Introduction
============

The field of gene-based medicine has witnessed great progress since the first somatic gene therapy performed in 1990. Several thousand patients have been involved in clinical trials all over the world with majority focusing on cancer (67%), followed by vascular diseases (8.9%) and monogenic diseases (8.6%) (<http://www.wiley.co.uk/genetherapy/clinical>). Gene therapy works on the basic concept that the delivery of polynucleotides to the cells will alter the expression of a given protein resulting in therapeutic benefit. Gene therapy involves delivering polynucleotides such as DNA, RNA, anti-sense oligonucleotides and small interfering RNA, either locally or systemically. Although Vitravene, an antisense oligonucleotide-based product, is the only gene delivery product approved so far by US-FDA, there are several other products in late stages of clinical trials. Gendicine, an adenovirus encoding tumor suppressor p53 gene, developed by SiBiono GeneTech Co., Ltd., was recently approved by China's state food and drug administration, for the treatment of head and neck squamous cell carcinoma.

Delivering functional polynucleotides into cells is the first and most critical step towards efficient gene therapy. The administration of naked DNA resulted in local transient expression in skeletal muscle tissue ([@b77-ijn-2-353]). Efficient transfection levels have also been obtained on direct application of naked DNA to the liver ([@b27-ijn-2-353]). To obtain systemic effect with the injection of naked DNA is difficult, however, as the intravenous injection of naked DNA results in low levels of gene expression in all major organs.

In order to enhance uptake of genes into cells, they have to be delivered using a carriers or vectors. The vectors can be broadly classified as viral and non-viral. Viral vectors account for nearly 75% of all clinical trials conducted so far (<http://www.wiley.co.uk/genetherapy/clinical>). They are essentially viruses that have been stripped of their gene for replication while preserving their ability to transfect cells. The gene of interest is then incorporated into the viral genome. Most commonly used viral vectors are retrovirus, adenovirus, adeno-associated virus and herpes simplex virus ([@b59-ijn-2-353]; [@b75-ijn-2-353])). Virus based approaches are highly efficient as viruses have a highly evolved and specific mechanism for inserting their genome into that of host cell. Despite the high efficiency of viral vectors, their use has been limited by their pathogenicity, immunogenicity and potential for insertional mutagenesis.

Incidences of severe adverse reactions using viral vectors during clinical trials have caused a gradual shift towards non-viral vectors. Non-viral vectors mostly include use of polymers and lipids to deliver genetic material intracellularly protecting it from extracellular and intracellular degradative enzymes and blood components ([@b13-ijn-2-353]). Non-viral vectors are mostly non-immunogenic, less expensive to produce, relatively safer, and can carry higher amounts of genetic material as compared to viruses. However, transfection efficiency using non-viral vectors remains lower as compared to viral vectors.

Challenges associated with non-viral gene delivery
==================================================

Non-viral vectors face a multitude of barriers at systemic, tissue and cellular levels that prevent efficient gene delivery to the nucleus of cells. A significant portion of DNA is lost at each step, resulting in a several fold decrease in expression of the encoded protein. Most non-viral delivery vectors display colloidal instability which results in aggregation of the complexes thereby hindering cellular internalization. Systemic delivery of charged vector may result in interaction with blood components which may lead to opsonisation by the reticulo-endothelial system ([@b46-ijn-2-353]). Problems such as steric instability and rapid plasma clearance have been partly overcome by shielding the surface charge of the vectors ([@b36-ijn-2-353]). Properties of vector such as size, shape, and surface characteristics can also have a major impact on its pharmacokinetic properties and delivery efficiency.

Non-viral vectors, also encounter several extracellular and intracellular barriers before reaching its final destination, the nucleus ([@b61-ijn-2-353]; [@b76-ijn-2-353]). Typical pathway of a nanoparticulate vector inside the cell is depicted in [Figure 1](#f1-ijn-2-353){ref-type="fig"}. The presence of nucleases in the extracellular compartment may lead to extensive degradation of the DNA. Cellular internalization is another major barrier as DNA is a polyanionic molecule with a large hydrodynamic volume which makes its entry through the negatively charged plasma membrane extremely difficult. Following uptake into the cell, the vector may become trapped within the endosomal compartment, from which it must escape. Failure to do so may result in degradation of DNA by lysosomal degradative enzymes. Surviving passage through the cytoplasm is a crucial step, because of the presence of cytosolic endonucleases, which may further lead to DNA fragmentation. Lastly, the DNA has to traverse the nuclear envelope to get transcribed. In order to enhance gene therapy, specialized design features are required for delivery vector to overcome each of these barriers, and ensure efficient DNA delivery to the nucleus.

