Biocompatible gelatin nanoparticles for tumor-targeted delivery of polymerized siRNA in tumor-bearing mice

Structural modifications of the siRNA backbone improved its physiochemical properties for incorporating in gene carriers without loss of gene-silencing efficacy. These modifications provide a wider variety of choice of vector systems for siRNA delivery. We developed a tumor-targeted siRNA delivery system using polymerized siRNA (poly-siRNA) and natural polymer gelatin. The polymerized siRNA (poly-siRNA) was prepared through self-polymerization of thiol groups at the 5′-end of sense and anti-sense strands of siRNA and was encapsulated in the self-assembled thiolated gelatin (tGel) nanoparticles (NPs) with chemical cross-linking. The resulting poly-siRNA-tGel (psi-tGel) nanoparticles (average of 145 nm in diameter) protect siRNA molecules from enzymatic degradation, and can be reversibly reduced to release functional siRNA molecules in reductive conditions. The psi-tGel NPs presented efficient siRNA delivery in red fluorescence protein expressing melanoma cells (RFP/B16F10) to down-regulate target gene expression. In addition, the NPs showed low toxicity at a high transfection dose of 125 μg/ml psi-tGel NPs, which included 1 μM of siRNA molecules. In tumor-bearing mice, the psi-tGel NPs showed 2.8 times higher tumor accumulation than the naked poly-siRNA, suggesting tumor-targeted siRNA delivery of psi-tGel NPs. Importantly, the psi-tGel NPs induced effective tumor RFP gene silencing in vivo without remarkable toxicity. The psi-tGel NPs have great potential for a systemic siRNA delivery system for cancer therapy, based on their characteristics of low toxicity, tumor accumulation, and effective siRNA delivery.     

Lipoplex-mediated gene delivery to the lung occurs within 60 minutes of intravenous administration.

We have defined the critical time period for gene delivery in the lung after intravenous administration of cationic lipoplex. We accomplished this through the displacement of intravenously injected cationic lipoplexes from the lungs by the subsequent administration of anionic liposomes. When reporter gene-bearing lipoplexes were injected intravenously and followed by anionic liposomes 5 min later, reporter gene expression was reduced up to 400-fold compared with animals into which lipoplex alone was administered. Administration of anionic liposomes 60-90 min after lipoplex injection yielded no significant reduction in lung transfection. When lipoplexes were disrupted 5 min after administration, the pulmonary distribution of the cationic lipid and DNA components was reduced by 80%. Lipids subsequently accumulated primarily in the liver, while the plasmid DNA constituent distributed into the blood and liver. As the interval between lipoplex and anionic liposome injection increased, the degree of lipoplex displacement from the lung decreased to such a point that, 60 min after lipoplex injection, the anionic liposome injection did not displace significant quantities of the lipoplex. We conclude that cationic lipid-DNA complexes can be disrupted in vivo via the administration of anionic liposomes; moreover, we have employed this phenomenon to demonstrate that transfectionally active DNA is taken up within 60 min of systemic lipoplex administration.     

Transfection of shRNA-encoding Minivector DNA of a few hundred base pairs to regulate gene expression in lymphoma cells

This work illustrates the utility of Minivector DNA, a non-viral, supercoiled gene therapy vector incorporating short hairpin RNA from an H1 promoter. Minivector DNA is superior to both plasmid DNA and small interfering RNA (siRNA) in that it has improved biostability while maintaining high cell transfection efficiency and gene silencing capacity. Minivector DNAs were stable for over 48?h in human serum, as compared with only 0.5 and 2?h for siRNA and plasmid, respectively. Although all three nucleic acids exhibited similar transfection efficiencies in easily transfected adhesion fibroblasts cells, only Minivector DNAs and siRNA were capable of transfecting difficult-to-transfect suspension lymphoma cells. Minivector DNA and siRNA were capable of silencing the gene encoding anaplastic lymphoma kinase, a key pathogenic factor of human anaplastic large cell lymphoma, and this silencing caused inhibition of the lymphoma cells. Based on these results, Minivector DNAs are a promising new gene therapy tool.     

A Mini-intronic Plasmid (MIP): A Novel Robust Transgene Expression Vector In Vivo and In Vitro

The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene expression cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic expression cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene expression compared with minicircle vectors containing the same expression cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.     

