PEGylated polyplex with optimized PEG shielding enhances gene introduction in lungs by minimizing inflammatory responses.

Safety is a critical issue in clinical applications of nonviral gene delivery systems. Safe and effective gene introduction into the lungs was previously achieved using polyplexes from poly(ethyleneglycol) (PEG)-block-polycation [PEG-block-PAsp(DET)] and plasmid DNA (pDNA). Although PEGylated polyplexes appeared to be safe, an excess ratio of polycation to pDNA was needed to obtain sufficient transgene expression, which may cause toxicities shortly after gene introduction. In the present study, we investigated the combined use of two polymers, PEG-block-PAsp(DET) (B) and homo PAsp(DET) (H) across a range of mixing ratios to construct polyplexes. Although transgene expressions following in vitro transfections increased in parallel with increased proportions of H, polyplexes with B/H = 50/50 formulation produced the highest expression level following in vivo intratracheal administration. Higher proportions of H elicited high levels of cytokine induction with significant inflammation as assessed by histopathological examinations. Based on the aggregation behavior of polyplexes in bronchoalveolar lavage fluids (BALFs), we suggested that rapid aggregation of polyplexes in the lung induced acute inflammatory responses, resulting in reduced transgene expression. B/H formulation of polyplex can help to improve gene therapy for the respiratory system because it achieves both effective PEG shielding of polyplexes and functioning of PAsp(DET) polycations to enhance endosomal escape.

Liver-targeted siRNA delivery by polyethylenimine (PEI)-pullulan carrier.

Recently, small interfering RNA (siRNA)-based therapeutics have been used to treat diseases. Efficient and stable siRNA delivery into disease cells is important in the use of this agent for treatment. In the present study, pullulan was introduced into polyethylenimine (PEI) for liver targeting. PEI/siRNA or pullulan-containing PEI/siRNA complexes were delivered into mice through the tail vein either by a hydrodynamics- or non-hydrodynamics-based injection. The incidence of mortality was found to increase with an increase in the nitrogen/phosphorus (N/P) ratio of PEI/siRNA complexes. Moreover, the hydrodynamics-based injection increased mice mortality. Introduction of pullulan into PEI dramatically reduced mouse death after systemic injection. After systemic injection, the PEI/fluorescein-labeled siRNA complex increased the level of fluorescence in the lung and the PEI-pullulan/siRNA complex led to an increased fluorescence level in the liver. These results suggest that the PEI-pullulan polymer may be a useful, low toxic means for efficient delivery of siRNA into the liver.

Intratracheal gene transfer of adrenomedullin using polyplex nanomicelles attenuates monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive PAH and right ventricular failure. Despite recent advances in therapeutic approaches using prostanoids, endothelin antagonists, and so on, PAH remains a challenging condition. To develop a novel therapeutic approach, we have established a nonviral gene delivery system of poly(ethylene glycol) (PEG)-based block catiomers, which form a polyplex nanomicelle with a nanoscaled core-shell structure in the presence of DNA. The polyplex nanomicelle from PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-P[Asp(DET)]), having ethylenediamine units at the side chain, showed ~100-fold increase in luciferase transgene expression activity in mouse lung via intratracheal administration with a minimal toxicity compared with the polyplex from linear poly(ethylenimine) (LPEI). The transfection activity was highest on day 3 after administration and remained detectable until day 14. PEG-b-P[Asp(DET)] polyplex nanomicelles were formulated with a therapeutic plasmid bearing the human adrenomedullin (AM) gene and intratracheally administered to rats with monocrotaline-induced pulmonary hypertension. The right ventricular pressure significantly decreased 3 days after administration as confirmed by a notable increase of pulmonary human AM mRNA levels. Intratracheal administration of PEG-b-P[Asp-(DET)] polyplex nanomicelles showed remarkable therapeutic efficacy with PAH animal models without compromising biocompatibility.

Photo-control of the polyplexes formation between DNA and photo-cation generatable water-soluble polymers.

