Cationic Polymers and its Uses in Non-viral Gene Delivery Systems: A Conceptual Research
Sarder Nasir Uddin
The main objective in gene delivery system is the development of efficient, non-toxic gene carriers that can encapsulate and deliver foreign genetic materials into specific cell types To perform gene transfer two types of vectors are available, (i) viral vectors (ii) nonviral vectors. Among the vectors, nonviral vectors are proved less toxic and safe compare to viral vectors through clinical trial. No single vector is proved suitable for every gene transfection experiment. Cationic polymers such as polyethylenimine (PEI) and poly L-lysine (PLL) and their complexes are experimentally established as non-viral vector with higher transfection efficiency. Identifying the barriers for transfection and the possible solution, the stability improvement research is needed for better clinical performance of cationic polymers. In future it is also possible to find new conjugates as cationic polymers bearing many properties, suitable to bound with DNA and penetrate cell. Considering all these properties, these works reviews the most recent studies highlighting cationic polymers used in nonviral gene delivery systems.
Gene Delivery System represents a new and promising therapeutic modality.
The underlying principle is based on the introduction of genetic material into
cells to generate a curative biological effect (Miller, 1992; Mulligan, 1993).
Gene therapy is not limited to hereditary diseases but can be used for a broad
variety of different acquired diseases, such as infections, degenerative disorders
and cancer. The most challenging issues for successful application of gene therapy
to human diseases concern: (1) the choice of the relevant therapeutic gene,
(2) the choice of promoter and regulatory sequences driving expression of the
transgene and (3) the vector used for delivery of the transgene into cells (target
cells). Promoter, regulatory elements and vector characteristics determine transduction
efficacy (the number of target cells expressing the transgene and the intensity
of gene expression per cell), specificity of the transduction, time of transgene
expression, the host's immune response against the vector and eventually undesired
side effects. At present, important efforts are being focused on the search
for vectors with less toxicity and prolonged and controlled transgene expression,
thereby widening the potential application of gene therapy to a high spectrum
of medical fields. In recent years, gene therapy has emerged as a new and promising
method to treat human diseases. To perform gene delivery two types of vectors
||Viral vectors (retorviruses, adeneviruses, herpesviruses,
lentiviruses and hybrid/retor-adeno viruses etc).
||Nonviral vectors (cations e.g., polymers and lipids, naked
DNA, artificial chromosomes, plasmid, conjugates and complexes).
Despite of the impressive progress in biomedical sciences during the last decades, the therapy of many liver diseases remains unsatisfactory. This applies not only to primary and metastatic liver tumours, hepatic cirrhosis and hereditary metabolic diseases but also to a high proportion of cases with chronic viral hepatitis that do not respond to current antiviral therapy. There is evidently an urgent need for efficient alternative therapeutic approaches and in recent years gene therapy has emerged as a new and promising method to treat human diseases (Schmitz et al., 2005)
Gene Therapy Vectors
Gene therapy vehicles can be categorized into two groups: biological and
non-biological systems. Each group has its own advantage and limitations. The
limitations of biological delivery system are less encountered in non-viral
gene carrier namely cationic polymers, cationic peptides and cationic lipids
(liposomes). The main objective in gene therapy is the development of efficient,
non-toxic gene carriers that can encapsulate and deliver foreign genetic materials
into specific cell types. During the past two decades, enormous research in
the area of gene delivery has been conducted worldwide, in particular for cancer
gene therapy application. Viral vectors are biological systems derived from
naturally evolved viruses capable of transferring their genetic materials into
the host cells. Many viruses including retrovirus, adenovirus, herpes simplex
virus (HSV), adeno-associated virus (AAV) and pox virus have been modified to
eliminate their toxicity and maintain their high gene transfer capability. The
limitations associated with viral vectors, however, in terms of their safety,
particularly immunogenicity and in terms of their limited capacity of transgenic
materials, have encouraged researchers to focus on non-viral vectors as an alternative
to viral vectors. Non-viral vectors are generally cationic in nature. They include
cationic polymers such as polyethylenimine (PEI) and poly L-lysine (PLL), cationic
peptides and cationic liposomes. The newly described liposomal preparation LPD
(liposomes/protamine/DNA), for example, has shown superiority over conventional
liposomes/DNA complexes (lipoplexes)(Aneed, 2004). However, physical properties
such as size and zeta potential play a critical role in their efficiency. In
either delivery system, selected modifications that can produce safe efficient
and targetable gene carriers are desirable. Although non-viral vectors are less
efficient than viral ones, they have the advantages of safety, simplicity of
preparation and high gene encapsulation capability. This article reviews the
most recent studies highlighting the advantages and the limitations of nonviral
cationic types of gene delivery systems used in gene therapy (Aneed, 2004).
Both viral and non-viral vectors are used to transfer genetic material to the inside of target cells. In general, non-viral vectors have lower transduction efficiency and allow for shorter duration of transgene expression than viral vectors. The ideal vector should have (i) low antigenic potential, high capacity, high transduction efficiency, (ii) allow controlled and targeted transgene expression, (iii) reasonable expense and (iv) safe for both patient and the environment. Gene delivery vehicles must be selected according to the specific therapeutic aim. But the perfect vector covering all therapeutic and safety requirements does not exist and much work is needed in this important research field (Prince, 1998).
Non-viral systems are cationic in nature. They interact with negatively
charged DNA through electrostatic interactions. The total charge however maintains
a positive net value. This will enable the carrier of efficiently interacting
with the negatively charged cell membranes and internalizes into the cell, which
occurs mainly through the endocytosis pathway (Behr, 1994).
The cationic natures of the non-viral vectors help the vector to interact with the negatively charged DNA through electrostatic interactions. The reaction complex also bears positive net values which enable the carrier to react efficiently with negatively charged cell membranes.
Nonviral gene transfer vectors have been actively studied in the past years in order to obtain structural entities with minimum size and defined shape. The final size of a gene transfer vector, which is compacted into unimolecular complexes, is directly proportional to the mass of the nucleic acid to be compacted. To perform gene delivery ssDNA (SS-Single stranded)/poly-L-lysine complex was found significantly smaller than their double-stranded counterparts. Expression of compacted ssDNA was observed in hepatoma cell lines. Firstly, galactosylated ssDNA complexes were successfully delivered into cells and then expression of the asialoglycoprotein receptor via receptor-mediated endocytosis. The reduced size and biophysical behaviour of ssDNA vectors may provide an advantage for transfection of eukaryotic cells (Behr, 1994).