A novel environment-sensitive biodegradable polydisulfide with protonatable pendants for nucleic acid delivery

Clinical application of nucleic acid-based therapies is limited by the lack of safe and efficient delivery systems. The purpose of this study is to design and evaluate novel biodegradable polymeric carriers sensitive to environmental changes for efficient delivery of nucleic acids, including plasmid DNA and siRNA. A novel polydisulfide with protonatable pendants was synthesized by the oxidative polymerization of a dithiol monomer, which was readily prepared by solid phase chemistry. The polydisulfide exhibited good buffering capacity and low cytotoxicity. It formed stable complexes with both plasmid DNA and siRNA. The particle sizes of the complexes decreased with the increase of the N/P ratios in the range of 100 to 750 nm. The complexes were stable in the presence of salt and heparin under normal physiological conditions, but dissociated to release nucleic acids in a reductive environment similar to cytoplasm. The polydisulfide demonstrated N/P ratio dependent transfection efficiency for plasmid DNA and gene silencing efficiency for siRNA. The presence of an endosomal disrupting agent, chloroquine, did not affect the DNA transfection efficiency of the polydisulfide. The transfection or gene silencing efficiency of the polydisulfide/DNA or siRNA complexes was comparable to or slightly lower than that of corresponding PEI complexes. Moreover, the polydisulfide showed better serum-friendly feature than PEI when delivering either DNA or siRNA in the presence of 10% FBS. This novel polydisulfide is a promising lead for further design and development of safe and efficient delivery systems for nucleic acids.     

Shock Waves and DNA-Cationic Lipid Assemblies: A Synergistic Approach to Express Exogenous Genes in Human Cells

Cationic lipid/DNA complexes (lipoplexes) represent a powerful tool for cell transfection; however, their use is still limited by important concerns, including toxicity and poor internalization into deep tissues. In this work, we investigated the use of shock wave-induced acoustic cavitation in vitro for the transfection of lipoplexes in human embryo kidney 293 cells. We selected shock waves with the ability to internalize 10-kDa fluorescein isothiocyanate-dextran into cells while maintaining survival rates above 50%. Cell transfection was tested using the green fluorescent protein-encoding plasmid pCX::GFPGPI2. Confocal microscopy and fluorescence-assisted cell sorting analyses revealed successful transfection after treatments ranging from 1 to 3 min using 60 to 180 shock waves at peak amplitudes of 12.3 ± 1.5 MPa. Interestingly, the combination of shock waves and lipoplexes induced a 3.1- and 3.8-fold increase in the expression of the reporter gene compared with the use of lipoplexes or shock waves alone, respectively. These results indicate that cationic DNA assembly and shock waves act in a synergistic manner to promote transfection of human cells, revealing a potential approach for non-invasive site-specific gene therapy.     

Investigating siRNA delivery to chronic myeloid leukemia K562 cells with lipophilic polymers for therapeutic BCR-ABL down-regulation

RNAi represents a new alternative for treatment of chronic myeloid leukemia (CML) to overcome the difficulties of current drug treatments such as the acquired resistance. However, potent carriers that can overcome delivery barriers to RNAi agents and have therapeutic efficacy especially in difficult-to-transfect CML cells are needed. Here, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2 and 2.0 kDa) in K562 cells and showed that the delivery efficiency was dependent on the type of lipid used for polymer modification, degree of lipid substitution and polymer molecular weight. Among the lipid-substituted polymers investigated, palmitic acid (PA)-substituted 1.2 kDa PEI (~ 2 lipids/PEI) has proven to be highly efficient in delivering siRNA and silencing of the reporter gene green fluorescent protein (GFP). The silencing efficacy achieved with this polymer was found to be higher than the 25 kDa PEI and is similar to commercial reagent Lipofectamine™ 2000. Moreover, when BCR-ABL protein was targeted in K562 cells, a reduction in the corresponding mRNA levels was observed, as well as an induction of early and late stage apoptosis. The results of this study demonstrated that PA-substitutions on low MW polymers could be useful for siRNA delivery in CML cells for therapeutic purposes.     

Novel dextran-spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

Recently, a novel cationic polymer, dextran-spermine (D-SPM) was developed for gene delivery. An efficient transfection was obtained using this polycation for a variety of genes and cell lines in serum-free or serum-poor medium. However, transfection using the water-soluble D-SPM-based polyplexes decreased with increasing serum concentration in cell culture in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium. In order to overcome this obstacle, oleyl derivatives of D-SPM (which form micelles in aqueous phase) were synthesized at 1, 10, and 20 mol% of oleyl moiety to polymer epsilon-NH2 to form N-oleyl-D-SPM (ODS). Polyplexes based on ODS transfected well in medium containing 50% serum. Comparison with polyplexes based on well-established polymers (branched and linear polyethyleneimine) and with DOTAP/Cholesterol lipoplexes showed that regarding beta-galactosidase transgene expression level and cytotoxicity in tissue culture, the D-SPM and ODS compare well with the above polyplexes and lipoplexes. Intracellular trafficking using FITC-labeled ODS and Rhodamine-labeled pGeneGrip plasmid cloned with hBMP2 monitored by confocal microscopy revealed that during the transfection process the fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus, while the cell cytoplasm was free of fluorescent particles, suggesting that the polyplexes move in the cell toward the nucleus by vesicular transport through the cytoplasm and not by a random diffusion. We found that the plasmids penetrate the cell nucleus without the polymer. Preliminary results in zebra fish and mice demonstrate the potential of ODS to serve as an efficient nonviral vector for in vivo transfection.     