Photo-cation generatable water-soluble polymers (Mw: approximately 1x10(5)) were prepared by radical copolymerization of N,N-dimethylacrylamide and vinyl monomer of triphenylmethane leucohydroxide (malachite green), which generate a cation upon ultraviolet light (UV) irradiation at wavelengths of between 290 and 410 nm. The malachite green contents of the copolymers were 3.6 (0.4 mol%), 7.9 (0.7 mol%), and 15.0 (2.7 mol%) per molecule. When an aqueous solution of the photo-cationized copolymers generated by UV irradiation was mixed with a Tris-HCl buffer (pH 7.4) of DNA (pGL3-control plasmid), dynamic light scattering (DLS) measurements showed the formation of polyplexes between the cationic copolymers and anionic DNA by non-specific electrostatic interaction, which was visualized with a confocal laser scanning microscopy (CLMS). Their mean cumulant diameter was about 150 nm with low polydispersity irrespective of the malachite green content in the copolymers. In the copolymer with the lowest malachite green content, almost all of the amount of the polyplexes was maintained by repeated UV irradiation, but the amount gradually decreased in the dark at 37 degrees C due to dissociation of the polyplexes, synchronized with the neutralization of the cation form of malachite green. The photo-cation generatable copolymers designed here can undergo photo-induced formation of the polyplexes with DNA and thermal polyplex dissociation, which may be used as a model for a novel photo-induced gene delivery system into cells.

High performance gene delivery polymeric vector: nano-structured cationic star polymers (star vectors).

Nano-structured hyperbranched cationic star polymers, called star vectors, were molecularly designed for a novel gene delivery non-viral vector. The linear and 3, 4 or 6 branched water-soluble cationic polymers, which had same molecular weight of ca. 18,000, were synthesized by iniferter (initiator-transfer agent-terminator)-based photo-living-radical polymerization of 3-(N,N-dimethylamino)propyl acrylamide, initiated from respective multi-dithiocarbamate-derivatized benzenes as an iniferter. All polymers produced polyion complexes 'polyplexes' by mixing with pDNA (pGL3-control plasmid), in which the particle size was ca. 250 nm in diameter [the charge ratio < 2/1 (vevtor/pDNA)] and ca. 150 nm (the charge ratio > 2.5/1), and the zeta-potential was ca. +10 mV (the charge ratio > 1/1). When COS-1 cells were incubated with the polyplexes 12 h after preparation under the charge ratio of 5/1, higher gene expression was obtained as an increase in branching, with a little cytotoxicity. The relative gene expression to the linear polymer was about 2, 5, and 10 times in 3-, 4-, and 6-branched polymers, respectively. The precise change in branching of polymers enabled the control of the gene transfer activity.

Elevated atherogenic index following oral administration of quaternized polyamine nanogels.

Quaternized polyamine nanogels possessing poly(ethylene glycol) (PEG)-tethered chains as the surface layer were prepared by redox-initiated emulsion polymerization of 2-(N,N-diethylamino)ethyl methacrylate (EAMA) in the presence of vinylbenzyl-ended poly(ethylene glycol) (PEG-CH2PhCHCH2), followed by quaternization with methyl iodide (QNG-I). QNG-I absorbed taurocholic acid regardless of environmental pH, because of the fixed positive charge of QNG-I. Oral administration of polyamine nanogels into mice tended to cause intestinal retention, with accumulation of up to 70% of the initial dose. Levels of low-density lipoprotein cholesterol (LDL-C) in hyperlipidemic mice effectively decreased following oral administration of QNG-I. Interestingly, oral administration of QNG-I increased the levels of high-density lipoprotein cholesterol (HDL-C), resulting in an extremely high atherogenic index. Iodide counter-anions in QNG-I played an important role in the increase in HDL-C levels.

Quick nuclear transportation of siRNA and in vivo hepatic ApoB gene silencing with galactose-bearing polymeric carrier.

