. The
following work-plan was chalked out to fulfill the above mentioned objective:
1. Design and synthesis of novel PEI-based nanocomposites/nanoparticles as non-
viral gene delivery vectors,
2. Characterization of these nanocomposites/nanoparticles by spectroscopic
techniques, analytical methods and in terms of zeta potential, particle size (DLS
and TEM) and surface morphology (AFM),
25
Introduction
3. Evaluation of nanocomposites/nanoparticles for DNA binding ability, in vitro
transfection, cytotoxicity and intracellular trafficking, and
4. In vivo examination of nanocomposites/nanoparticles in mice and rabbits.
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