Influence of lipoplex surface charge on siRNA delivery: application to the in vitro downregulation of CXCR4 HIV-1 co-receptor

Objective: Cationic lipidic formulations have been successfully used to deliver small interfering RNA (siRNA) into cells but they show limitations for in vivo application due to their cytotoxicity and instability in the presence of serum. To overcome these limitations, the authors developed an anionic lipid-based carrier named Neutraplex (Nx). Here, they wanted to investigate the influence of the lipoplex (Lx) surface charge on cytotoxicity, delivery and silencing activity of siRNAs.

Methods: The efficiency of three Nx formulations (cationic, close to neutrality and anionic) to deliver anti-CXCR4 siRNAs in MAGI cells was investigated and compared with the cationic commercial transfection reagent Lipofectamine RNAiMAX. Cellular uptake and intracellular localization of a fluorescent siRNA was monitored in live cells using fluorescence microscopy and silencing activity was measured by flow cytometry and RT-PCR analysis.

Results: The authors found that the Lx surface charge influenced cellular uptake and silencing activity of siRNA in cell cultures. Although cationic Lx formulations were the most efficient carriers to deliver active silencing siRNAs, negatively charged lipoplexes were taken up by cells, delivered active siRNAs and presented low cytotoxicity.

Conclusions: Altogether, the findings support further investigation for in vivo delivery of therapeutic siRNAs using Nx. Furthermore, this study indicates that anionic delivery systems may have potential for in vivo RNAi therapeutics.     

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.     

Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import

Although transfections are routinely used in the laboratory, the mechanism(s) by which exogenous DNA is transported into the nucleus is poorly understood. By improving our understanding of how vectors circumvent the numerous cellular barriers to gene transfer, more efficient gene delivery methods can be devised. We have begun to design plasmid constructs that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing levels of expression. We have shown that inclusion of specific DNA sequences in plasmid constructs mediates nuclear import both in vitro and in vivo. Here, we use plasmid affinity chromatography, mass spectrometry (MS), and live-cell pulldowns of transfected plasmid constructs to identify protein cofactors that interact in a sequence-specific manner with these DNA nuclear targeting sequences (DTSs). Importin β₁, importin 7, and the small guanosine triphosphatase Ran all demonstrate DTS-specific interaction in both MS and pull-down assays, consistent with our model of plasmid nuclear import. In addition, knockdown of importin β₁ with small interfering RNA (siRNA) abrogates plasmid nuclear import, indicating that it is a necessary cofactor. Our discovery that specific karyopherins mediate plasmid nuclear import can be used to design more effective vectors for gene delivery.     

靶向wnt-1基因的RNA干涉质粒构建及抑制U251细胞增殖的实验研究

目的构建靶向wnt-1基因的RNA干涉表达载体,转染人胶质瘤细胞U251中,研究该质粒的抑瘤效果。方法根据siRNA设计原则,通过软件设计,选取含有21个核苷酸(21nt)靶序列,形成siRNA的DNA模板并克隆到pGPU6/GFP/Neo载体中,获得靶向抑制wnt-1基因表达的siRNA表达载体,并经酶切测序鉴定。用该干涉质粒转染U251细胞,通过逆转录PCR、Western blotting方法检测wnt-1基因mRNA和蛋白表达水平,用MTT法、流式细胞术初步评价对U251细胞的增生、细胞周期影响。结果成功构建针对靶向wnt-1基因的pGPU6/GFP/Neo-shRNA-wnt-1 RNA干涉质粒,可显著抑制U251细胞wnt-1基因的mRNA和蛋白表达。MTT法分析转染细胞增生明显抑制,流式细胞仪分析细胞周期证实转染后比空白对照组受到阻滞。结论靶向wnt-1基因的pGPU6/GFP/Neo-shRNA-wnt-1 RNA干涉质粒构建成功,并可特异性的沉默wnt-1基因蛋白表达,使细胞增长减慢,细胞周期阻滞,本实验通过wnt-1的RNA干涉,为由wnt-1介导的wnt信号通路在胶质瘤发病机制中的抑瘤作用提供了依据,并为进一步该基因的基因治疗临床实验提供了可靠基础。     

