O-Carboxymethyl-chitosan/organosilica hybrid nanoparticles as non-viral vectors for gene delivery

Polymeric non-viral vectors, such as chitosan nanoparticles show good biocompatibility, but low transfection efficiency. The objective of this study was to improve the transfection efficiency of chitosan based non-viral vectors by using o-carboxymethyl-chitosan which is a kind of water-soluble chitosan derivative and also has good biocompatibility. O-Carboxymethyl-chitosan-organosilica hybrid nanoparticles (CMG NPs) were synthesized through a rapid one-step aqueous synthetic approach for gene delivery. The size of nanoparticles was 276 ± 25 nm and zeta potential was 31.6 ± 0.4 mV in deionized water. Zeta potential increased with the decrease of pH, and it had been discovered that pH = 5.5 is the best point for CMG NPs to bond with plasmid DNA. DNA inclusion and integrity was evaluated by gel electrophoresis, and it is indicated that CMG NPs could protect DNA against DNase I and serum degradation. The results of MTT for cell viability and in vitro transfection also support the idea that CMG NPs could be used as efficient and safe vectors for gene delivery.     

Nanoscale cell adhesion ligand presentation regulates nonviral gene delivery and expression

It is hypothesized that the nanoscale organization of cell adhesion ligands in a synthetic ECM regulates nonviral gene delivery. This hypothesis was examined with pre-osteoblasts cultured on substrates which present varied density and spacing of synthetic adhesion ligands. The levels of gene transfer and expression were increased with the density of adhesion ligands, but decreased with the spacing of ligands, due to changes in the cell growth rate. This study provides a material-based control point on the nanometer scale for nonviral gene based therapies.     

Cell response to collagen-calcium phosphate cement scaffolds investigated for nonviral gene delivery

Collagen-hydroxyapatite (HA) scaffolds for the non-viral delivery of a plasmid encoding the osteoinductive protein bone morphogenetic protein (BMP)-7 were developed. The collagen-HA was obtained by the combination of calcium phosphate cement in a collagen template. The effect on cell behavior of increasing amounts of HA in the scaffolds was evaluated. Collagen-HA scaffolds containing 13, 23 or 83 wt% HA were prepared. Cell proliferation was reduced in the 83% HA scaffold after 1 day compared to 13 and 23% HA, but by 14 days the number of cells in 83% HA considerably increased. Alkaline phosphatase (ALP) activity was 8 times higher for the 83% HA scaffolds. BMP-7 plasmid was incorporated into the 83% HA scaffold. The transfection was low, although significant levels of BMP7 were expressed, associated with an increase in cell proliferation.     

Emerging links between surface nanotechnology and endocytosis: impact on nonviral gene delivery

Significant effort continues to be exerted toward the improvement of transfection mediated by nonviral vectors. These endeavors are often focused on the design of particulate carriers with properties that encourage efficient accumulation at the membrane surface, particle uptake, and endosomal escape. Despite its demonstrated importance in successful nonviral transfection, relatively little investigation has been done to understand the pressures driving internalized vectors into favorable nondegradative endocytic pathways. Improvements in transfection efficiency have been noted for complexes delivered with a substrate-mediated approach, but the reasons behind such enhancements remain unclear. The phenotypic changes exhibited by cells interacting with nano- and micro-featured substrates offer hints that may explain these effects. This review describes nanoscale particulate and substrate parameters that influence both the uptake of nonviral gene carriers and the endocytic phenotype of interacting cells, and explores the molecular links that may mediate these interactions. Substrate-mediated control of endocytosis represents an exciting new design parameter that will guide the creation of efficient transgene carriers.     

Polymeric micelles comprising stearic acid-grafted polyethyleneimine as nonviral gene carriers

Non-viral vectors composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, 10 k PEI was modified by grafting stearic acid (SA) and formulated to polymer micelles with positive surface charge and evaluated for pDNA delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil in water (o/w) solvent evaporation method. The success of PEI-SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential determined by photon correlation spectroscopy was 149.6 +/- 1.2 nm and 64.1 +/- 1.5 mV, respectively. These self-assemble positive charge micelles showed effective binding to pDNA for transfection. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results demonstrated that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Stearic acid grafted PEI micelles can be promising polymer carriers in genetic therapy.     

Uptake pathways and subsequent nucleus targeting of tat-conjugated gelatin-siloxane nanoparticles as nonviral gene vector

Novel hybrid biomaterial of Tat peptide modified gelatin-siloxane nanoparticles (Tat-GS NPs), with positive surface potential was synthesised through a two-step sol-gel process. The particles were subsequently tested in vitro with HeLa cells by fluorescent activitated cell sorter analysis, confocal laser scanning microscopy, and transmission electron microscopy to determine whether the functionalisation with Tat peptide allowed particles to transfer across the cell membrane and locate in the nucleus. Our current results indicated that the internalisation of Tat-GS NPs in HeLa cells is time- and concentration- dependent. Moreover, Tat-GS NPs could penetrate the nucleus membrane and enter into nucleus. The Tat-GS/pDNA nanocomplexes were formulated with higher encapsulation efficiency, and exhibited efficient transfection in vitro. Tat-GS NPs are thus considered as novel non-viral vectors will be widely used in further study.     