Synthetic cationic polymers combined with DNA at physiological pH and form
a particulate complex (polyplex), capable of gene transfer into the targeted
cells. As they are synthetic compound many modifications e.g., molecular weight
and ligand attachment can be easily achieved. The most widely studied polymers
for gene therapy include poly L-lysine (PLL) and polyethylenimine (PEI) (Aneed,
2004). Several polycations possessing sustainable buffering capacity below physiological
pH, such as lipopolyamines and polyamidoamine polymers are efficient transfection
agents, without the addition of membrane disruption agents. Indeed, every third
atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network
an effective proton sponge at virtually any pH. Cytotoxicity was low and seen
only at concentrations well above those required for optimal transfection. The
PEI cation/anion balance for in vitro transfection is only slightly on
cationic side, which is advantageous for in vivo delivery. Together
these properties make PEI a promising vector for gene therapy and an outstanding
core for the design of more sophisticated devices (Boussif et al., 1995).
Basic Nature and Transfection Mechanisms
PLL polymers are one of the first cationic polymers employed for gene transfer
(Wu and Wu, 1987). They are linear polypeptides with the amino acid lysine as
the repeat unit; thus, they possess a biodegradable nature. This property is
very useful for in vivo applications. Poly-L-lysine polyplexes are, however,
rapidly bound to plasma proteins and cleared from the circulation (Ward
et al., 2001; Dash et al., 1999). In addition, successful transfection
requires co-application of chloroquine, a lysosomotropic agent, which reduces
the lysosomal degradation of lipoplexes (Pouton et al., 1998; Shewring
et al., 1997). The exact mechanism in which chloroquine acts is not well
understood. Chloroquine can be substituted with fusogenic peptides, which undergo
pH-related conformational changes perturbing the lysosomal/endosomal membranes,
thus the DNA is successfully delivered into the cell cytoplasm (Lee et al.,
2002; Wagner et al., 1992).
Poly-l-lysine Modified Complexes with Other Compounds
a) Poly-l-lysine Condensed Dna and Galactosylated Bovine Serum Albumin (Galbsa)
Liver targeting of DNA delivery could be enhanced via asialoglycoprotein
receptors using a complex of poly-L-lysine (PLL) condensed DNA and galactosylated
bovine serum albumin (GalBSA) via charge interaction (GalBSA:PLL:DNA = 3:0:5:1,
w/w/w) (Han et al., 1999).
b) Poly-l-lysine Modified Iron Oxide Nanoparticles (IONP-PLL)
Another nonviral gene carrier is developed, which is poly-L-lysine modified
iron oxide nanoparticles (IONP-PLL) and is formed by modifying poly-L-lysine
to the surface of iron oxide nanoparticles. IONP-PLL could bind and protect
DNA. In contrast to Poly-L-lysine and cationic liposomes, ION-PLL proved less
cytotoxic in a broad range of concentrations. Poly-L-lysine modified iron oxide
nanoparticles can deliver exogenous gene to cells in vitro and in
vivo. After intravenous injection, ION-PLL transferred reporter gene EGFP-C2
to lung, brain, spleen and kidney. Also it transfers exogenous DNA across the
blood-brain barrier to the glial cells and neuron of brain. It is found a potential
vector for gene therapy against CNS diseases (Xiang et al., 2003).
c) The Terplex Complex
A new DNA delivery vector (the terplex system) based on a balanced hydrophobicity
and net surface charge between stearyl-poly-L-lysine, low density lipoprotein
(LDL) and genetic material (i.e., plasma DNA or antisense oligonucleotide) was
developed. The pSV-beta-gal plasmid in terplex system showed a 2-5 fold increase
in betagalactosidase expression on murine smooth muscle cells (A7R5) compared
to lipofection. Delivery of unmodified cmyb antisense oliogoneucleotide to A7R5
cells was also facilitated significantly by the terplex system requiring as
little as 5.4 nm of antisense oligoneucleotide to achieve a 50% antiproliferative
effect (Kim et al., 1998). After characterization of physical properties
it is found that:
||Plasmid DNA was condensed by addition of stearyl-PLL and LDL,
resulting in the terplex system of about 100 nm in diameter as shown by
atomic force microscopy.
||A strong hydrophobic interaction between stearyl-poly-L-lysine
and LDL was registered by 1H-NMR spectrometry, showing a significant decrease
in the epsilon-methylene signal of poly-L-lysine backbone when stearyl-poly-L-lysine
was mixed with LDL; however, this phenomena was not observed with unmodified
||Agarose gel electrophoresis revealed that electrophoretic
mobility of the terplex system decreased with increasing amounts of stearyl-poly-L-lysine,
indicating that the surface charge of the terplex system became more positive
by addition of stearyl-poly-L-lysine.
||Zeta potential measurement showed that the terplex system
exerted a slightly positive charge (+2 mV) at a 1:1:1 weight ratio of plasmid
DNA: LDL: stearyl-poly-L-lysine.
By conjugating poly-L-lysine (PLL) to pluronic, poly-L-lysine-g-pluronic
is synthesized. Pluronic is partially functionalized with para-nitrophenyl carbonate
groups. This pluronic grafted poly-L-lysine is tested as a new synthetic gene
carrier. Compared with unmodified PLL, PLL-g-pluronic showed about two fold
increases in transfection efficiency with similar cytotoxicity specifically
at the 1:1 weight ratio of polymer: DNA. The in vitro transfection efficiency
was measured in Hela cells by using the O-nitrophenyl-beta-D-galactopyranoside
assay (Jeon et al., 2003).