Nanoparticulate vectors for polynucleotide delivery
===================================================

Nanotechnology has been at the forefront of drug and gene delivery in the last few years. Several types of nanometer scale devices such as nanoparticles, nanospheres, nanotubes, nanogels and molecular conjugates are being investigated ([@b45-ijn-2-353]; [@b4-ijn-2-353]; [@b57-ijn-2-353]; [@b26-ijn-2-353]; [@b54-ijn-2-353]). Sub-micron size of delivery systems confers distinct advantages as compared to large sized systems such as higher and deeper tissue penetrability, greater cellular uptake, greater ability of cross blood-brain barrier, and greater ability to target specific cell types ([@b39-ijn-2-353]; [@b12-ijn-2-353]; [@b72-ijn-2-353]; [@b73-ijn-2-353]). Although this review, for most part, deals with polymeric nanoparticles, commonly used non-polymeric nanovectors have also been discussed briefly. A summary of all the nanoparticulate vectors discussed in the review is presented in [Table 1](#t1-ijn-2-353){ref-type="table"}.

Polymeric nanoparticles
-----------------------

Polymeric nanoparticles or nanospheres are the most commonly used type of nano-scale delivery systems. They are mostly spherical particles, in the size range of 1--1000 nm, carrying the genetic material of interest. The mechanism of incorporation of polynucleotides into polymeric nanoparticles depends on the nature of the polymer. Most cationic polymers have the ability to condense plasmid DNA into nanometer size complexes (polyplexes). DNA can also be entrapped into the polymeric matrix or can be adsorbed or conjugated on the surface of nanoparticles. A variety of natural and synthetic polymers have been used for gene delivery. In this section, we have discussed the most extensively used polymers for nanoparticle-based gene delivery.

### Poly lactide-co-glycolide (PLGA) and Poly lactic acid (PLA)

Biodegradable polyesters, PLGA and PLA are amongst the most commonly used polymers for delivering drugs and biomolecules. They consist of units of lactic acid and glycolic acid connected through ester linkage. These biodegradable polymers undergo bulk hydrolysis thereby providing sustained delivery of the therapeutic agent. The rate of degradation of polymer, and the release rate of drug, can be controlled by changing the polymer molecular weight and copolymer composition. The degradation products, lactic acid and glycolic acid, are removed from the body through citric acid cycle ([@b66-ijn-2-353]). The release of therapeutic agent from these polymers occurs by diffusion and polymer degradation.

PLGA and PLA nanoparticles are mostly matrix-type systems prepared by emulsification followed by evaporation of the organic phase ([@b60-ijn-2-353]). Several studies have shown higher uptake of nanoparticles into cells as compared to larger size particles ([@b15-ijn-2-353]; [@b16-ijn-2-353]; [@b14-ijn-2-353]; [@b55-ijn-2-353]). Nanoparticles have the ability to escape endo-lysosomal compartments and release the therapeutic molecule inside the cell at a sustained rate ([@b55-ijn-2-353]; [@b35-ijn-2-353]). The release pattern can be modulated according to the dosing requirement by changing various formulation parameters. The poly-nucleotide can be incorporated in PLGA/PLA nanoparticles by either entrapment into the polymeric matrix ([@b58-ijn-2-353]) or by surface-adsorption by using a cationic polymer or surfactant ([@b34-ijn-2-353]). PLGA and PLA nanoparticles have been used for the delivering plasmid DNA ([@b10-ijn-2-353]), siRNA ([@b79-ijn-2-353]), and aptamers ([@b7-ijn-2-353]).

### Cationic polymers

Cationic polymers by virtue of their positive charge can efficiently condense the anionic polynucleotides into nano-meter range complexes (polyplexes) thereby masking their negative charge ([@b17-ijn-2-353]; [@b81-ijn-2-353]). Polynucleotides are polyanionic molecules with a large hydrodynamic diameter which presents a significant barrier towards efficient cellular uptake. Cationic polymers, apart from condensing it to a several fold smaller size, also provide a net positive charge to the complex which helps in attachment on cellular membrane. Also, most cationic polymers bear amine groups that are protonable at acidic pH. Thus, once inside the endosome, these polymers accept proton thereby resisting a drop in pH. This causes influx of counterions (chloride ions) resulting in osmotic swelling and subsequent rupture of endosome. This phenomenon, first explained by Behr, is known as proton sponge effect ([@b3-ijn-2-353]). The efficiency of polyplexes has been found to be dependent on the ratio of nitrogen atoms of the polymer to the phosphate groups present in the polynucleotide (N/P ratio) ([@b23-ijn-2-353]). A variety of natural and synthetic cationic polymers have been used for gene delivery. Some of the most commonly used cationic polymers have been discussed below.

