Lipid coated chitosan-DNA nanoparticles for enhanced gene delivery

Chitosan as a polycationic non-viral vector for gene delivery has the advantage of being a biocompatible and biodegradable polymer. However, without laborious chemical modifications to its structure, it is of limited use as a gene delivery vehicle due to its low ability to efficiently transfect under physiological conditions. To address this problem, we developed novel liposome encapsulated chitosan nanoparticles; lipochitoplexes (LCPs). Chitosan nanoparticles (CsNPs) were obtained using the ionic gelation technique. For this purpose, an ultrapure low molecular weight chitosan with a high degree of deacetylation was cross-linked using polyanionic tripolyphosphate resulting in efficient entrapment of plasmid DNA (pDNA) inside the nanoparticles. LCPs were prepared by incubating chitosan nanoparticles together with anionic liposomes (DPPC/Cholesterol). The LCPs offered better pDNA protection, reduced cytotoxicity and at least twofold increase in the transfection efficiency under physiological conditions. The efficiency of our delivery vehicle was also proved in vivo in the chorioallantoic membrane model (CAM). LCPs were able to transfect the CAM without traumatising the surrounding blood vessels. This new biocompatible composite system devoid of chemical modifications, organic solvents and harsh production conditions makes it an optimal gene delivery vehicle for in vivo applications offering new insights into the field of non-viral gene therapy.     

Recent progress in copolymer-mediated siRNA delivery

RNAi-mediated gene silencing has great potential for treating various diseases, including cancer, by delivering a specific short interfering RNA (siRNA) to knock down pathogenic mRNAs and suppress protein translation. Although many researchers are dedicated to devising polymer-based vehicles for exogenous in vitro siRNA transfection, few synthetic vehicles are feasible in vivo. Recent studies have presented copolymer-based vectors that are minimally immunogenic and facilitate highly efficient internalizing of exogenous siRNA, compared with homopolymer-based vectors. Cationic segments, organelle-escape units, and degradable fragments are essential to a copolymer-based vehicle for siRNA delivery. The majority of these cationic segments are derived from polyamines, including polylysine, polyarginine, chitosan, polyethylenimines and polyamidoamine dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Degradable fragments of copolymers must be derived from various polyelectrolytes to release the siRNA once the complexes enter the cytoplasm. This review describes recent progress in copolymer-mediated siRNA delivery, including various building blocks for biocompatible copolymers for efficient in vitro siRNA delivery, and a useful basis for addressing the challenges of in vivo siRNA delivery.     

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Abstract

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ～57% and ～70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.     

Low-molecular-weight polyethylenimine enhanced gene transfer by cationic cholesterol-based nanoparticle vector

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.     

Recent progress in copolymer-mediated siRNA delivery

RNAi-mediated gene silencing has great potential for treating various diseases, including cancer, by delivering a specific short interfering RNA (siRNA) to knock down pathogenic mRNAs and suppress protein translation. Although many researchers are dedicated to devising polymer-based vehicles for exogenous in vitro siRNA transfection, few synthetic vehicles are feasible in vivo. Recent studies have presented copolymer-based vectors that are minimally immunogenic and facilitate highly efficient internalizing of exogenous siRNA, compared with homopolymer-based vectors. Cationic segments, organelle-escape units, and degradable fragments are essential to a copolymer-based vehicle for siRNA delivery. The majority of these cationic segments are derived from polyamines, including polylysine, polyarginine, chitosan, polyethylenimines and polyamidoamine dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Degradable fragments of copolymers must be derived from various polyelectrolytes to release the siRNA once the complexes enter the cytoplasm. This review describes recent progress in copolymer-mediated siRNA delivery, including various building blocks for biocompatible copolymers for efficient in vitro siRNA delivery, and a useful basis for addressing the challenges of in vivo siRNA delivery.     

