Takashi Kitahara

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.

The Essential Role of p53 in Hyperpigmentation of the Skin via Regulation of Paracrine Melanogenic Cytokine Receptor Signaling

Hyperpigmentation of the skin is characterized by increases in melanin synthesis and deposition. Although considered a significant psychosocial distress, little is known about the detailed mechanisms of hyperpigmentation. Recently, the tumor suppressor protein p53 has been demonstrated to promote ultraviolet B-induced skin pigmentation by stimulating the transcription of a melanogenic cytokine, POMC (pro-opiomelanocortin), in keratinocytes. Given that p53 can be activated by various kinds of diverse stresses, including sun exposure, inflammation, and aging, this finding led us to examine the involvement of p53 in cytokine receptor signaling, which might result in skin hyperpigmentation. Immunohistochemical and reverse transcription-PCR analyses revealed the increased expression and phosphorylation of p53 in the epidermis of hyperpigmented spots, accompanied by the higher expression of melanogenic cytokines, including stem cell factor, endothelin-1, and POMC. The involvement of p53 in hyperpigmentation was also indicated by the significantly higher expression of p53 transcriptional targets in the epidermis of hyperpigmented spots. Treatment of human keratinocytes and melanocytes with known p53 activators or inhibitors, including pifithrin-α (PFT), demonstrated significant increases or decreases, respectively, in the expression of melanogenic factors, including cytokines and their receptors. Additionally, PFT administration abolished stem cell factor-induced phosphorylation of mitogen-activated protein kinase in human melanocytes. Furthermore, when organ-cultured hyperpigmented spots, in vitro human skin substitutes, and mouse skin were treated with PFT or p53 small interfering RNA, the expression of melanogenic cytokines and their receptors was significantly decreased, as were levels of tyrosinase and melanogenesis. Taken together, these data reveal the essential role of p53 in hyperpigmentation of the skin via the regulation of paracrine-cytokine signaling, both in keratinocytes and in melanocytes.

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.

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

PURPOSEnIn this study, we developed various ternary complexes of encapsulated polyplexes and lipoplexes using chondroitin sulfate (CS) and investigated their universal usefulness for gene delivery.nnnMETHODSnTo 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.nnnRESULTnThe 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.nnnCONCLUSIONnThese results indicate that CS can contribute to polyplex-mediated gene delivery systems for effective and safe gene therapy.

Therapeutic index of methotrexate depends on circadian cycling of tumour necrosis factor‐α in collagen‐induced arthritic rats and mice

Objectives Rheumatoid arthritis is an autoimmune disorder of unknown aetiology. Morning stiffness, a characteristic feature of rheumatoid arthritis, shows a 24‐h rhythm. Noticing this rhythm, we hypothesized the presence of a similar rhythm for a rheumatoid arthritis indicator, in addition to dosing‐time dependency of the anti‐rheumatic effect of methotrexate in arthritis induced by collagen in rats and mice, which reflect the symptomatology of rheumatoid arthritis patients.

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

PURPOSEnIn 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.nnnMETHODSnTransdermal 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.nnnRESULTSnIn 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.nnnCONCLUSIONnIn 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.