### Polyethylenimine (PEI)

Polyethyleneimine is the most commonly used cationic polymer and is widely regarded as a gold standard, amongst non-viral vectors, in order to compare transfection efficiencies. Transfection efficiency of PEI has been found to be dependent on a multitude of factors such as molecular weight, degree of branching, N/P ratio, complex size etc ([@b69-ijn-2-353]). PEI has a high density of protonable amino groups, every third atom being amino nitrogen, which imparts it a high buffering ability at practically any pH. Hence, once inside the endolysosomal compartment, PEI can efficiently destabilize the endosome releasing the polynucleotide in the cytoplasm. PEI can also be used in combination with PLGA/PLA for preparing matrix-type nanoparticles to deliver DNA by adsorption/complexation on the surface ([@b34-ijn-2-353]). Toxicity of PEI, however, has been a concern. Acute toxicity has been observed in several studies both in vitro and in vivo ([@b9-ijn-2-353]; [@b52-ijn-2-353]). Investigators have studied conjugation of polyethylene glycol (PEG) to PEI to form diblock or triblock copolymers to reduce PEI-associated toxicity ([@b56-ijn-2-353]; [@b82-ijn-2-353]; [@b8-ijn-2-353]). PEG also shields the positive charge of the polyplexes, thereby providing steric stability to the complex. Such stabilization prevents non-specific interaction with blood components during systemic delivery ([@b40-ijn-2-353]).

### Polymethacrylates

Polymethacrylates are cationic vinyl-based polymers that possess the ability to condense polynucleotides into nano-meter size particles. Several polymethacrylates such as poly \[2-(dimethylamino) ethyl methacrylate\] (DMAEMA) and its co-polymers have been used for polynucleotide delivery. Presence of protonable tertiary amine groups in their structure provides buffering ability similar to that of PEI. A range of Polymethacrylates, differing in molecular weights and chemical structures, have been evaluated for their potential as gene delivery vector ([@b18-ijn-2-353]; [@b19-ijn-2-353]). Polymethacrylates containing only tertiary amine groups were found to be similar to PEI in terms of transfection efficiency while displaying much better biocompatibility profile ([@b19-ijn-2-353]). Nanoparticles with a methacrylate core and PEI shell prepared via graft copolymerization have also been employed lately for gene delivery ([@b46-ijn-2-353]; [@b21-ijn-2-353]). Such conjugation resulted in nanoparticles with a higher transfection efficiency and lower toxicity as compared to PEI alone. We have recently formulated cationic nanoparticles with commercially available polymethacrylate Eudragit^®^ E100 in combination with PLGA/PLA using cationic surfactant, cetyltrimethyl-ammonium bromide (CTAB), and achieved much improved transfection efficiency as compared to PLA/CTAB and PLGA/CTAB nanoparticles (Unpublished data).

### Poly-L-Lysine (PLL)

Poly-l-lysine is amongst the first cationic polymers investigated for gene delivery. It is a biodegradable polymer synthesized by polymerization of N-carboxy-anhydride of lysine ([@b81-ijn-2-353]). Biodegradability is a highly desirable property for gene delivery applications in vivo. Poly-l-lysine is able to form nanometer size complexes with polynucleotides owing to the presence of protonable amine groups on the lysine moiety. Complexation of PLL with polynucleotide results in formation of toroidal or rod-shaped polyplexes less than 100 nm in size ([@b41-ijn-2-353]). Although PLL polyplexes have shown good cellular uptake, their transfection efficiency is several folds lower than PEI polyplexes ([@b51-ijn-2-353]). Possible reason for this may be reduced endosomal escape due to lack of amine groups that can be protonated at acidic pH ([@b1-ijn-2-353]). Resultant lack of buffering ability leads to degradation of polyplexes by lysosomal enzymes. Conjugation of PLL with poly (ethylene glycol (PEG) has been performed to shield charge on the surface thereby providing in vivo stability, delaying body clearance, and protecting DNA from nuclease degradation ([@b32-ijn-2-353]; [@b41-ijn-2-353]). Enhanced transfection efficiency has been observed by conjugating the PLL polyplexes with membrane disruptive peptides and fusion peptides ([@b74-ijn-2-353]; [@b44-ijn-2-353]). Delivery of siRNA using PLL has also been reported ([@b53-ijn-2-353]).

