The use of myristic acid as a ligand of polyethylenimine/DNA nanoparticles for targeted gene therapy of glioblastoma

To establish a gene delivery system for brain targeting, a low molecular weight polyethylenimine (PEI(10?K)) was modified with myristic acid (MC), and complexed with DNA, yielding MC-PEI(10?K)/DNA nanoparticles successfully. The nanoparticles were observed to be successfully taken up by the brains of mice. The transfection efficiency of the nanoparticles was then investigated, and both the in?vitro and in?vivo gene expression of MC-PEI(10?K)/DNA nanoparticles is significantly higher than that of unmodified PEI(10?K)/DNA nanoparticles. The anti-glioblastoma effect of MC-PEI(10?K)/pORF-hTRAIL was demonstrated by the survival time of intracranial U87 glioblastoma-bearing mice. The median survival time of the MC-PEI(10?K)/pORF-hTRAIL group (28 days) was significantly longer than that of the PEI(10?K)/pORF-hTRAIL group (24 days), the MC-PEI(10?K)/pGL(3) group (21 days) and the saline group (22 days). Therefore, our results suggested that MC-PEI(10?K) could be potentially used for brain-targeted gene delivery and in the treatment of glioblastoma.     

Kidney-specific peptide-conjugated poly(ester amine) for the treatment of kidney fibrosis

Kidney gene therapy using the hepatocyte growth factor (HGF) gene may offer new strategies for the treatment of chronic renal disease such as kidney fibrosis, because HGF has the potential to promote tubular repair and to inhibit tissue fibrosis. As a non-viral vector for gene delivery, polyethylenimine (PEI) exhibits high gene expression due to its buffering capacity with cytotoxicity, although its cytotoxicity depends on its molecular weight. In this study, to minimize the cytotoxicity of PEI with a high transfection efficiency, biodegradable poly(ester amine) (PEA) based on glycerol dimethacrylate (GDM) and low molecular weight PEI (LMW PEI) was synthesized and kidney targeting peptide was conjugated to the PEA (PEP-PEA) to give it kidney cell specificity. The PEP-PEA showed good physicochemical properties as a gene delivery carrier, such as DNA condensation ability, protection of the DNA in the complexes from enzyme degradation, and formation of nanosized complexes with spherical shapes. Higher transfection efficiency in 293T cells was achieved with the PEP-PEA than with the PEA and the PEI 25 kDa with lower cytotoxicity. Also, the HGF gene that was complexed with the PEP-PEA was specifically delivered to the obstructed kidney in the unilateral ureteral obstruction (UUO) model rats. The delivered HGF gene exhibited potency in recovering renal functions, which indicates the potential of the PEP-PEA as a safe and efficient carrier for the treatment of kidney fibrosis.     

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.     

Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles

Dendritic cells (DCs) are professional antigen presenting cells that induce, sustain, and regulate immune responses. Gene modification of DCs is of particular interest for immunotherapy of diseases where the immunes system has failed or is abnormally regulated, such as in cancer or autoimmune disease. Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA. Among various non-viral vectors, chitosan is considered to be a good candidate for gene delivery system, however, lack of cell specificity and low transfection of chitosan need to be overcome prior to clinical use. In this study, mannosylated chitosan (MC) was prepared to induce the receptor-mediated endocytosis and targeting into antigen presenting cells (APCs), especially DCs having mannose receptors. MC showed great ability to form complexes with DNA and showed suitable physicochemical properties for gene delivery system. It had low cytotoxicity and exhibited much enhanced gene transfer efficiency on the macrophage cell line than chitosan itself. Also, MC/DNA complex was more efficient for transferring IL-12 gene into DCs rather than water-soluble chitosan (WSC)/DNA one, which resulted in better induction of INF-gamma from DCs. Therefore, MC is a promising gene delivery system for repeated administration to maintain sustained gene expression, thereby opening the possibility for immunotherapy.     

Preparation of dexamethasone-based cationic liposome and its application to gene delivery in vitro

In this study, dexamethasone was conjugated to PAMAM dendrimer (generation 0) and its gene transfection efficiency was investigated. To make a liposomal solution for gene delivery, DOPE was used as a fusogenic helper lipid. In gel retardation assay, PAMAM-dexamethasone conjugate (PAM-Dex)/DOPE liposome/DNA complex was completely retarded at 8:1 N/P (nitrogen/phosphate) ratio. The physicochemical characteristics are studied by measuring the average size distribution and zeta-potential values of the complexes. In vitro transfection assay showed that the PAM-Dex/DOPE liposome/DNA complex displayed higher gene delivery efficiency compared to PAMAM/DNA complex. In addition, PAM-Dex/DOPE liposome showed the lowest toxicity compared to PAMAM, PEI 25 kD and Lipofectamine. These results indicate that PAM-Dex/DOPE liposome has a potential to be used as an efficient gene carrier for gene therapy.     