Efficiency Influencing Factors and Actions for Improvement
In fact, PLL has poor transfection ability when applied alone or without
modifications (Pouton et al., 1998; Brown et al., 2000). One popular
modification that can increase both the transfection ability and the circulation
half-life of these vectors is coating with PEG (Lee et al., 2002; Ward
et al., 2002). Targeting ligands were also linked to the polymerase chain
(even in early studies) resulting in enhanced transfection (Wu and Wu, 1987;
Suh et al., 2001). A common efficient strategy implies the addition of
both PEG and a targeting ligand to PLL polymer to optimize transfection (Faraasen
et al., 2003; Nah et al., 2002). Another approach can create the
desirable proton sponge effect similar to that of PEI polyplexes by introducing
histidine residues to PLL backbone (Pichon et al., 2001). Histidylated
PLL showed better transfection efficiency than PLL/chloroquine mixture (Midoux
and Monsigny, 1999).
Many PLL polymers with different molecular weight were tested and evaluated for gene transfer (Ward et al., 2002; Mannisto et al., 2002; Nishikawa et al., 1998). It has been shown that DNA condensation and transfection efficiency increased with high molecular weight PLL, which was also associated with undesirable high toxicity (Wolfert et al., 1999). The creation of amphiphilic PLL, by linking both PEG and palmitoyl groups to the polymer, reduced toxicity without compromising the gene delivery efficiency (Brown et al., 2000).
Basic Nature and Transfection Mechanism
PEI was used in gene delivery more recently than PLL. PEI is obtained by
acid-catalyzed polymerization of aziridine (Dick and Ham, 1970; Klotz et
al., 1969) yielding a highly branched network with a high cationic charge-density
potential that can ensnare DNA. Since 1995, PEI has been found to be a versatile
polymeric vector for gene delivery that tightly condenses plasmid DNA and is
able to promote transgene delivery to the nucleus of mammalian cells (Boussif
et al., 1995; Pollard et al., 1998). A merely mechanistic study
(Wiethoff et al., 2001) has shown that cationic lipid-DNA complexes can
dock and interact with proteoglycans expressed on the surface of mammalian cells,
promoting their intracellular uptake, a possible route that might also be followed
by PEI/DNA complexes. The high transfection efficiency of PEI in vitro
has been ascribed to its ability to act as a proton sponge that buffers the
low pH in the endolysosomal compartments and potentially induces ruptures of
the endolysosomal membrane, resulting in the release of PEI/DNA complex into
the cytoplasma (Boussif et al., 1995). It is usually branched with every
third amino nitrogen atom being protonated such that PLL has a buffer capacity
virtually at any pH value (proton sponge) (Kichler et al., 2001). This
feature and PEI ability to destabilize lysosomal membranes enables PEI polyplexes
to efficiently escape the degradation within the acidic endosomal environment
(Kichler et al., 2001; Klemm et al., 1998).
Furthermore, Lechardeur et al. (1999) have shown that upon entry into the cytoplasm, naked plasmid DNA (pDNA) undergoes a rapid turnover because of degradation by cytosolic nucleases. Moret et al. (2001) have shown that PEI is able to protect pDNA against degradation by serum DNases. Although the proton-sponge effect and the ability to deliver DNA to the nucleus enhance transgene expression, the presence of PEI in the cellular nucleus may interfere with transcriptional and translational processes and even induce cell death (Godbey et al., 2001). Efforts have been undertaken to diminish the high cationic charge density of PEI to a magnitude that promotes DNA delivery but decreases the adverse effects of PEI on cell viability (Putnam et al., 2001; Bogdan et al., 2002).
PEI/DNA/plasmid and Other Conjugate and Complexes
a) Plasmid and PEI Complex
In case of gene delivery to adult neural stem cells plasmids can be delivered
in vivo when complexed with linear polyethylenimine and gene expression
can be targeted specifically to neural stem or progenitor cells by the use of
specific promotors. These techniques may be utilize both to study the function
of various genes in the differentiation of neural stem cells to specific cell
fates and ultimately for gene therapy or to generate specific differentiated
progeny for cell transplantation (Falk et al., 2002).
b) Galactosylated PEI
Galactosylated PEIs (Gal-PEI) could be use for gene transfer. To optimize
a receptor mediated and cell selective gene transfer with polyethylenimine (PEI)
based vector three galactosylated PEIs (Gal-PEI) with different molecular weight
were tested on asialglycoprotein receptor-positive cells (Morimoto et al.,
c) PEI Conjugated to Antibody
Tumor-targeted gene delivery is performed via anti-HER2 antibody (trastuzumab,
Herceptin) conjugated polyethylenimine. Vectors were DNA polycation complexes
(polyplexes) prepared by mixing, at vector ratios, plasmid DNA carring a luciferase
reporter gene to Her PEI, which is a conjugate of linear polyethylenimine(PEI),
a cationic polymer and trastuzumab (Herectin), a HER2-specific monoclonal antibody.
HerPEI polyplexes has shown promising HER2 receptor specific gene transfer properties
and warrant further evaluation as a tumor-targeted vector for gene therapy (Chiu
et al., 2004).
d) PEI conjugated to Cholesterol
Non-viral polymer/plasmid DNA complexes were formed using linear polyethylenimine
(LPEI) Mw 25k conjugated to cholesterol in a T-shaped geometry (LPC-T) and pDNA
encoding murine interleukin-12 (pmlL-12e). These complexes were subsequently
injected weekly into mice (BALB/C) intravenously and locally for the treatment
of murine renal cell adenocarcinoma (Renca) induced pulmonary metastases and
subcutaneous (SC) Renca tumors respectively. Thus it was concluded that LPC-T
is an effective carrier for passive targeting of the pulmonary tissue, treatment
of Renca-induced pulmonary metastases and local administration of Renca cell
SC tumors (Furgeson et al., 2004).
e) Cetyl PEI and DNA Complexes
A novel gene transfer system utilizing polycation liposomes (PCLs), obtained
by modifying liposomes with cetylpolyethylenimine (PEI), is found. PCLs show
notable transfection efficiency with low cytotoxicity. PCLs effectively transfer
DNA to endosomes and release cetyl PEI-DNA complexes into the cytosol. Furthermore,
cetyl PEI also contributes to gene entry into the nucleus (Sugiyama et al.,
Another report describes gene transfer in vitro as well as in vivo using cetylated low-molecular mass (600 Da) polyethylenimine (28% of amine groups substituted with cetyl moieties), termed CT-PEI. This compound is hydrophobic and has to be incorporated into liposomes in order to be suitable for gene transfer studies. Serum-induced plasmid DNA degradation assay demonstrated that CT-PEI-containing liposomal carriers could protect complexed DNA (probably via condensation). In vitro luciferase gene expression achieved using medium supplemented with 10% serum was comparable to that achieved in serum-reduced medium and was highest for CT-PEI/cholesterol liposomes, followed by CT-PEI/dioleoylphosphatidylcholine liposomes and PEI 600 Da (uncetylated) carrier. In vivo systemic transfer into mice was most efficient when liposome formulations contained CT-PEI and cholesterol. Higher luciferase expression was then observed in lungs than in liver. In conclusion: liposomes containing cetylated polyethylenimine and cholesterol are a suitable vehicle for investigating systemic plasmid DNA transfer into lungs (Aleksander et al., 2004).