Nanoparticles for intracellular-targeted drug delivery

Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.     

Evaluation of PEG-transferrin-PEI nanocomplex as a gene delivery agent

Polyethylenimine (PEI) has been shown to be an efficient nonviral delivery vector. To improve its specificity and reduce its cytotoxicity, PEI should be modified. Transferrin (Tf) is a cell-binding ligand and Tf-receptors are expressed in malignant cells. Modification of cationic polymer by polyethylene glycol (PEG) can reduce the protein interaction and cell cytotoxicity of delivery vectors. We have synthesized PEG-Tf-PEI conjugate as an efficient and safe carrier of plasmid DNA (pDNA). Nanocomplexes of conjugates with pDNA were characterized by measuring the particle size and the surface charge. Transfection efficiency of nanocomplexes in Jurkat cells was improved and cytotoxicity was decreased compared with those of PEI complex. This was due to a reduction in the membrane damaging effect via shielding of the positive charge on the nanocomplex surface by PEG.     

Novel gelatin-siloxane nanoparticles decorated by Tat peptide as vectors for gene therapy

In principle, the technique of gene delivery involves taking complete or parts of genes that can code specific messages and delivering them to selected cells in the body. Such a transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. A series of gelatin-siloxane nanoparticles (GS NPs) with controlled size and surface charge were synthesized through a two-step sol-gel process. In order to increase the efficiency of cellular uptake, HIV-derived Tat peptide was further grafted to GS NPs. In vitro co-location and endocytosis inhibition experiments suggested that the as-synthesized TG NPs may enter HeLa cells via a combined pathway of lipid-raft-?and receptor-dependent endocytosis, and only cause little cell damage. Moreover, this study shows the encapsulation of a plasmid DNA in TG NPs to be obtained as a non-viral gene vector. This kind of encapsulation provides complete protection to the plasmid DNA from the external DNase and serum environment, and generates the hope that the resulting formulation can be developed into a potential vector for effective gene delivery. In order to check this potential, the reporter gene pSVβ-gal was encapsulated, and in vitro transfection efficiency of this system was found to be nearly 130% compared to the commercially available transfection reagent Lipofectamine?.     

Nanoparticles for cellular drug delivery: mechanisms and factors influencing delivery

Polymeric nanoparticles have demonstrated enormous potential as cellular drug delivery vehicles. Nanoparticles improve drug's stability as well as its availability and retention at the target intracellular site of action. Therapeutic efficacy of nanoparticles can be further enhanced by conjugating specific ligands to nanoparticle surface. Ligand conjugation can also be used to favorably modify the intracellular disposition of nanoparticles. A number of ligands are available for this purpose; use of a specific ligand depends on the target cell, the material used for nanoparticle formulation, and the chemistry available for ligand-nanoparticle conjugation. Cellular drug delivery using nanoparticles is also affected by clearance through the reticuloendothelial system. In this paper, we review the recent progress on our understanding of physicochemical factors that affect the cellular uptake of nanoparticles and the different cellular processes that could be exploited to enhance nanoparticle uptake into cells.     

Cholesterol tethered bioresponsive polycation as a candidate for gene delivery

The efficient unpacking of viral protein shell gave the inspiration for the synthesized vectors. In this research, novel cholesterol tethered bioresponsive polyethylenimine (PEI) was specially designed via disulfide-containing cross-linker. The cholesterol lipid had proved to increase the permeability of gene vector through cell membrane. The acid–base titration indicated that the synthesized polycation possessed efficient proton sponge effect, which was suggested to increase endosomal release of pDNA complexes into the cytoplasm. The cholesterol tethered polycation could effectively induce DNA condensation and form spherical particles with diameter about 200 nm at N/P ratio of 10. At glutathione concentration of 3 mM, the polyplexes were unpacked due to the bioresponsive cleavage of the disulfide bonds. The in-vitro experiment indicated that the polyplexes showed efficient transfection efficiency to HEK293T cells. All the results indicated that the bioresponsive polycation could be served as an effective trigger to control the release of DNA at the intracellular environment. The novel bioresponsive polycation might have great potential in non-viral gene delivery research and application.     

Dendrosome-based gene delivery

Gene transfer to humans requires carriers for the plasmid DNA, which can efficiently and safely carry the gene into the nucleus of the desired cells. The purpose of the present study was to design dendrosomes as a novel, non-viral, vesicular, gene delivery vector and to carry out a comparative study of the relative transfection efficiencies of dendrosomes with standard non-viral, gene delivery vectors.

