Tomoaki Kurosaki

Ternary complexes of pDNA, polyethylenimine, and γ-polyglutamic acid for gene delivery systems

We discovered a vector coated by gamma-polyglutamic acid (gamma-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic-polycytidylic acid, alpha-polyaspartic acid, alpha-polyglutamic acid, and gamma-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive zeta-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/gamma-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/gamma-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of gamma-PGA significantly inhibited the uptake of pDNA/PEI/gamma-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/gamma-PGA complex was taken up by gamma-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/gamma-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.

γ-Polyglutamic acid-coated vectors for effective and safe gene therapy.

In the present study, we developed some novel gene delivery vectors, coated cationic complexes with gamma-polyglutamic acid (gamma-PGA) for effective and safe gene therapy. Cationic complexes were constructed with pDNA and cationic vectors, such as poly-L-arginine hydrochloride (PLA), poly-L-lysine hydrobromide (PLL), N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol (Chol) liposomes, and DOTMA-dioleylphosphatidylethanolamine (DOPE) liposomes. The cationic complexes showed high gene expression with strong cytotoxicity in melanoma B16-F10 cells. The cationic complexes were also strongly toxic to erythrocytes. On the other hand, the gamma-PGA was able to coat all cationic complexes and form stable nano-sized particles with negative charges. These gamma-PGA-coated complexes had high gene expression without cytotoxicity and toxicities to the erythrocytes. In in vivo transfection experiments, polyplexes showed high transfection efficiency over 10(5) RLU/g in the lung tissue after intravenous injection, although gamma-PGA-coated polyplexes showed a high value in the spleen. High transfection efficiency in lipoplexes and gamma-PGA-coated lipoplexes was observed in the spleen and lung. Thus, gamma-PGA-coated vectors are useful for clinical gene therapy.

The development of a gene vector electrostatically assembled with a polysaccharide capsule.

The purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. The pDNA/PEI complex was electrostatically encapsulated with various polysaccharides such as fucoidan, lambda-carrageenan, xanthan gum, alginic acid, hyaluronic acid, and chondroitin sulfate (CS). The pDNA/PEI complex was shown as nanoparticles with positive zeta-potential, although the ternary complexes encapsulated with polysaccharides were shown as nanoparticles with negative zeta-potential. The pDNA/PEI complex showed high agglutination activity and cytotoxicity, although the ternary complexes encapsulated with polysaccharides had no agglutination activities and lower cytotoxicities. The pDNA/PEI complex showed high uptake and high transgene efficiency in B16-F10 cells. On the other hand, most of the ternary complexes show little uptake and gene expression. The ternary complex encapsulated by CS, however, showed comparable transgene efficiency to the pDNA/PEI complex. The uptake and gene expression of the ternary complex encapsulated by CS were significantly inhibited by hypothermia and the addition of CS, suggesting that the ternary complex was taken by CS-specific receptor-mediated energy-dependent process.

Nanoparticles electrostatically coated with folic acid for effective gene therapy.

We developed a novel vector, electrostatically coated poly(ethylenimine) (PEI)/pDNA complexes with folic acid (FA). Without covalent binding, the FA molecules could coat the PEI/pDNA complexes, and stable anionic nanoparticles were formed at a charge ratio greater than 60. The addition of FA markedly decreased the cytotoxicity of the cationic PEI/pDNA complexes to the melanoma cell line, B16-F10 cells, which regularly expressed FA-specific receptor (FR). Furthermore, the anionic FA60/PEI/pDNA complexes showed high transgene efficiency via the FR-mediated pathway in B16-F10 cells. The FA60/PEI/pDNA complexes did not show agglutination with erythrocytes. After the intravenous injection of FA60/PEI/pDNA complexes into mice, a higher transgene efficiency than PEI/pDNA complexes was observed in the liver, kidney, spleen, and lung with FR. The gene expressions of FA60/PEI/pDNA complexes were significantly inhibited by preadministration of FA. Thus, the FA60/PEI/pDNA complexes were useful for effective gene therapy.

Immunogenicity of novel nanoparticle-coated MSP-1 C-terminus malaria DNA vaccine using different routes of administration.

An important aspect in optimizing DNA vaccination is antigen delivery to the site of action. In this way, any alternative delivery system having higher transfection efficiency and eventual superior antibody production needs to be further explored. The novel nanoparticle, pDNA/PEI/γ-PGA complex, is one of a promising delivery system, which is taken up by cells and is shown to have high transfection efficiency. The immunostimulatory effect of this novel nanoparticle (NP) coated plasmid encoding Plasmodium yoelii MSP1-C-terminus was examined. Groups of C57BL/6 mice were immunized either with NP-coated MSP-1 plasmid, naked plasmid or NP-coated blank plasmid, by three different routes of administration; intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c). Mice were primed and boosted twice at 3-week intervals, then challenged 2 weeks after; and 100%, 100% and 50% mean of survival was observed in immunized mice with coated DNA vaccine by i.p., i.v. and s.c., respectively. Coated DNA vaccine showed significant immunogenicity and elicited protective levels of antigen specific IgG and its subclass antibody, an increased proportion of CD4(+) and CD8(+) T cells and INF-γ and IL-12 levels in the serum and cultured splenocyte supernatant, as well as INF-γ producing cells in the spleen. We demonstrate that, NP-coated MSP-1 DNA-based vaccine confers protection against lethal P. yoelii challenge in murine model across the various route of administration and may therefore, be considered a promising delivery system for vaccination.

Biodegradable nanoparticles composed of dendrigraft poly-l-lysine for gene delivery

We developed novel gene vectors composed of dendrigraft poly-L-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-L-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, γ-polyglutamic acid (γ-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/γ-PGA complexes) were shown to be stable nanoparticles, and those with γ-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/γ-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/γ-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/γ-PGA complexes for clinical gene therapy.