Effect of cationic carriers on the pharmacokinetics and tumor localization of nucleic acids after intravenous administration

Nucleic acid based therapeutics are currently being studied for their application in cancer therapy. In this study, the effect of different cationic delivery systems on the circulation kinetics, tumor localization, and tissue distribution of short interfering RNA (siRNA) and plasmid DNA (pDNA) was examined, after intravenous administration in mice bearing a s.c. Neuro 2A tumor. Nanosized particles were formed upon complexation of siRNA with the cationic liposome formulation DOTAP/DOPE and the targeted, cationic polymer RGD-PEG-PEI. Both the circulation kinetics and the overall tumor localization of the siRNA complexes were similar to non-complexed siRNA. Importantly, the different carriers changed the intratumoral distribution of siRNA within the tumor. pDNA was effectively condensed with linear polyethylenimine (PEI), PEGylated linear PEI (PEG-PEI) or poly(2-dimethylamino ethylamino)phosphazene. Only PEG-PEI was able to improve the pDNA circulation kinetics. All pDNA complexes yielded similar pDNA tumor localization (1% of the injected dose, 60 min after administration). We conclude that the level of nucleic acid tumor localization is independent on the type of formulation used in this study. Therefore, the value of carrier systems for the intravenous delivery of nucleic acids cannot be solely attributed to benefits relevant during the transport towards the tumor. Rather, the benefits are arising from carrier-induced changes in the intratumoral fate of the nucleic acids.     

Development and characterization of a new plasmid delivery system based on chitosan-sodium deoxycholate nanoparticles

Chitosan is one of the most promising polymers for drug delivery through the mucosal routes because of its polycationic, biocompatible, and biodegradable nature, and particularly due to its mucoadhesive and permeation-enhancing properties. Bile salts are known to interact with lipid membranes, increasing their permeability. The addition of bile salts to chitosan matrices may improve the delivery characteristics of the system, making it suitable for mucosal administration of bioactive substances. In the present study we have developed chitosan nanoparticles using sodium deoxycholate as a counter ion and evaluated their potential as gene delivery carriers. Chitosan-sodium deoxycholate nanoparticles (CS/DS) obtained via a mild ionic gelation procedure using different weight ratios were used to encapsulate plasmid DNA (pDNA) expressing a "humanized" secreted Gaussia Luciferase as reporter gene (pGLuc, 5.7 kDa). Mean particle size, polydispersity index and zeta potential were evaluated in order to select the best formulation for further in vitro studies. The nanoparticles presented an average size of 153-403 nm and a positive zeta potential ranging from +33.0 to +56.9 mV, for nanoparticles produced with CS/DS ratios from 1:4 to 1:0.6 (w:w), respectively. The pDNA was efficiently encapsulated and AFM studies showed that pDNA-loaded nanoparticles presented a more irregular surface due to the interaction between cationic chitosan and negatively charged pDNA which results in a more compact structure when compared to empty nanoparticles. Transfection efficiency of CS/DS-pDNA nanoparticles into moderately (AGS) and well differentiated (N87) gastric adenocarcinoma cell lines was determined by measuring the expression of luciferase, while cell viability was assessed using the MTT reduction. The CS/DS nanoparticles containing encapsulated pDNA were able to transfect both AGS and N87 cell lines, being more effective with AGS cells, the less differentiated cell line. The highest enzymatic activity was achieved with 20% pDNA encapsulated and after 24 h of transfection time. Low cytotoxicity was observed for the CS/DS nanoparticles either with or without pDNA, suggesting this could be a new potential vehicle for mucosal delivery of pDNA.     

Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex

Safe and efficient systems capable of specifically targeting brain tumour cells represent a promising approach for the treatment glioblastoma multiforme. Neuropilin-1 (NRP-1) is over-expressed in U87 glioma cells. In the current study, the tumour specific peptide RGERPPR, which binds specifically to NRP-1, was used as a targeting ligand in a gene delivery strategy for glioblastoma. The RGERPPR peptide was coupled to branched polyethylenimine (PEI, 25 kDa) using heterobifunctional Mal–PEG–NHS, resulting in a novel gene delivery polymer. Polymer/plasmid DNA (pDNA) complexes were formed and their sizes and zeta potentials were measured. Compared with the unmodified mPEG–PEI/pDNA complexes, the RGERPPR–PEG–PEI/pDNA complex led to a significant enhancement in intracellular gene uptake and tumour spheroid penetration. Furthermore, the RGERPPR–PEG–PEI/pDNA complex facilitated enhanced transfection efficiency levels, as well as a reduction in cytotoxicity when tested in U87 glioma cells in vitro. Most significantly of all, when complexes formed with pDsRED-N1 were injected into the tail vein of intracranial U87 tumour-bearing nude mice, the RGERPPR–PEG–PEI complexes led to improved levels of red fluorescence protein expression in the brain tissue. Taken together, the results show that RGERPPR–PEG–PEI could be used as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene delivery.     