Fourth-generation PAMAM dendrimers were synthesized by double the Michael addition reaction and extensively characterized. The dendrimer–DNA complex was prepared and was confirmed by CD spectroscopy. The dendrosomes were prepared by the reverse phase evaporation method and the entrapment efficiency of the dendrosomal formulation was estimated. In vitro toxicity of the formulation was evaluated by hemolytic toxicity and cytotoxicity studies. Transfection efficiency of the dendrosomal formulations was compared to standard non-viral gene delivery vectors in HEK-293 cell.

The results of hemolytic toxicity cytotoxicity studies demonstrated that the dendrosomes possess negligible toxicity as compared to the other formulations and are suitable for in vivo administration. The results of transfection of HEK-293 cell with PGL2 showed that the dendrosomal formulation DF3 possesses superior transfection efficiency against other delivery systems under study.

Dendrosomes possess tremendous potential as a novel non-viral and non-toxic gene delivery vector.     

Synthetic polymeric vectors in gene therapy

The synthetic polymer vectors in gene therapy have opened a very prospective field for research in gene therapy and the area in the use of these synthetic polymers as vectors in targeting oncogenes is developing very fast. With the completion of Human Genome Project, the list of genetic targets is growing very fast. These diseases sparked the initiative to create such gene based therapeutics. The low levels of transfection and expression of the gene held non-viral methods are at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies, similar to those of viruses. Amongst these synthetic polymers, polyethylenimine (PEI) and the poly(amidoamine) (PAMAM) dendrimers and poly(β-amino ester) are used extensively as vectors in gene therapy.     

Chitosan-functionalized graphene oxide as a nanocarrier for drug and gene delivery

The covalent functionalization of graphene oxide (GO) with chitosan (CS) is successfully accomplished via a facile amidation process. The CS-grafted GO (GO-CS) sheets consist of about 64 wt.% CS, which imparts them with a good aqueous solubility and biocompatibility. Additionally, the physicochemical properties of GO-CS are studied. As a novel nanocarrier, GO-CS is applied to load a water-insoluble anticancer drug, camptothecin (CPT), via π-π stacking and hydrophobic interactions. It is demonstrated that GO-CS possesses a superior loading capacity for CPT, and the GO-CS-CPT complexes show remarkably high cytotoxicity in HepG2 and HeLa cell lines compared to the pure drug. At the same time, GO-CS is also able to condense plasmid DNA into stable, nanosized complexes, and the resulting GO-CS/pDNA nanoparticles exhibit reasonable transfection efficiency in HeLa cells at certain nitrogen/phosphate ratios. Therefore, the GO-CS nanocarrier is able to load and deliver both anticancer drugs and genes.     

Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery

Polyethylenimine (PEI) functionalized magnetic nanoparticles were synthesized as a potential non-viral vector for gene delivery. The nanoparticles could provide the magnetic-targeting, and the cationic polymer PEI could condense DNA and avoid in vitro barriers. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray powder diffraction, dynamic light scattering measurements, transmission electron microscopy, vibrating sample magnetometer and atomic force microscopy. Agarose gel electrophoresis was used to asses DNA binding and perform a DNase I protection assay. The Alamar blue assay was used to evaluate negative effects on the metabolic activity of cells incubated with PEI modified magnetic nanoparticles and their complexes with DNA both in the presence or absence of an external magnetic field. Flow cytometry and fluorescent microscopy were also performed to investigate the transfection efficiency of the DNA-loaded magnetic nanoparticles in A549 and B16-F10 tumor cells with (+M) or without (?M) the magnetic field. The in vitro transfection efficiency of magnetic nanoparticles was improved obviously in a permanent magnetic field. Therefore, the magnetic nanoparticles show considerable potential as nanocarriers for gene delivery.     

Protein nanocapsules based vectors for efficient gene transfection

Gene therapy employs exogenous nucleic acids to treat genetic diseases and disorders.The insufficient cytosolic delivery of genetic materials remains a major hurdle for effective gene therapy.To address this challenge,we have designed and synthesized various cationic protein nanocapsules that can efficiently condense nucleic acids via self-assembly.Through systematically investigating the gene transfection efficiency of these nanocapsules as delivery vectors,we find that nanocapsules,which were synthesized with hydrolyzable polymers containing tertiary amine groups,afford the highest transfection efficiency(?80%),resulting in stable protein expression for over four days.The mechanistic study reveals that tertiary amine groups facilitate the endosomal escape of the nucleic acid-nanocapsule complexes after their cell internalization via endocytosis.The subsequent hydrolysis of the polymers triggers the cytosolic release of the nucleic acids,thereby prompting gene expression.Our results not only provide a new class of gene delivery vectors but also detail the parameters for future vector design.