Self-assemble gene delivery system for molecular targeting using nucleic acid aptamer.

We have developed a novel vector constructed with pDNA, polyethylenimine (PEI), and mucin 1 (MUC1) aptamer for tumor-targeted gene delivery. The MUC1 aptamer and non-specific aptamer were employed to coat the pDNA/PEI complexes electrostatically and stable nanoparticles were formed. The addition of a non-specific aptamer to the pDNA/PEI complex decreased gene expression in the human lung cancer cell line, A549 cells expressing MUC1 regularly. At the same time, the pDNA/PEI/MUC1 aptamer complex showed higher gene expression than pDNA/PEI/non-specific aptamer complex. Furthermore, the pDNA/PEI/MUC1 aptamer complex showed markedly high gene expression in tumor-bearing mice; thus, pDNA/PEI/MUC1 aptamer complexes are useful as a tumor-targeted gene delivery system with high transfection efficiency.

Chondroitin Sulfate Capsule System for Efficient and Secure Gene Delivery

PURPOSE In this study, we developed various ternary complexes of encapsulated polyplexes and lipoplexes using chondroitin sulfate (CS) and investigated their universal usefulness for gene delivery. METHODS To prepare the cationic complexes, pDNA was mixed with some cationic vectors such as poly-L-arginine, poly-L-lysine, N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol liposomes, and DOTMA- dioleylphosphatidylethanolamine (DOPE) liposomes. CS was added to the cationic complexes for constructions of ternary complexes. We examined in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of the ternary complexes. RESULT The cationic polymers and cationic liposomes bound to pDNA and formed stable cationic polyplexes and lipoplexes, respectively. Those cationic complexes showed high transgene efficiency in B16-F10 cells; however, they also had high cytotoxicity and strong agglutination with erythrocytes. CS could encapsulate the polyplexes and lipoplexes and form stable anionic particles without disrupting their structures. The ternary complexes encapsulated by CS showed high transgene efficiency in B16-F10 cells with low cytotoxicity and agglutination. As the result of animal experiments, the polyplexes had little transgene efficiency after intravenous administration in mice, whereas polyplexes encapsulated by CS showed specifically high transgene efficiency in the spleen. The capsulation of CS, however, reduced the high transgene efficiency of the lipoplexes. CONCLUSION These results indicate that CS can contribute to polyplex-mediated gene delivery systems for effective and safe gene therapy.

Exploitation of De Novo Helper-Lipids for Effective Gene Delivery.

PURPOSE In gene delivery, a fusogenic lipid such as dioleyl phosphatidylethanolamine (DOPE) which is a component of cationic liposomal vector is important factor for effective transfection efficiency. We investigated the effect of penetration enhancers as alternative helper-lipids to DOPE. METHODS Transdermal penetraion enhancers such as N-lauroylsarcosine (LS), (R)-(+)-limonene (LM), vitamin E (VE), and phosphatidyl choline from eggs (EggPC) were used in this experiments as helper-lipids with N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethlylammonium chloride (DOTMA) and cholesterol (CHOL). We examined in vitro transfection efficiency, cytotoxicity, hematotoxicity, and in vivo transfection efficiency of plasmid DNA/cationic liposomes complexes. RESULTS In transfection experiments in vitro, the cationic lipoplexes containing LS had highest transfection efficiency among the other lipoplexes independently of FBS. Furthermore, the lipoplexes containing LS had lowest cell toxicity among the other lipoplexes in the presence of FBS. As the results of erythrocytes interaction experiment, DOTMA/LS/CHOL, DOTMA/VE/CHOL, and DOTMA/EggPC/CHOL lipoplexes showed extremely lower hematotoxicity. On the basis of these results, the in vivo transfection efficiencies of the lipoplexes were examined. The lipoplexes containing LS had the highest transfection activity among the other lipoplexes. CONCLUSION In conclusion, several transdermal penetration enhancers are available for alternative helper-lipids to DOPE in cationic liposomal vectors. Among them, DOTMA/LS/CHOL lipoplexes showed superior characteristics in in vitro transfection efficiency, cell toxicity, hematotoxicity, and in vivo transfection efficiency.

Hybrid vector including polyethylenimine and cationic lipid, DOTMA, for gene delivery.

We developed polyethylenimine (PEI) lipopolyplexes with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA) and pDNA to investigate their usefulness for in vitro and in vivo gene delivery. The charge ratio of the complex to pDNA was calculated with molar values of nitrogen of PEI, and nitrogen of DOTMA to phosphate of pDNA. The polyplexes were prepared at charge ratio 2 (polyplex 2P) and 8 (polyplex 8P). DOTMA solution was added to polyplex 2P to prepare lipopolyplexes at charge ratio 1 (lipopolyplex 2P-1D), 2 (lipopolyplex 2P-2D), and 4 (lipopolyplex 2P-4D). The particle size of the complex was significantly reduced by the addition of DOTMA and settled to 74-114nm, indicating pDNA compaction. The addition of DOTMA to polyplex 2P decreased pDNA dissociation from the complex and degradation in serum. The addition of DOTMA to polyplex 2P remarkably increased gene expression in HepG2 cells in the absence or presence of FBS. These lipopolyplexes showed little cytotoxicity in the presence of FBS. After intravenous injection of the lipopolyplexes into mice, high-gene expression in the liver, spleen, and lung was observed with lipopolyplex 2P-2D, lipopolyplex 2P-4D, and polyplex 8P. In particular, lipopolyplex 2P-4D showed the highest gene expression.