### Poly (β-amino ester)s

Poly (β-amino ester) (PBAE) is a novel, charge inducible, biodegradable, and non-toxic polymer. PBAE polyplexes have been prepared and optimized in terms of polymer molecular weight, polymer end groups, complex size, and DNA/polymer ratio ([@b50-ijn-2-353]; [@b2-ijn-2-353]). The polyplexes displayed transfection efficiencies comparable to PEI and commercial lipid-based transfection reagents and were able to transfect a variety of cell types ([@b2-ijn-2-353]). The complexes displayed significantly low cytotoxicity as compared to PEI polyplexes in vitro ([@b50-ijn-2-353]). PBAE in combination with PLGA was used for DNA vaccination and led to significant enhancement in transfection efficiency ([@b47-ijn-2-353]; [@b48-ijn-2-353]). PBAE/PLGA microparticles showed high immunogenic potential and led to a significant reduction of tumor size in mice ([@b47-ijn-2-353]). PBAE/PLGA particles were also able to delay the release of plasmid DNA for several days similar to PLGA particles thereby prolonging bioavailability ([@b48-ijn-2-353]).

### Chitosan

Chitosan, a naturally derived polycation, is amongst most widely investigated polymers for gene delivery. Mucoadhesive property of Chitosan makes it suitable for oral and nasal delivery of DNA ([@b22-ijn-2-353]). Chitosan can deliver polynucleotides, both, by encapsulation and by complexation, forming positively charged particles ([@b5-ijn-2-353]; [@b38-ijn-2-353]). Chitosan nanoparticles efficiently protect DNA from nuclease degradation ([@b6-ijn-2-353]). Chitosan nanoparticles have also been evaluated for siRNA delivery ([@b28-ijn-2-353]; [@b31-ijn-2-353]). Transfection efficiency of chitosan polyplexes is found to be dependent on charge ratio, pH, cell type, molecular weight of chitosan, and degree of deacetylation ([@b63-ijn-2-353]; [@b38-ijn-2-353]; [@b43-ijn-2-353]). Transfection efficiency of optimized chitosan polyplexes was similar to PEI but the onset of expression was found to be slower than PEI which may be due to slower endosomal escape of chitosan nanoparticles ([@b37-ijn-2-353]). Recently, oligosaccharides derived from chitosan were evaluated for gene delivery after conjugation with stearic acid ([@b29-ijn-2-353]). These nanoparticles displayed transfection efficiency comparable to that of commercial transfection reagent, Lipofectamine™ 2000, at higher time points post-transfection, while showing low cytotoxicity. Chitosan polyplexes physically combined with PLGA microparticles have also been evaluated for sustained gene delivery ([@b80-ijn-2-353]).

Cationic liposomes
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Liposomes are spherical vesicles made of phospholipids used to deliver drugs or genes inside the cells. They can range in size from 20 nm to a few microns. The use of cationic liposomes to deliver DNA into cells was first reported in 1987 ([@b20-ijn-2-353]). Since then, liposomes have been routinely used in gene delivery research. Positively charged liposomes combine with negatively charged polynucleotides to form liposome/polynucleotide complexes (lipoplexes). Although the positive charge of liposomes is a requirement for preparing lipoplexes, neutral lipids dioleoylphosphatidyl-ethanolamine is also commonly incorporated. It facilitates endosomal destabilization apart from providing structural stability to the liposome. Liposomes destabilize the lipid bilayer membranes by promoting the formation of non-bilayer lipid structures ([@b25-ijn-2-353]). Cellular entry of lipoplexes is shown to be primarily via clathrin-mediated endocytosis ([@b11-ijn-2-353]). Liposomes have also been employed for cell-targeting using a variety of targeting ligands ([@b68-ijn-2-353]; [@b70-ijn-2-353]). Although a variety of high-efficiency liposomes are commercially available for transfecting cells in vitro and in vivo, their toxicity is still a concern. Several lipoplex formulations have caused moderate to severe toxicities in animal models ([@b67-ijn-2-353]; [@b71-ijn-2-353]). At cellular level, lipoplexes have been reported to cause cell shrinking, reduced mitoses, and vacuolization of cytoplasm ([@b42-ijn-2-353]).