Factors That Influence the Improvement of Efficiency and Actions
Many factors affect the efficiency profile of PEI polyplexes (and almost
any non-viral vector) such as molecular weight, degree of branching, ionic strength
of the solution, zeta potential and particle size (Kunath et al., 2003;
Kircheis et al., 1999). One study, for instance, showed that low molecular
weight (10 kDa), moderately branched polymer resulted in efficient delivery
with low toxicity in comparison with commercial high molecular weight PEI (Fischer
et al., 1999). Another study demonstrated that linear PEI (22 kDa) was more
efficient in both salt and salt-free buffers than branched polymers (25 and
800 kDa) (Wightman et al., 2001). Lethal side effects in mice, however,
were observed when the linear PEI (22 kDa) polyplex was injected intravenously
within its therapeutic window (Chollet et al., 2002). Therefore, more
studies are needed to produce optimum PEI carriers with respect to efficiency/toxicity
(PEI) Recent progress in consequent rational vector improvement is highlighted by our finding of polyethylenimine derivatives more potent and yet less cytotoxic than the 25-kDa polyethylenimine (one of the most effective non-viral vector). Such vectors could be further modified with cell targeting ligands to enhance their utility for in vivo applications (Thomas et al., 2003).
The nature of these polymers enables the researchers of successfully introducing targeting ligands and/or polyethylene glycol (PEG) (that produces sterically stabilized gene carriers) to their surfaces. Pegylated PEI polyplexes, for instance, were linked to tumor specific ligand transferrin, an asialoglycoprotein and then applied intravenously, resulting in five-fold increase in the transfection efficiency with lower toxicity in comparison with pegylated (transferrin-free) PEI polyplexes (Kircheis et al., 1999).
In vitro instability of a polycationic vector limits its efficacy
after systemic administration. Conjugation of hydrophilic polymers with neural
charge onto polycationic vectors has been used to improve the stability by reducing
the interaction between the vectors and the blood components, such as serum
albumin. In an experiment, dextrans of molecular weight 10000 (dex-10000) and
1500 (dex-1500) were used to produce various degrees of grafting on linear and
branched polyethylenimines (PEI) and the dextran-grafted polymers were used
to prepare DNA-polymer complexes. The changes in size and in zeta potential
and the extent of DNA release after the exposure of the complexes to bovine
serum albumin (BSA) were used to evaluate the stability of the complexes prepared
at various ratios of DNA to polymer. Finally it was found that the dextran-grafted
branched PEI improved the stability of the DNA-polymer complexes and showed
potential to conjugate with ligands for in vivo targeted gene delivery
(Tseng and Jong, 2003).
Chitosan as Gene Delivery Vector
Chitosan, naturally occurring cationic polysaccharides, has been shown to
excel in transcellular transport (Guang et al., 2002). It is a candidate
nonviral vector for gene delivery because of its high positive charges
and low cytotoxicity. Unlike high molecular weight chitosan (HMWC), low molecular
weight chitosan (LMWC) is highly water soluble and can form complex with plasmids
in physiological buffer. The plasmid DNA was completely retarded at a weight
ratio of 1:2 (plasmid : LMWC) in 1% agarose gel. DNase1 protection assay showed
that plasmids were protected from DNase1 over 60 min. The most efficient transfection
was obtained at a weight ratio of 1:3 (plasmid: LMWC). The transfection efficiency
of LMWC was significantly higher than poly-L-lysine (PLL). MTT assay showed
that LMWC was less cytotoxic than PLL. Therefore, LMWC will be useful in the
development of safe gene carriers (Lee et al., 2001). The formulations
with high molecular weight (HMW) chitosan can be an effective nonviral method
of gene vector in animal studies. Two different preparation methods ( the solvent
evaporation method and the complex coacervation method ) an the encapsulation
of a model plasmid with chitosan was performed. The ability of different molecular
weights of chitosan to form nanoparticles with a plasmid and particulate polymers
to stabilize a plasmid in a supercoiled form, were examined by agarose gel electrophoresis.
The efficiency of nanoparticles mediated transformation to Escherichia coli.
cells was significantly higher than naked DNA or poly-L-lysine(PLL)-DNA polycation
complexes. The transfection studies were performed in cos7 cells. A three fold
increase in gene expression was produced by nanoparticles as compared to the
same amount of naked plasmid DNA (Bozkir et al., 2004).
Chitosan also presents some charateristics favourable for gene delivery, such as the ability to condanse DNA and form small discrete particles in defined conditions. In controlled conditions, when plasmid DNA formulated with chitosan, produce homologous populations of complexes which were stable and had a diameter of approximately 50-100 nm. Discrete particles of nicely condensed DNA had a donut, rod or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum and did not require an added endosomolytic agent. In addition gene expression generally increased over time, from 24 to 96 h whereas, in the same conditions the efficiency of polyethylenimine mediated transfection dropped by two orders of magnitude. At 96 h, chitosan was found to be 10 times more efficient than PEI. However, chitosan mediated transfection depended on cell type (Erbacher et al., 1998).
Poly (D, L-lactide-co-4-hydroxy-L-proline) (PLHP)
A new polymeric gene carrier, poly (D, L-lactide-co-4-hydroxy-L-proline)
(PLHP) was synthesized and characterized to prove non-toxic. PLHP is a promising
candidate for long term gene delivery with good biocompatibility and biodegradability
while gene expression mediated by a non-viral vector usually lasts only a few
days (Li and Huang, 2004). Degradation of PLHP was examined by changing the
pH of the medium and molecular weight (MW) of the remaining polymer. The polymer
was characterized by (1) Nuclear Magnetic Resonance (NMR) and Gel Permeation
Chromatography (GPC). The cytotoxicity of PLHP is significantly lower than polyethylenimine
(PEI) and poly-L-lysine hydrochloride (PLL). When attached with plasmid DNA
gene transfer efficiency of PLHP/pDNA delivery system showed a sustained activity
(over a week) when compared with PEI and PLL. It can be further improved by
the addition of cationic liposomes.