Effective endogenous gene silencing mediated by pH responsive peptides proceeds via multiple pathways

Cationic amphipathic histidine rich peptides possess high plasmid DNA and siRNA delivery capabilities. To further understand the pH responsive siRNA delivery process and evaluate the capabilities of such peptides we have investigated their ability to mediate specific silencing of endogenous GAPDH gene activity in MCF-7 and A549 cells and compared this with plasmid DNA delivery. A substantial and selective reduction of both GAPDH activity and expression was achieved using pH responsive peptide vectors, which compared favourably with that mediated by commercially available non-viral vectors in terms of efficacy and toxicity. Furthermore, by comparing the efficacy of both gene delivery and silencing mediated by a series of such peptides, their sensitivities to known inhibitors of endocytotic processes, and their route of uptake via confocal live cell imaging, we show that both plasmid DNA and siRNA are internalised via endocytosis. However siRNA entry facilitated by LAH4-L1, proceeds via a cholesterol dependent mechanism, in contrast to DNA transfer which is associated with clathrin dependent endocytosis. Furthermore, using peptides that respond at increasingly acidic pH, we demonstrate that the route of entry for the siRNA that ultimately mediates silencing is peptide specific and whilst some pH responsive peptides promote the escape of labelled siRNA from endosomes, others may promote entry via alternative mechanisms.     

Formation and intracellular trafficking of lipoplexes and polyplexes.

Cationic lipid/DNA lipoplexes and cationic polymer/DNA polyplexes represent an attractive alternative to viral vectors for cell transfection in vitro and in vivo but still suffer from a relatively low efficiency. Optimization of their transfection efficiency may be attempted by using a trial and error approach consisting of synthesizing and testing a large number of derivatives. On the other hand, rational design of highly efficient cationic lipids and polymers requires a deeper understanding of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved in DNA entry into the cell and ultimately the nucleus. In the present review, the pathways and mechanisms involved in lipoplex- and polyplex-mediated transfection are comparatively addressed and unresolved questions are highlighted.     

Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes

There is mounting interest in developing antisense and siRNA oligonucleotides into therapeutic entities; however, this potential has been limited by poor access of oligonucleotides to their pharmacological targets within cells. Transfection reagents, such as cationic lipids and polymers, are commonly utilized to improve functional delivery of nucleic acids including oligonucleotides. Cellular entry of large plasmid DNA molecules with the assistance of these polycationic carriers is mediated by some form of endocytosis; however, the mechanism for delivery of small oligonucleotide molecules has not been well established. In this study, splice-shifting oligonucleotides have been formulated into cationic lipoplexes and polyplexes, and their internalization mechanisms have been examined by using pharmacological and genetic inhibitors of endocytosis. The results showed that intercellular distribution of the oligonucleotides to the nucleus governs their pharmacological response. A mechanistic study revealed that oligonucleotides delivered by lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for the functional induction of the target gene. In contrast, polyplexes are internalized by unconventional endocytosis pathways that do not require dynamin or caveolin. These studies may help rationally design novel delivery systems with superior transfection efficiency but lower toxicity.     

Pyridylthiourea-grafted polyethylenimine offers an effective assistance to siRNA-mediated gene silencing in vitro and in vivo

Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10 nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100 nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4 days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments.     

Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates

The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.     

Enhanced transfection efficiency of PAMAM dendrimer by surface modification with L-arginine.

We designed a novel type of arginine-rich dendrimer, with a structure based on the well-defined dendrimer, polyamidoamine dendrimer (PAMAM). Further characterization was performed to prove that the polymer is a potent nonviral gene delivery carrier. The primary amines located on the surface of PAMAM were conjugated with L-arginine to generate an L-arginine-grafted-PAMAM dendrimer (PAMAM-Arg). For comparison, an L-lysine-grafted-PAMAM dendrimer (PAMAM-Lys) was also generated and compared as a control reagent. The polymers were found to self-assemble electrostatically with plasmid DNA, forming nanometer-scale complexes. From dynamic light scattering experiments, the mean diameter of the polyplexes was observed to be around 200 nm. We used PicoGreen reagent as an efficient probe for assaying complex formation of polymers with plasmid DNA. The complex composed of PAMAM-Arg/DNA showed increased gene delivery potency compared to native PAMAM dendrimer and PAMAM-Lys. The cytotoxicity and transfection efficiencies for 293, HepG2, and Neuro 2A cells were measured by comparison with PEI and PAMAM. In addition, transfection experiments were performed in primary rat vascular smooth muscle cells, and PAMAM-Arg showed much enhanced transfection efficiency. These findings suggest that the L-arginine-grafted-PAMAM dendrimer possesses the potential to be a novel gene delivery carrier for gene therapy.     

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.