Smart Carbon Nanotubes with Laser‐Controlled Behavior in Gene Delivery and Therapy through a Non‐Digestive Trafficking Pathway

Near‐infrared (NIR) laser‐controlled gene delivery presents some benefits in gene therapy, inducing enhanced gene transfection efficiency. In this study, a “photothermal transfection” agent is obtained by wrapping poly(ethylenimine)‐cholesterol derivatives (PEI‐Chol) around single‐walled carbon nanotubes (SWNTs). The PEI‐Chol modified SWNTs (PCS) are effective in compressing DNA molecules and protecting them from DNaseI degradation. Compared to the complexes formed by PEI with DNA (PEI/DNA), complexes of PCS and DNA that are formed (PCS/DNA) exhibit a little lower toxicity to HEK293 and HeLa cells under the same PEI molecule weight and weight ratios. Notably, caveolae‐mediated cellular uptake of PCS/DNA occurs, which results in a safer intracellular transport of the gene due to the decreased lysosomal degradation in comparison with that of PEI/DNA whose internalization mainly depends on clathrin rather than caveolae. Furthermore, unlike PEI/DNA, PCS/DNA exhibits a photothermal conversion ability, which promotes DNA release from PCS under NIR laser irradiation. The NIR laser‐mediated photothermal transfection of PCS_10K/plasmid TP53 (pTP53) results in more apoptosis and necrosis of HeLa cells in vitro than other groups, and achieves a higher tumor‐growth inhibition in vivo than naked pTP53, PEI_25K/pTP53, and PCS_10K/pTP53 alone. The enhanced transfection efficiency of PCS/DNA can be attributed to more efficient DNA internalization into the tumor cells, promotes detachment of DNA from PCS under the mediation of NIR laser and higher DNA stability in the cells due to caveolae‐mediated cellular uptake of the complexes.     

Degradable poly(amino ester) based on poly(ethylene glycol) dimethacrylate and polyethylenimine as a gene carrier: molecular weight of PEI affects transfection efficiency

The aim of the research is to study the effect of polyethylenimine (PEI) molecular weight on the gene transfection efficiency of degradable poly(amino ester) based on poly(ethylene glycol) dimethacrylate (PEGDMA) and polyethylenimine (PEG-cr-PEI) as a gene carrier. Various low molecular weight (LMW) branched PEI based PEG-cr-PEI was synthesized via Michael addition. The degradation half-life of PEG-cr-PEI was longer at pH 5.6 than that at pH 7.4. The plasmid condensation and protection ability of the PEG-cr-PEI were confirmed by agarose gel electrophoresis assay. PEG-cr-PEI/DNA nanoparticles showed high positive zeta potential (>+20 mV), narrow size distribution, and spherical shapes with size below 250 nm when N/P ratios of PEG-cr-PEI to DNA were above 10, suggesting that they have endocytosis potential. The cytotoxicity of PEG-cr-PEI/DNA complexes was lower than that of PEI 25K/DNA complexes, and the transfections mediated by PEG-cr-PEI were checked in 293T, HeLa and HepG2 cell lines. The report gene expression was increased with increasing the molecular weight of LMW PEI. The “proton sponge effect” was proposed as the mechanism of PEG-cr-PEI mediated gene transfection.     

Enhanced gene transfer with multilayered polyplexes assembled with layer-by-layer technique

Successful gene therapy asks for multifunctional vectors which can not only protect DNA from degradation but also transfer it into nuclear and subsequently express the loaded gene. Here we reported a novel multilayered delivery system constructed with DNA, protamine (Pro) and polyethylenimine (PEI) via lay-by-layer (LbL) technique, which posed multifunctions. DNA was previously condensed into a compact core with Pro which also contained nuclear localisation signals (NLS) domains for nuclear transfer. Then additional DNA was deposited as the first layer onto the cationic core via the electrostatic attraction which would increase the loading dose of DNA. At last, PEI was absorbed as the outmost layer to achieve the endosomal escape. Therefore a quaternary polyplexes which offered high loading of DNA, nuclear transfer ability and endosomal escape capability was constructed with the LbL technique. The obtained quaternary polyplexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA protection capability. Compared with commercially available PEI/DNA complexes, the novel multifuctional vector exhibited not only lower cytotoxicity (P<0.05) but also higher transfection efficiency in HepG2 and HeLa cells (P<0.05) in vitro test.     

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.     

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.     

Non-viral gene delivery regulated by stiffness of cell adhesion substrates

Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.     

Co-delivery of drug and DNA from cationic dual-responsive micelles derived from poly(DMAEMA-co-PPGMA)

The synthesis and gene transfection efficiency of a series of amphiphilic copolymers with poly(dimethylaminoethyl methacrylate) (PDMAEMA), and poly(propylene glycol methacrylate) (PPGMA) segments is reported. The hydrophobic PPGMA interior allows a cell-sensitizing drug such as paclitaxel to be incorporated while the cationic and hydrophilic PDMAEMA corona allows the complexation of anionic DNA to form a nano-sized polyplex. These drug-encapsulated copolymers display excellent gene transfection efficiency as compared to PEI or PDMAEMA homopolymers.     

