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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