Enhancement of gene transfection into human dendritic cells using cationic PLGA nanospheres with a synthesized nuclear localization signal

Effective delivery of DNA encoding antigen into the dendritic cells (DCs), which are non-dividing cells, is very important for the development of DNA vaccines. In a previous study, we developed the PLGA nanospheres that contained a cationic nanomaterial and showed high transfection efficiency in COS7 cells, which divide. In the present study, to produce an effective vector for the DNA vaccines, the gene expression and intracellular trafficking of pDNA complexed with PLGA/PEI nanospheres, in combination with an NF-κB analog as a nuclear localization signal (NLS) and electroporation were evaluated in human monocyte-derived DCs (hMoDCs). Cellular uptake of pDNA both in COS7 cells and hMoDCs was enhanced using the PLGA/PEI nanospheres. On the other hand, the PLGA/PEI nanospheres significantly promoted the transfection in COS7 cells, but had almost no effect on transfection in hMoDCs. The intranuclear transport of pDNA by PLGA/PEI nanospheres in COS7 cells was significantly higher than that in hMoDCs. These results indicate that pDNA complexed with PLGA/PEI nanospheres cannot enter into the nuclei of non-dividing cells. However, PLGA/PEI nanospheres combinated with NLS and electroporation (experimental permeation enhancer) greatly elevated the transfection efficiency by improvement of not only intracellular uptake but also intranuclear transport of pDNA in the hMoDCs. Thus, this delivery system using nanospheres combined with synthesized NLS might be applicable to DC-based gene vaccines when much non-invasive application such as needle-free injector should be required.     

Gelucire-stabilized nanoparticles as a potential DNA delivery system

Clinical viability of gene delivery systems has been greatly impacted by potential toxicity of the delivery systems. Recently, we reported the nanoparticle (NP) preparation process that employs biocompatible materials such as Gelucire® 44/14 and cetyl alcohol as matrix materials. In the current study, the NP preparation was modified for pDNA loading through: (i) inclusion of cationic lipids (DOTAP or DDAB) with NP matrix materials; or (ii) application of cationic surfactants (CTAB) to generate NPs with desired surface charges for pDNA complexation. Colloidal stability and efficiency of loading pGL3-DR4X2-luciferase plasmid DNA in NPs were verified by gel permeation chromatography. Compared to pDNA alone, all the NPs were effective in preserving pDNA from digestion by DNase. While pDNA loading using CTAB-NPs involved fewer steps compared to DOTAP-NPs and DDAB-NPs, CTAB-NPs were greatly impacted by elevated cytotoxicity level which could be ascribed to the concentrations of CTAB in NP formulations. In vitro transfection studies (in HepG2 cells) based on luciferase expression showed the ranking of cell transfection efficiency as DOTAP-NPs?>?DDAB-NPs?>?CTAB-NPs. The overall work provided an initial assessment of gelucire-stabilized NPs as a potential platform for gene delivery.     

Polyxylitol-based gene carrier improves the efficiency of gene transfer through enhanced endosomal osmolysis

Endosomal escape is one of the important processes for efficient non-viral gene delivery. In this study, we synthesized a novel non-viral vector called polyxylitol-based gene carrier (XGC) through a Miachael addition reaction between xylitol diacrylate as a crosslinking agent and low molecular weight polyethylenimine (PEI 1.2 kDa). The small amount of xylitol integrated into XGC (3.9% w/w) contributed 50% of the osmotic pressure of XGC, and enhaned the osmolysis of endosome cooperatively with the proton sponge effect, thus improving endosomal escape. Furthermore, XGC showed higher transfection efficiency in vivo in muscle tissue than pDNA alone or PEI 25 kDa. In conclusion, our results show that XGC enhanced transfection efficiency compared with PEI 25 kDa, the golden standard non-viral gene carrier, by enhancing endosomal escape without increasing the number of transfected cells.From the Clinical EditorEnhanced gene delivery methods would greatly facilitate the development of gene therapies. These authors demonstrate that a polyxylitol-based gene carrier enhanced the transfection efficiency compared with the gold standard non-viral gene carrier, as a result of enhancing endosomal escape without increasing the number of transfected cells, warranting further studies of this method.     

Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose

The successful control of intracellular trafficking (i.e., endosomal escape and nuclear delivery) is prerequisite for the development of a gene delivery system. In the present study, we developed an in vivo hepatic gene delivery system using a plasmid DNA (pDNA)-encapsulating lipid envelope-type nanoparticle, to which we refer as a multifunctional envelope-type nanodevice (MEND). The critical structural elements of the MEND are a DNA/protamine condensed core coated with lipid bilayers including serum-resistant cationic lipids. Intravenous administration of bare MEND represents minimal transfection activity. For the surface modification of functional devices, hydrophobic moieties were chemically attached, which are shed in the spontaneous orientation outward from the MEND surface by anchoring to the lipid bilayers. Modification of the pH-dependent fusogenic peptide GALA as an endosome escape induced transfection activity by 1 and 2 orders of magnitude. In an attempt to induce the nuclear delivery of pDNA, maltotriose, a recently characterized nuclear localization signal, was additionally modified. As a result, transfection activity further enhanced by 1 order of magnitude, and it reached to the higher level obtained for a conventional lipoplex and an in vivo jetPEI-Gal, with less hepatic toxicity. The data show that the combination of GALA and maltotriose results in a highly potent functional device that shows an enhanced endosomal escape and nuclear delivery in vivo.     

PEG grafting of polyethylenimine (PEI) exerts different effects on DNA transfection and siRNA-induced gene targeting efficacy

BACKGROUND: Gene targeting by RNA interference (RNAi) is mediated through small interfering RNA (siRNA), which, as plasmid DNA molecules, can be delivered into cells by polyethylenimines (PEI). Grafting with poly(ethylene glycol) has been introduced previously to improve PEI biocompatibility; however, data on the effects of PEGylation have been somewhat contradictory and various PEI(-PEG) need to be evaluated independently for DNA transfection and siRNA gene targeting efficacies. AIM: We directly compare plasmid DNA transfection and siRNA-mediated gene targeting efficacies, employing a larger set of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG; PEI(-PEG)) with different molecular weights and degrees of PEG substitution. METHOD: We performed tissue culture-based bioassays on DNA transfection and siRNA-mediated targeting efficacies as well as on toxicity and cellular nucleic acid uptake, and, using sensitive assays based on radioactive labelling, physicochemically characterize the complexes regarding the degree of nucleic acid complexation and complex stabilities under various conditions. RESULTS: In contrast to the DNA transfection efficacy, siRNA-mediated gene targeting is much less dependent on the PEGylation of PEI or on the N/P ( = PEI nitrogen/nucleic acid phosphate) ratio. A more detailed analysis reveals that, in order to define optimal N/P ratios for DNA transfection, complex toxicities and nucleic acid uptake are the most critical parameters. In contrast, at optimal N/P ratios, complex stabilities and complexation efficacies determine PEI(-PEG)/DNA transfection efficacies and the major differences between various PEI(-PEG) are observed. All these parameters are less critical for PEI(-PEG)/siRNA gene targeting efficacy. Thus, our data lead to the distinction between three PEI(-PEG) groups, which relies on the differences in transfection rather than gene targeting efficacies, and which is correlated with the molecular weights and degrees of PEG substitution. CONCLUSION: In contrast to PEI(-PEG)/DNA complexes, a broader panel of PEI-PEG are capable of siRNA-mediated gene targeting. Thus, PEG grafting of PEI requires a separate evaluation of siRNA and DNA complexes, which expands the portfolio of available PEI(-PEG) for the preparation of non-toxic, biocompatible siRNA delivery reagents for the induction of RNAi.     