Network Type Poly Amino Ester (n-PAE)
Efficient transfection and nontoxicity are two of the most important requirements
of an ideal gene delivery vector. Artificially constructed polymeric vectors
should have simultaneous multiple functions, i.e., controlled degradation and
endosome disruptive function and positive changes. Conceding all these functions
a network type poly amino ester (n-PAE) is found more suitable than PEI, which
is one of the most effective polymeric gene delivery vectors, reported to date.
n-PAE biodegradation and successive events of effective endosome escape result
in high transfection efficiency and favorable cell viability response. The n-PAE
mediated transfection is also very effective on the presence of serum (Lim et
Self-assembling polycation/DNA complexes represent a promising synthetic
vector for gene delivery. However, despite considerable versatility and transfectional
activity in vitro, such materials are quickly eliminated from the blood
stream following intravenous injection (plasma alpha half-life typically less
than 5 min). For targeted systemic delivery a more prolonged plasma circulation
of the vector is essential. It is found that rapid plasma elimination of polycation/DNA
complexes results from their binding serum albumin and other proteins, perhaps
due to aggregation and phagocytic capture or accumulation of the ternary complexes
in fine capillary beds (Dash et al., 1999).
Adenovirus (Ad)/polycation Complexes
Complexing recombinant adenovirus (Ad) vector with various polycations has
been shown to enhance transduction of cell lines. The activity of Ad/polycation
complexes was tested in vivo in the mouse lung. Several polycations were
capable of enhancing transduction of mouse respiratory epithelium, leading to
a 1-2 log increase in levels of transgene expression. Poly-L-lysine (PLL) and
DEAE-dextran were found to increase Ad-mediated gene transfer without any additional
toxicity as assessed histologically or through the measurement of inflammatory
cytokines in bronchoalveolar lavages. So, complexing Ad vectors with polycations
has the potential to improve the therapeutic index by increasing transgene expression
while reducing unwanted responses associated with high doses of vector (Kaplan
et al., 1998 ).
Folate Targeted Transfection Complex
A folate targeted transfection complex that is internalized by certain cancer
cells and displays several properties reminiscent of enveloped viruses has been
developed. These liposomal vectors are comprised of a polycation condensed DNA
plasmid associated with a mixture of neutral and anionic lipids supplemented
with folate polyethyleneglycol dioleylphatidylethanolamine for tumor cell-specific
targeting N-Citraconyl- dioleylphosphatidylethanolamine is also included for
pH dependent release of endosome-entrapped DNA into the cytoplasm and a novel
plasmid containing a 366 bp segment from SV40 DNA has been employed to facilitate
transport of the plasmid into the nucleus. It was found that complexation of
plasmid DNA with high molecular weight polymers such as acylated polylysine
and cationic dendrimers leads to higher folate mediated transfection efficiency
than DNA complexed with unmodified polylysine. In contrast, compaction of plasmid
DNA with small cationic molecules such as spermine, spermidine or gramicidin
S yields only weakly active folate-targeted liposomal vectors (Reddy et al.,
Barriers of Using Cations in non Viral Gene Therapy
Extracellular and Intracellular Barriers
Complexes of DNA with cationic lipids and cationic polymers are frequently
used for gene transfer. Extracellular interactions of the complexes with anionic
glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of
GAGs with carrier DNA complexes have been studied using tests for DNA relaxation
(ethidium bromide intercalation), DNA release (electrophoresis) and transfection
(pCMVbGal transfer into RAA smooth muscle cells). Several cationic lipid formulations
(DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured
dendrimer, polyethylene imines 25 and 800 kDa, polylysines 20 and 200 kDa) were
tested. Polycations condensed DNA more effectively than monovalent lipids. Hyaluronic
acid did not release or relax DNA in any complex, but it inhibited transfection
by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by other carriers
was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin
sulfates B and C) completely blocked transfection, except in the case of liposomes
with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these
events were not prerequisites for the inhibition of transfection. Furthermore,
preliminary results suggest that cell surface GAGs, particularly heparan sulfate,
inhibit gene transfer by cationic lipids and polymers (Ruponen et al.,
The additional hydrogen bonding or covalent interactions of the headgroup with the plasmid DNA, leading to higher binding affinity of the cationic lipids to pDNA, results in higher transfection. This hypothesis is supported by TEM observations where elongated complexes were observed and more lipid was seen associated with the DNA (Narang et al., 2005).
Effect of Human Plasma Proteins
In situ gene expression assay indicated that both the ratio of DNA and liposome
and the dose of DNA could affect the gene transfection efficiency. Naked endostatin
plasmid intratumoral injection can get a similar gene transfection efficiency
to liposome-DNA complex when used in situ (Ma et al., 2004).
Interaction of cationic lipid/DNA complex with the plasma is a limiting step
for the cationic lipid-mediated intravenous gene transfer and expression process.
Most of the plasma components that interact with the complex and inhibit its
transfection efficiency are still unknown. In an experiment, human plasma proteins
and lipoproteins that bind to a cationic lipid/DNA complex were isolated on
a sucrose density gradient and identified by 2-D gel electrophoresis. Protein
binding did not result in complex dissociation or DNA degradation. The effects
of several complex-binding plasma components on the transfection efficiency
were studied using lung endothelial cells cultured in vitro. Lipoprotein
particles caused a drastic loss of the transfection efficiency of the complex.
Surprisingly, fibrinogen was found to activate the transfection process. The
roles of these complex-binding plasma components on the complex uptake efficiency
were quantitatively assessed using radiolabeled plasmid DNA and qualitatively
evaluated using fluorescence microscopy. A good correlation was found between
the effects of the complex-binding plasma components on the transfection and
on cell uptake efficiencies. In contrast to what was generally believed, our
data suggest that disruption of the complex does not occur when it is in contact
with the plasma and therefore could not be responsible for the loss of transfection
activity. Instead, coating of complexes with plasma components seems to be responsible
for reduced uptake by cells, which in turn results in reduced transfection (Tandia
et al., 2003).