Since previous studies have linked the genetic mutations of Apolipoprotein B (ApoB) to the low density lipoprotein (LDL) cholesterol levels, it can be believed that the knockdown of ApoB by siRNA silencing is a useful method to reduce the cardiovascular disease. However, the spontaneous uptake of siRNA is hindered, and thus vectors are necessary to aid its transfer into the cells. Among the synthetic non-viral vectors, cationic polymers are extensively investigated as possible candidates for efficient and specific gene delivery, because they can be easily modified to get different set of properties. Therefore, in this work a set of random copolymers with different molecular weight and composition were synthesized. These vectors present 2-(dimethylamino)ethyl methacrylate, as cationic monomer, and galactose units as liver-targeting moieties. From in vitro experiments, copolymers with monomer ratio and molecular weight about 0.1 and 80kDa, respectively, showed adequate transfection capabilities and displaying good cell viability, independently of the nature of the saccharides units. However, in the in vivo experiments in C57BL/6 high-fat-fed mice, a better blood compatibility and protection against degradation leading to better transfection by the random copolymers bearing galactose units was confirmed.

Efficient reduction of serum cholesterol by combining a liver-targeted gene delivery system with chemically modified apolipoprotein B siRNA.

Apolipoprotein B (Apo B) is a key amphipathic glycoprotein compound in the metabolism of plasma lipoproteins (mainly very low-density lipoprotein (VLDL) and LDL). Inhibition of Apo B synthesis by short interfering RNA (siRNA) targeting Apo B (Apo B siRNA) is very efficient for serum LDL reduction. In the present study, the chemically modified Apo B siRNA (Apo B-siBNA) with the increased enzymatic stability was selected. We developed a cationic conjugate for efficient delivery of Apo B-siBNA into the liver by introducing pullulan with different molecular weights (MWs) (5900 and 107,000) into polyethylenimine (PEI). Introduction of pullulan into PEI dramatically decreased mortality and lung damage after systemic injection of the conjugate/Apo B-siBNA complexes into mice. The PEI-pullulan carrier prepared with high MW pullulan (107,000) was more stable in the blood stream and showed higher fluorescence levels in the liver for a longer time than the carrier prepared with low MW pullulan (5900). Moreover, efficient reduction of serum LDL and Apo B mRNA in the liver was observed in mice injected with PEI-pullulan (MW, 107,000)/Apo B-siBNA, whereas there was no or little change in serum LDL and Apo B mRNA in livers of mice treated with Apo B-siBNA alone, PEI/Apo B-siBNA, and PEI-pullulan (MW, 5900)/Apo B-siBNA. These results suggest that combining a liver-targeted gene delivery system with chemically modified Apo B siRNA efficiently reduces the level of serum LDL and Apo B mRNA in the liver.

Combination of chondroitin sulfate and polyplex micelles from Poly(ethylene glycol)-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} block copolymer for prolonged in vivo gene transfection with reduced toxicity.

Nonviral polycation-based gene carriers (polyplexes) have attracted attention as safe and efficient gene delivery systems. Polyplex micelles comprised of poly(ethyleneglycol)-block-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-PAsp(DET)) and plasmid DNA (pDNA) have shown high transfection efficiency with low toxicity due to the pH-sensitive protonation behavior of PAsp(DET), which enhances endosomal escape, and their self-catalytic degradability under physiological conditions, which reduces cumulative toxicity during transfection. In this study, we improved the safety and transfection efficiency of this polyplex micelle system by adding an anionic polycarbohydrate, chondroitin sulfate (CS). A quantitative assay for cell membrane integrity using image analysis software showed that the addition of CS markedly reduced membrane damage caused by free polycations in the micelle solution. It also reduced tissue damage and subsequent inflammatory responses in the skeletal muscle and lungs of mice following in vivo gene delivery with the polyplex micelles. Subsequently, this led to prolonged transgene expression in the target organs. This combination of polyplex micelles and CS holds great promise for safe and efficient gene introduction in clinical settings.