Gold nanoparticles
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Gold nanoparticles have lately been investigated as an alternative to lipid-mediated and polymer-mediated gene delivery. Gold nanoparticles can be easily prepared, display low toxicity, and the surface can be modified using various chemical techniques. The gold nanoparticles functionalized using quarternary ammonium chains can efficiently transfect mammalian cells ([@b62-ijn-2-353]). Up to 8 times higher transfection efficiency compared to PEI was observed using optimized formulations. Systemic delivery of plasmid DNA using PEG-modified gold nanoparticles resulted in improved stability and increased circulation time of DNA in the blood ([@b33-ijn-2-353]). Surface functionalization of gold nanoparticles using a PEG spacer also resulted in rapid cellular uptake and internalization ([@b65-ijn-2-353]).

Magnetic nanoparticles
----------------------

Delivery of polynucleotides using magnetic nanoparticles, also known as magnetofection, has been reported ([@b64-ijn-2-353]). In this technique, superparamagnetic iron oxide nanoparticles are prepared, and are coated with a polyelectrolyte such as PEI, to produce particles in size range of 400-1000 nm. Surface coating of PEI facilitates complexation of DNA on the surface. These nanoparticles are then delivered to the cells under the influence of a magnetic field. The mechanism of cellular uptake of magnetofectins was shown to be analogous to PEI polyplexes ([@b30-ijn-2-353]). Magnetofection for 10 minutes resulted in up to several hundred-fold increase in protein expression, compared to standard transfection, using a variety of transfection vectors in different cell lines ([@b64-ijn-2-353]). Another study reported an increase in transfection efficiency in primary airway epithelial cells using megetofection ([@b24-ijn-2-353]).

A recent study, investigating non-viral gene transfer in vivo has, however, reported a decrease in transfection efficiency using magnetic nanoparticles as compared to non-magnetic particles ([@b78-ijn-2-353]). The versatility and potential applications of this technique thus remain to be seen.

Conclusions
===========

The ultimate goal of gene therapy is to deliver genes with high transfection efficiency to specific cells without causing any adverse effects. Viral vectors are efficient at delivering genes but are inherently unsafe. Nanotechnology-based gene delivery vectors have shown tremendous potential at overcoming physiological and biochemical barriers towards efficient gene delivery. Vector modifications at molecular level have enabled scientists to develop a variety of nanosystems with high efficiency, high specificity and low toxicity. With increase in the information available about cellular and molecular mechanisms involved in the uptake and transport of these carriers inside the cells, there is great hope for the future of nanoparticulate systems for gene delivery.

![Schematic diagram of the entry of non-viral vector inside the nucleus of cell.](ijn-2-353f1){#f1-ijn-2-353}

###### 

Polymeric and non-polymeric nanoparticulate vectors for gene delivery

                  **Nanoparticles**                                             **Properties**                                                                                                **References**
  --------------- ------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------
                  Poly lactide-co-glycolide (PLGA) and Poly lactic acid (PLA)   Biocompatible and biodegradable, can provide sustained delivery of polynucleotides.                           [@b34-ijn-2-353]; [@b58-ijn-2-353]
                  Polyethyleneimine (PEI)                                       High transfection efficiency owing to faster endosomal escape. High toxicity reported in vitro and in vivo.   [@b52-ijn-2-353]; [@b69-ijn-2-353]
  Polymeric       Polymethacrylate                                              Lower toxicity as compared to PEI. Endosomal buffering ability similar to PEI.                                [@b18-ijn-2-353]; [@b21-ijn-2-353]
                  Poly-L-Lysine (PLL)                                           Biodegradable. Slow endosomal escape resulting in lower transfection efficiency                               [@b51-ijn-2-353]; [@b81-ijn-2-353]
                  Poly (β-amino ester) (PBAE)                                   High transfection efficiency comparable to PEI. Prolonged release of polynucleotide.                          [@b50-ijn-2-353]; [@b48-ijn-2-353]
                  Chitosan                                                      Mucoadhesive property desired for oral and nasal delivery. Slow onset of expression.                          [@b22-ijn-2-353]; [@b37-ijn-2-353]
                  Cationic liposomes                                            Extensively used for in vitro transfections. High in vivo toxicity                                            [@b20-ijn-2-353]; [@b71-ijn-2-353]
  Non-polymeric   Gold Nanoparticles                                            Highly inert and non-toxic. Surface functionalization can be easily performed.                                [@b33-ijn-2-353]; [@b62-ijn-2-353]
                  Magnetic nanoparticles                                        High transfection efficiency in variety of cell lines.                                                        [@b30-ijn-2-353]; [@b64-ijn-2-353]