Improvement of Transfection Efficiency
Efficiency Increasing by Surface Coating
Systemic gene delivery systems are needed for therapeutic applications.
For systemic circulation, masking the surface charge of DNA complexes has to
be accomplished to avoid interactions with plasma components, erythrocytes and
the reticuloendothelial system. Polyplexes based on polyethylenimine (PEI),
shielded with polyethylene glycol (PGE) and linked to the receptor binding ligands
transferrin (Tf) or epidermal growth factor (EGF) have been developed. Complexes
were found to mediate efficient gene transfer into tumor cell lines in a receptor
dependent and cell-cycle dependent manner. Systemic administration of surface
shielded Tf-PEI polyplexes into the tail vein of mice resulted in preferential
gene into distantly growing subcutaneous tumors. In contrast, application of
positively charged PEI polyplexes directed gene transfer primarily to the lung
(Ogris and Wagner, 2002).
Delivery Efficiency and Gene Expression Efficiency Increasing Action
Tumor targeting is an important issue in cancer gene therapy. A gene transfection
method, based on light inducible photochemical internalization (PCI) of a transgene,
is already developed to improve gene delivery and expression selectively in
illuminated areas, for example in tumors. PCI improve the non-viral vector polyethylenimine
(PEI)-mediated transfection of therapeutic gene, the suicide gene encoding herpes
simplex virus thymidine kinase (HSV+K). Photochemical transfection allows selective
enhancement in gene expression and gene mediated biological effects (cell killing
by the HSV+K/GCV approach) in response to illumination (Prasmickaite et al.,
Systemic gene therapy vectors must be designed to safety and efficiently escort DNA from outside the cell to the nucleus and to overcome several physiological barriers that are obstacles to internalization, escape from endocytic vecicles, movement through the cytoplasm and transport into the nucleus. Chloroquine appears to facilitate PLL-mediated gene delivery by a mechanism other than buffering of endocytic compartments. Additionally, PEI does not appear to buffer endocytic compartments but requires exposure to an acidic environment for efficient gene delivery (Forrest and Pack, 2002).
Gene delivery/therapy is one of the youngest and most promising fields of medicine. In the course of one decade the basic research in recombinant DNA has been translated into a number of applied projects aiming at curing the diseases at its root, the gene. Though no clinically acceptable protocols have yet been developed, the researchers ability to solve many technical problems of gene therapy has been greatly improved.
Several viral and nonviral gene therapy vectors have been evaluated for a range of conditions in animal models and in clinic. Cationic polymers and lipids showed a great potential among the vector implied for gene therapy and it also proved that they are safer than viral vectors. Many types of cationic polymers and lipids are tested but it is still a vital field of research to invent new one.
From this review, it has emerged that a single vector is highly unlikely to be optimal for all gene therapy applications. To improve gene transfer efficiency, the appropriate vector must be selected for transfection of the required cell type. Ultimately all disease targets will benefit from an improved understanding of the biology of vector delivery, uptake and expression. Still the perfect vector covering all therapeutic and safety requirements does not exist and much work is needed in this important research field.
Improvements in vector design will also reap rewards in the clinic. Research
into vector design and targeting needs to proceed in parallel with early clinical
studies, where proof of concept and identification of barriers to clinical gene
transfer can inform the next step to basic research.
1: Aleksander, S., T. Cichon, M. Makselon, M. Stroiyk, R. Smolarczyk, J.J. Rakus and S. Szala, 2004. In vivoGene transfer using cetylated polyethylenimine. Acta Biochimica Polonica, 51: 693-702.
Direct Link |
2: Aneed, E.A., 2004. An overview of current delivery systems in cancer gene therapy. J. Control Release, 94: 1-14.
3: Behr, J.P., 1994. Gene transfer with synthetic cationic amphiphiles prospects for gene therapy. Bioconjug. Chem., 5: 382-389.
4: Bogdan, I.F., C. Meaney, H.E. Junginger and G. Borchard, 2002. Transfection efficiency and toxicity of polyethylenimine in differentiated calu 3 and nondifferentiated cos 1 cell cultures. AAPS Pharm. Sci., 4: 12-12.
5: Boussif, O., F. Lezoualch, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix and J.P. Behr, 1995. Versatile vector for gene and oligonucleotide transfer into cells in culture and In vivo polyethylenimine. Proc. Natl. Acad. Sci. USA., 92: 7297-7301.
6: Bozkir, A. and O.M. Saka, 2004. Chitosan DNA nanoparticles effect on DNA integrity bacterial transformation and transfection efficiency. J. Drug Target, 12: 281-288.
7: Brown, M.D., A. Schatzlein, A. Brownlie, V. Jack and I.F. Uchegbu et al., 2000. Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents. Bioconjug. Chem., 11: 880-891.
8: Chiu, S.J., N.T. Ueno and R.J. Lee, 2006. Tumor targeted gene delivery via anti HER2 antibody (Trastuzuma hberceptin) conjugated polyethylenimine. J. Control Release, 97: 357-369.
9: Chollet, P., M.C. Favrot, A. Hurbin and J.L. Coll, 2002. Side effects of a systemic injection of linear polyethylenimine DNA complexes. J. Gene. Med., 4: 84-91.
10: Dash, P.R., M.L. Read, L.B. Barrett, M.A. Wolfert and L.W. Seymour, 1999. Factors affecting blood clearance and In vivodistribution of polyelectrolyte complexes for gene delivery. Gene. Ther., 6: 643-650.
Direct Link |
11: Dick, C.R. and G.E. Ham, 1970. Characterisation of polyethylenimine. J. Macromol. Sci., 4: 1301-1314.
Direct Link |
12: Erbacher, P., S. Zou, T. Bettinger, A.M. Steffan and J.S. Remy, 1998. Chitosan based vector/DNA complexes for gene delivery biophysical characteristics and transfection ability. Pharm. Res., 15: 1332-1339.
13: Falk, A., N. Holmstrom, M. Carlen, R. Cassidy, C. Lundberg and J. Frisen, 2002. Gene delivery to adult neural stem cells. Exp. Cell. Res., 279: 34-39.