Spray-drying preparation of microparticles containing cationic PLGA nanospheres as gene carriers for avoiding aggregation of nanospheres

Preparation of nano-sized particles using lyophilization, which is a standard drying technique for high-molecular-weight compounds such as bioactive peptides, proteins, plasmid DNA and siRNA, often results in particle aggregation. In this study, spray-drying was applied for preparation of cationic PLGA nanospheres as gene delivery vectors in order to minimize aggregation and loss of gene transfection efficiency. PLGA nanoparticle emulsions were prepared by dropping an acetone/methanol mixture (2/1) containing PLGA and a cationic material, such as PEI, DOTMA, DC-Chol or CTAB, into distilled water with constant stirring. The PLGA nanosphere emulsion was dried with mannitol by spray-drying, and mannitol microparticles containing PLGA nanospheres were obtained. Mean particle diameter of spray dried PLGA particles was 100-250 nm, which was similar to that of the nano-emulsion before drying, whereas the lyophilized PLGA particles showed increased particle diameter due to particle aggregation. PEI, DOTMA and DC-Chol were useful for maintaining nanoparticle size and conferring positive charge to nanospheres. Transfection of pDNA (pCMV-Luc) using these spray-dried cationic PLGA nanospheres yielded high luciferase activity in COS-7 cells, particularly with PLGA/PEI nanospheres. The present spray-drying technique is able to provide cationic PLGA nanospheres, and may improve redispersal and handling properties.     

Establishing chitosan coated PLGA nanosphere platform loaded with wide variety of nucleic acid by complexation with cationic compound for gene delivery

The purpose of this paper was to establish the surface modified poly(d,l-lactide-co-glycolide) (PLGA) nanosphere platform with chitosan (CS) for gene delivery by using the emulsion solvent diffusion (ESD) method. The advantages of this method are a simple process under mild conditions without sonication. This method requires essentially dissolving both polymer and drug in the organic solvent. Therefore a hydrophilic drug such as nucleic acid is hardly applied to the ESD method. Nucleic acid can easily form an ion-complex with cationic compound, which can be dissolved in the organic solvent. Thereafter, nucleic acid solubility for organic solution can increase by complexation with cationic compound. We used DOTAP as a cationic compound to increase the loading efficiency of nucleic acid. By coating the PLGA nanospheres with CS, the loading efficiency of nucleic acid in the modified nanospheres increased significantly. The release profile of nucleic acid from PLGA nanospheres exhibited sustained release after initial burst. The PLGA nanospheres coated with chitosan reduced the initial burst of nucleic acid release and prolonged the drugs releasing at later stage. Chitosan coated PLGA nanosphere platform was established to encapsulate satisfactorily wide variety of nucleic acid for an acceptable gene delivery system.     

Optimized dextran-polyethylenimine conjugates are efficient non-viral vectors with reduced cytotoxicity when used in serum containing environments

Polyethylenimine (PEI) is a cationic polymer that is an efficient transfection reagent marred by high toxicity and a susceptibility to aggregate in the presence of serum. Dextran is a biodegradable natural polysaccharide that can be used to reduce the toxicity of PEI and increase its stability in the presence of serum. In this study, small branched PEI units (800/2000 Da) were attached to dextran (Dex; 15/100-200 kDa) to form dextran-polyethylenimine (Dex-PEI) conjugates. The Dex-PEI conjugates were then tested as a gene carrier in the model HEK293 cell line. Dex-PEI conjugates displayed significantly lower cytotoxicity than PEI (25k). Both Dex-PEI and PEI efficiently delivered firefly luciferase encoded plasmid DNA (pDNA) to the HEK293 cells. Dex-PEI resulted in moderately lower transfection efficiencies than PEI 25k when the transfection was carried out in media without serum for 4h. However, in the presence of serum, which more accurately predicts the anticipated environment of non-viral vectors in vivo, Dex-PEI and unmodified PEI generated similar transfection efficiencies when incubated with the cells for 4h. When the incubation time of the vectors was increased to 48h, significantly higher transfection efficiencies were generated by Dex-PEI in comparison to PEI. Turbidity measurements showed that complexes formed between plasmid DNA and unmodified PEI were more susceptible to aggregation in serum-containing media than complexes formed from pDNA and Dex-PEI. Dex-PEI conjugates are therefore believed to have greater potential for translational applications because of lower cytotoxicity characteristics and improved stability in serum containing environments.     

