When polycations are used as a non-virus vector, they induces DNA condensation by hydrophobic interaction due to electric charge neutralization and can protect DNA from nuclease and so on. However, it is concerned that the excessive DNA condensation leads to a drop of the gene expression. To improve the stability against serum proteins of a head-tail type polycation (PAMAM dendron-PLL), which is composed of polyamidoamine (PAMAM) dendron head and poly(L-lysine) (PLL) tail, we have designed multi-arm poly (ethylene glycol) (PEG) installed PLL (maPEG-PLL) by PEGlation at the periphery of the PAMAM dendron head. As a result, not only effective stabilization of the PAMAM dendron-PLL polyplexes through PEGlation of the PAMAM dendron block, but also the interesting behavior as the vector was comfirmed. In AFM observation, the polyplexes prepared using maPEG-PLL with the largest exclusion volume of maPEG took nanofiber-like morphology with uncondensed state of pDNA. Furthermore, The cell-free gene expression of nanofiber-polyplexes is higher than those of the polyplexes with the other condensed morphologies. It is thought that there is a strong association between morphology of polyplex, that is, DNA condensation state and gene expression from this results. On the other hand, gene expression of nanofiber-polyplexes was remarkbly low in cultured cell because of low ability of endosomal escape of nanofiber-polyplexes. In this study, we designed the hybrid vectors composed of nanofiber-polyplexes and lipofectaminewhich had the ability of endosomal escape. When we optimized quantity of lipofectamine and N/P ratio of nanofiber-polyplexes, the hybrid vector was took up by HeLa cell via endocytosis pathway, then carried to nucleus nearby. Furthermore, the hybrid vector showed higher transfection efficiency compared with the lipoplexes using only lipofectamine. Consequently, it is confirmed that creation of the hybrid vector which had both inhibition ability of DNA condensation and ability of endosomal escape.

Preparation of hybrid vector from nanofiber-polyplexes and lipofectamine for effective gene expression

247th National Meeting (2014)

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