| 研究実績の概要 |
Precursor polymers was reacted with endosomal escape moiety group (diethylenetriamine, DET) and various aliphatic amine groups (pentyl, heptyl, octyl, nonyl, decyl, 2-cyclohexylethyl (CHE)), 2-phenylethyl amines) to make amphiphilic polymers. All aliphatic amine groups were successfully introduced to the polymer at around 40% of total degree of polymerization. A series of amphiphilic polymers formed 100-150 nm sized nanoparticles with firefly or gaussian luciferase mRNA. These nanoparticles were transfected into cultured mouse myoblast cells and neuronal cancer cells. Amphiphilic polymers installed with DET and CHE moieties (CHE-polymer) showed the highest translation efficacy and exhibited 5 orders of magnitude higher than the other polymer with the least efficacy. The CHE-polymer also showed better Cas9 mRNA translation efficacy in luciferase-based gene editing assay, compared to commercially available lipofectamine. We confirmed that there is optimal hydrophobicity for efficient mRNA transfection in cultured cells, i.g. between particle stability and release of mRNA in cytoplasm. Thus, fine-tuning of hydrophobicity in the amphiphilic polymer dramatically increases in vitro transfection efficacy of mRNA.
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| 今後の研究の推進方策 |
1. In fiscal year 2019, a series of amphiphilic polymer will be investigated for delivery efficacy of mRNA in mouse animals. Firefly luciferase mRNA-loaded particles will be administered into mouse in the various injection manners and their luminescence intensities will be measured by in vivo imaging system. These experiments will provides the clues that the hydrophobicity tuning will also work in vivo condition.
2. The future work tries to find out the next parameter for the second round polymer synthesis. If possible, the second round amphiphilic polymers will be synthesized based on the new design parameter.
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| 次年度使用額が生じた理由 |
I successfully found amphiphilic polymer candidate with high performance for mRNA delivery earlier than I expected. I already finished particle characterization and mRNA transfection efficacy in cultured cells. Now, I want to investigate mRNA delivery efficacy of the amphiphilic polymer in mouse animal. High doses of polymer and mRNA amounts are necessary for animal experiments. In this reason, I want to transfer research fund in fiscal year 2018 into fiscal year 2019.
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