Gene delivery system of pDNA using the blood glycoprotein fetuin

Fetuin is a biocompatible plasma protein and strongly enhances phagocytosis of bacteria, DNA and apoptotic cells by peripheral blood cells such as monocytes, macrophages and dendritic cells. We developed a novel gene delivery system: ternary complexes constructed with pDNA, polyethylenimine (PEI) and fetuin. Without covalent binding, fetuin was able to coat pDNA–PEI complexes, and stable anionic nanoparticles formed at a weight ratio greater than 30. Optimised pDNA–PEI–fetuin complexes significantly decreased the cytotoxicity of pDNA–PEI complexes in the melanoma cell line B16F10. Furthermore, the pDNA–PEI–fetuin complexes had higher transgene efficiency compared to that of commercial lipofectin previously reported in B16F10 cells despite an anionic surface. The pDNA–PEI–fetuin complexes did not agglutinate with erythrocytes. The pDNA–PEI–fetuin complexes had high gene expression in the spleen after intravenous administration in mice. Thus, the pDNA–PEI–fetuin complexes were a useful in vivo gene delivery system with tropism for the spleen.     

Poly(L-lactic acid)/polyethylenimine nanoparticles as plasmid DNA carriers

Non-viral vectors such as liposomes, polycations, and nanoparticles have been used as gene delivery systems. In this study, we prepared and characterized biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) nanoparticles as gene carriers. pCMV/β-gal and pEGFP-C1 were utilized as model plasmid DNAs (pDNA). Nanoparticles were prepared using a double emulsion-solvent evaporation technique, and their pDNA binding capacity was assessed by agarose gel electrophoresis. Transfection was studied in HEK 293 and HeLa cell lines, and the transfection efficiencies were determined by β-galactosidase assay or flow cytometry. Three kinds of PLA/PEI systems were studied by varying the molecular weight of PEI. The PLA/PEI 25K system had a higher transfection efficiency than the PLA/PEI 0.8K or PLA/PEI 750K systems. The transfection efficiency was found to be dependent on the ratio of PLA/PEI nanoparticles to pDNA with an optimum ratio of 60:1 (w/w). The cytotoxicity was dependent on the quantity of PLA/PEI nanoparticles used, but it was comparable to that of commercial Lipofectin™. These results demonstrate the potential of PLA/PEI nanoparticles as gene carriers.     

Polyethylenimine as a promising vector for targeted siRNA delivery

Recent discovery of RNA interference (RNAi) technology for gene therapy has triggered explosive research efforts towards development of small interfering RNA (siRNA) as therapeutic modality for gene silencing. Owing to its large molecular weight (~13 kDa), polyanionic nature (~40 negative phosphate groups) and rapid enzymatic degradation, delivery of siRNA remains an unresolved issue. Hence, there arises a need of an appropriate delivery vector to overcome the intrinsic, poor intracellular uptake and limited in vitro and in vivo stability. Amongst the various non-viral delivery vectors, the application of polymeric vectors such as polyethylenimine (PEI) or its derivatives has attracted much attention due to its high transfection efficiency and ease of manipulation. PEI has been extensively investigated for DNA delivery, only recently this polymer has been employed for siRNA delivery. This review will focus on studies done on PEI to deliver siRNA, with emphasis on the targeted, self-assembled polymeric nanoparticles with promising potential to evolve as therapeutic tool in gene therapy.     

Chitosan-based formulations for delivery of DNA and siRNA

Among non-viral vectors, chitosan and chitosan derivatives have been developed in vitro and in vivo for DNA and siRNA delivery systems because of their cationic charge, biodegradability and biocompatibility, as well as their mucoadhesive and permeability-enhancing properties. However, the transfection efficiency of chitosan is too low for clinical application. Studies indicated that the transfection efficiency depends on a series of chitosan-based formulation parameters, such as the Mw of chitosan, its degree of deacetylation, the charge ratio of chitosan to DNA/siRNA (N/P ratio), the chitosan salt form used, the DNA/siRNA concentration, pH, serum, additives, preparation techniques of chitosan/nucleic acid particles and routes of administration. In this paper, chitosan-based formulations for the delivery of DNA and siRNA were reviewed to facilitate the process of chitosan vector development for clinical application. In addition to formulation optimization, chitosan structure modification or additive incorporation is an effective way to improve the stability of the polyplex in biological fluids, enhance targeted cell delivery and facilitate endo-lysosomal release of the complex. In summary, the transfection efficiency of chitosan-based delivery systems can be adjusted by changing formulation-related parameters.