Protamine and chloroquine enhance gene delivery and expression mediated by RNA-wrapped single walled carbon nanotubes

The use of non-viral vectors as delivery systems in gene therapy has been extensively studied recently owing to their advantages over viral vectors. Here, we propose a new gene delivery system based on the use of RNA-wrapped single-walled carbon nanotubes (SWCNTs) complexed with the cationic protein, protamine and the drug chloroquine. Protamine was selected as a cationic protein acting as bridge between negatively charged RNA-wrapped SWCNTs and plasmid DNA. Protamine also contains a nuclear localization signal which enhances the expression of the transfected gene. The drug chloroquine, a lysosomotropic compound which has been reported to increase the transfection efficiency, was attached to RNA-wrapped SWNTs by ionic interactions. The simultaneous delivery of the drug chloroquine with plasmid DNA clearly showed an enhanced gene delivery and expression. The levels of gene expression were quantified using the luciferase reporter gene as model. Optimal conditions for transfection and gene expression were obtained and cytoxicity of the carbon nanotube complexes measured. The optimal complexes were shown to efficiently deliver plasmid DNA for efficient gene expression and may thereby be useful as gene delivery systems for gene therapy.     

Gold Nanoparticles Capped with Polyethyleneimine for Enhanced siRNA Delivery

An efficient and safe delivery system for small interfering RNA (siRNA) is required for clinical application of RNA interfering therapeutics. Polyethyleneimine (PEI)‐capped gold nanoparticles (AuNPs) are successfully manufactured using PEI as the reductant and stabilizer, which bind siRNA at an appropriate weight ratio by electrostatic interaction and result in well‐dispersed nanoparticles with uniform structure and narrow size distribution. With siRNA binding, PEI‐capped AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA‐MB‐435s cells, though more internalized PEI/siRNA complexes in cells are evidenced by confocal laser scanning microscopy observation and fluorescence‐activated cell sorting analyses. PEI‐capped AuNPs/siRNA targeting endogenous cell‐cycle kinase, an oncogene polo‐like kinase 1 (PLK1), display significant gene expression knockdown and induce enhanced cell apoptosis, whereas it is not obvious when the cells are treated with PLK1 siRNA using PEI as the carrier. Without exhibiting cellular toxicity, PEI‐capped AuNPs appear to be suitable as a potential carrier for intracellular siRNA delivery.     

Galactosylation of chitosan-graft-spermine as a gene carrier for hepatocyte targeting in vitro and in vivo

Polyethyleneimine (PEI) has been described as a highly efficient gene carrier due to its efficient proton sponge effect within endosomes. However, many studies have demonstrated that PEI is toxic and associated with a lack of cell specificity despite high transfection efficiency. In order to minimize the toxicity of PEI, we prepared chitosan-graft-spermine (CHI-g-SPE) in a previous study. CHI-g-SPE showed low toxicity and high transfection efficiency. However, this compound also had limited target cell specificity. In the present study, we synthesized galactosylated CHI-g-SPE (GCS) because this modified GCS could be delivered specifically into the liver due to hepatocyte-specific galactose receptors. The DNA-binding properties of GCS at various copolymer/DNA weight ratios were evaluated by a gel retardation assay. The GCS copolymer exhibited significant DNA-binding ability and efficiently protected DNA from nuclease attack. Using energy-filtered transmission electron microscopy (EF-TEM), we observed dense spherical, nano-sized GCS/DNA complexes with a homogenous distribution. Most importantly, GCS was associated with remarkably low cytotoxicity compared to PEI in HepG2, HeLa, and A549 cells. Moreover, GCS carriers specifically delivered the gene-of-interest into hepatocytes in vitro as well as in vivo. Our results suggest that the novel GCS described here is a safe and highly efficient carrier for hepatocyte-targeted gene delivery.     

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.     

Charge shielding effects on gene delivery of polyethylenimine/DNA complexes: PEGylation and phospholipid coating

Polyethylenimine (PEI) is an efficient cationic polymer for gene delivery, but defective in biocompatibility. In this study, we developed two different strategies to shield the positively charged PEI/DNA complexes: PEGylation and lipid coating. The physicochemical properties, cytotoxicity and transfection efficiency of the two gene delivery systems were investigated. Both PEGylation and lipid coating succeeded in reducing the zeta-potential of the complexes. Lipid-coated PEI/DNA complexes (LPD complexes) and PEI/DNA complexes exhibited similar cytotoxicity, whereas PEG-PEI/DNA complexes showed lower cytotoxicity, especially at high N/P ratios. LPD complexes were less efficient in transfection compared to PEG-PEI/DNA complexes. The transfection efficiency was influenced remarkably by cytotoxicity and surface charge of the complexes. Intracellular processes studies revealed that endosomal release might be one of the rate-limiting steps in cell transfection with PEI as a gene delivery carrier.     

Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery

Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-βGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. β-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.