14: Faraasen, S., J. Voros, G. Csucs, M. Textor, H.P. Merkle and E. Walter, 2003. Ligand specific targeting of microspheres to phagocytes by surface modification with poly(L lysine) grafted poly(ethylene glycol) conjugate. Pharm. Res., 20: 237-246.
Direct Link |
15: Fischer, D., T. Bieber, Y. Li, H.P. Elsasser and T. Kissel, 1999. A novel non viral vector for DNA delivery based on low molecular weight branched polyethylenimine effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res., 16: 1273-1279.
16: Forrest, M.L. and D.W. Pack, 2002. On the kinetics of polyplex endocytic trafficking implications for gene delivery vector design. Mol. Ther., 6: 57-66.
17: Furgeson, D.Y., J.W. Yockman, M.M. Janat and S.W. Kim, 2004. Tumor efficacy and biodistribution of linear polyethylenimine cholesterol/DNA complexes. Mol. Ther., 9: 837-845.
18: Godbey, W.T., K.K. Wu and A.G. Mikos, 2001. Poly(ethylenimine) mediated gene delivery affects endothelial cell function and viability. Biomaterials, 22: 471-480.
19: Guang, L.W. and K.D. Yao, 2002. Chitosan and its derivatives a promising non viral vector for gene transfection. J. Control Release, 83: 1-11.
Direct Link |
20: Han, J., M. Lim and Y.I. Yeom, 1999. Receptor mediated gene transfer to cells of hepatic origin by galactosylated albumin polylysine complexes. Biol. Pharm. Bull., 22: 836-840.
Direct Link |
21: Jeon, E., H.D. Kim and J.S. Kim, 2003. Pluronic grafted poly (L) lysine as a new synthetic gene carrier. J. Biomed. Mater. Res. A., 66: 854-859.
Direct Link |
22: Kaplan, J.M., S.E. Pennington, J.A. St George, L.A. Woodworth and A.E. Smith et al., 1998. Potentiation of gene transfer to the mouse lung by complexes of adenovirus vector and polycations improves therapeutic potential. Hum. Gene. Ther., 9: 1469-1479.
23: Kichler, A., C. Leborgne, E. Coeytaux and O. Danos, 2001. Polyethylenimine mediated gene delivery a mechanistic study. J. Gene. Med., 3: 135-144.
24: Kim, J.S., B.I. Kim, A. Maruyama, T. Akaike and S.W. Kim, 1998. A new non viral DNA delivery vector the terplex system. J. Control Release, 53: 175-182.
25: Kircheis, R., S. Schuller, S. Brunner, M. Ogris, K.H. Heider, W. Zauner and E. Wagner, 1999. Polycation based DNA complexes for tumor targeted gene delivery in vivo. J. Gene. Med., 1: 111-120.
26: Klemm, A.R., D. Young and J.B. Lloyd, 1998. Effects of polyethyleneimine on endocytosis and lysosome stability. Biochem. Pharmacol., 56: 41-46.
27: Klotz, I.M., G.P. Royer and A.R. Sloniewsky, 1969. Macromolecule small molecule interactions strong binding and cooperativity in a model synthetic polymer. Biochemistry, 8: 4752-4756.
28: Kunath, K., A.V. Harpe, D. Fischer, H. Petersen, U. Bickel, K. Voigt and T. Kissel, 2003. Low molecular weight polyethylenimine as a non viral vector for DNA delivery comparison of physicochemical properties, transfection efficiency and In vivo distribution with high molecular weight polyethylenimine. J. Control. Release, 89: 113-125.
29: Lechardeur, D., K.J. Sohn, M. Haardt, P.B. Joshi and M. Monck et al., 1999. Metabolic instability of plasmid DNA in the cytosol a potential barrier to gene transfer. Gene. Ther., 6: 482-497.
Direct Link |
30: Lee, H., J.H. Jeong and T.G. Park, 2002. PEG grafted polylysine with fusogenic peptide for gene delivery high transfection efficiency with low cytotoxicity. J. Control Release, 79: 283-291.
31: Li, Z. and L. Huang, 2004. Sustained delivery and expression of plasmid DNA based on biodegradable polyester poly (D L lactide co 4 hydroxy L proline). J. Control Release, 98: 437-446.
Direct Link |
32: Lim, Y.B., S.M. Kim, H. Suh and J.S. Park, 2002. Biodegradable endosome disruptive and cationic network type polymer as a highly efficient and nontoxic gene delivery carrier. Bioconjug. Chem., 13: 952-957.
33: Ch, Z.Y.M., X.Y. Wang, L.F. Gao, H. Liu and W.S. Sun et al., 2004. Human endostatin gene transfer either naked or with liposome has the same inhibitory effect on growth of mouse liver tumor cells in vivo. World J. Gastroenterol., 10: 2874-2877.
34: Mannisto, M., S. Vanderkerken, V. Toncheva, M. Elomaa, M. Ruponen, E. Schacht and A. Urtti, 2002. Structure activity relationships of poly(L lysines) effects of pegylation and molecular shape on physicochemical and biological properties in gene delivery. J. Control Release, 83: 169-182.
35: Midoux, P. and M. Monsigny, 1999. Efficient gene transfer by histidylated polylysine/pDNA complexes. Bioconjug. Chem., 10: 406-411.
36: Miller, A.D., 1992. Human gene therapy comes of age. Nature, 357: 455-460.
37: Moret, I., J.E. Peris, V.M. Guillem, M. Benet and F. Revert et al., 2001. Stability of PEI/DNA and DOTAP/DNA complexes effect of alkaline pH heparin and serum. J. Control Release, 76: 169-181.
Direct Link |
38: Morimoto, K., M. Nishikawa, S. Kawakami, T. Nakano and M. Hashida et al., 2003. Molecular weight dependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver. Mol. Ther., 7: 254-261.
39: Mulligan, R.C., 1993. The basic science of gene therapy. Science, 260: 926-932.
40: Nah, J.W., L. Yu, S.O. Han, C.H. Ahn and S.W. Kim, 2002. Artery wall binding peptide poly(ethylene glycol) grafted poly(L lysine) based gene delivery to artery wall cells. J. Control Release, 78: 273-284.
41: Narang, A.S., L. Thoma, D.D. Miller and R.I. Mahato, 2005. Cationic lipids with increased DNA binding affinity for nonviral gene transfer in dividing and nondividing cells. Bioconjug. Chem., 16: 156-168.
Direct Link |
42: Nishikawa, M., S. Takemura, Y. Takakura and M. Hashida, 1998. Targeted delivery of plasmid DNA to hepatocytes In vivooptimization of the pharmacokinetics of plasmid DNA/galactosylated poly(L lysine) complexes by controlling their physicochemical properties. J. Pharmacol. Exp. Ther., 287: 408-415.
43: Ogris, M. and E. Wagner, 2002. Tumor targeted gene transfer with DNA polyplexes. Somat Cell Mol. Genet., 27: 85-95.
44: Pichon, C., C. Goncalves and P. Midoux, 2001. Histidine rich peptides and polymers for nucleic acids delivery. Adv. Drug Deliv. Rev., 53: 75-94.
45: Pollard, H., J.S. Remy, G. Loussouarn, S. Demolombe, J.P. Behr and D.E. cande, 1998. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells. J. Biol. Chem., 273: 7507-7511.
Direct Link |
46: Pouton, C.W., P. Lucas, B.J. Thomas, A.N. Uduehi, D.A. Milroy and S.H. Moss, 1998. Polycation DNA complexes for gene delivery a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids. J. Control Release, 53: 289-299.
47: Prasmickaite, L., A. Hogset, V.M. Olsen, O. Kaalhus, S.O. Mikalsen and K. Berg, 2004. Photochemically enhanced gene transfection increases the cytotoxicity of the herpes simplex virus thymidine kinase gene combined with ganciclovir. Cancer Gene. Ther., 11: 514-523.
48: Prince, H.M., 1998. Gene transfer a review of methods and applications. Pathology, 30: 335-347.
49: Putnam, D., C.A. Gentry, D.W. Pack and R. Langer, 2001. Polymer based gene delivery with low cytotoxicity by a unique balance of side chain termini. Proc. Natl. Acad. Sci. USA., 98: 1200-1205.
50: Reddy, J.A., D. Dean, M.D. Kennedy and P.S. Low, 1999. Optimization of folate conjugated liposomal vectors for folate receptor mediated gene therapy. J. Pharm. Sci., 88: 1112-1118.
51: Ruponen, M., P. Honkakoski, S. Ronkko, J. Pelkonen, M. Tammi and A. Urtti, 2003. Extracellular and intracellular barriers in non viral gene delivery. J. Control Release, 93: 213-217.
52: Schmitz, V., C. Qian, J. Ruiz, B. Sangro and J. Prieto et al., 2004. Gene therapy for liver diseases recent strategies for treatment of viral hepatitis and liver malignancies. J. Control Release, 99: 313-313.
53: Shewring, L., L. Collins, S.L. Lightman, S. Hart, K. Gustafsson and J.W. Fabre, 1997. A nonviral vector system for efficient gene transfer to corneal endothelial cells via membrane integrins. Transplantation, 64: 763-769.
54: Sugiyama, M., M. Matsuura, Y. Takeuchi, J. Kosaka, M. Nango and N. Oku, 2004. Possible mechanism of polycation liposome (PCL) mediated gene transfer. Biochim. Biophys. Acta, 1660: 24-30.
55: Suh, W., J.K. Chung, S.H. Park and S.W. Kim, 2001. Anti JL1 antibody conjugated poly(L lysine) for targeted gene delivery to leukemia t cells. J. Control Release, 72: 171-178.
Direct Link |
56: Tandia, B.M., M. Vandenbranden, R. Wattiez, Z. Lakhdar, J.M. Ruysschaert and A. Elouahabi, 2003. Identification of human plasma proteins that bind to cationic lipid/DNA complex and analysis of their effects on transfection efficiency implications for intravenous gene transfer. Mol. Ther., 8: 264-273.
57: Thomas, M. and A.M. Klibanov, 2003. Non viral gene therapy polycation mediated DNA delivery. Applied Microbiol. Biotechnol., 62: 27-34.
58: Tseng, W.C. and C.M. Jong, 2003. Improved stability of polycationic vector by dextran grafted branched polyethylenimine. Biomacromolecules, 4: 1277-1284.
59: Wagner, E., C. Plank, K. Zatloukal, M. Cotton and M.L. Birnsteil, 1992. Influenza virus hemagglutinin HA 2 N terminal fusogenic peptides augment gene transfer by transferringpolylysine DNA complexes toward a synthetic virus like gene transfer vehicle. Proc. Natl. Acad. Sci. USA., 89: 7934-7938.
60: Ward, C.M., M. Pechar, D. Oupicky, K. Ulbrich and L.W. Seymour, 2002. Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate mediated targeting in vitroand prolonged plasma circulation In vivo. J. Gene. Med., 4: 536-547.
61: Ward, C.M., M.L. Read and L.W. Seymour, 2001. Systemic circulation of poly (L lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA differential circulation in mice and rats and the implications for human gene therapy. Blood, 97: 2221-2229.
62: Wightman, L., R. Kircheis, V. Rossler, S. Carotta, R. Ruzicka, M. Kursa and E. Wanger, 2001. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J. Gene. Med., 3: 362-372.
63: Wiethoff, C.M., J.G. Smith, G.S. Koe and C.R. Middaugh, 2001. The potential role of proteoglycans in cationic lipid mediated gene delivery studies of the interaction of cationic lipid DNA complexes with model glycosami noglycans. J. Biol. Chem., 276: 32806-32813.
64: Wolfert, M.A., P.R. Dash, O. Nazarova, D. Oupicky and L.W. Seymour et al., 1999. Polyelectrolyte vectors for gene delivery influence of cationic polymer on biophysical properties of complexes formed with DNA. Bioconjug. Chem., 10: 993-1004.
65: Wu, G.Y. and C.H. Wu, 1987. Receptor mediated in vitrogene transformation by a solu. J. Biol. Chem., 262: 4429-4432.
66: Xiang, J.J., J.Q. Tang, S.G. Zhu, X.M. Nie and G.Y. Li et al., 2003. IONP PLL a novel non viral vector for efficient gene delivery. J. Gene. Med., 5: 803-817.