| 研究実績の概要 |
First, the structures of catiomers segment and ASO were carefully designed to strengthen their noncovalent interactions within the PIC membrane for fabrication of stable nanovesicles under a physiological condition. Thus, varying degrees of guanidino groups were introduced to the side chains of block catiomers to optimize noncovalent interactions with ASO. The block catiomers of poly(ethylene glycol) with degree of polymerization (DP) 45 and poly([5-aminopentyl]-α,β-aspartamide) with DP 60 (PEG-P(Asp-AP)) was synthesized by aminolysis reaction of the parent block copolymer of PEG and poly(β-benzyl-L-aspartate) (PEG-PBLA) with 1,5-diaminopentane (DAP). Then, guanidinylation of primary amines in PEG-P(Asp-AP) was performed at 40 °C and pH 9.5 in the presence of the guanidinylation reagent, 1-guanyl-3,5-dimethylpyrazole nitrate (GDMP). A series of guanidinylated PEG-P(Asp-AP)s (PEG-P(Asp-AP/G)s) were obtained by varying the reaction time.
PIC samples were prepared by vortex mixing of PEG-P(Asp-AP/G) with a chemically modified ASO comprising the PS backbone and the locked nucleic acid (LNA) at terminal nucleotides under a charge-stoichiometric condition. Particularly, the PIC prepared with PEG-P(Asp-AP/G) and PS-Gapmer showed a sharp single peak in the DLS histogram with a cumulant diameter of ~100 nm, a low PDI of ~0.1, and zeta-potential of ~-20 mV. The morphology of PICs was confirmed to be similar to those of conventional PICsomes by transmission electron microscopy (TEM), demonstrating the successful fabrication of ASOsome.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
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理由
It is proceeding as planned. About the synthesis of varying polymers, guanidinylation rate was confirmed form 1H NMR spectra and size exclusion chromatography (SEC) charts for PEG-P(Asp-AP/G20), PEG-P(Asp-AP/G50), and PEG-P(Asp-AP/G80), where GX represents X% guanidinylation of primary amines. Note that PEG-P(Asp-AP/G90) and PEG-P(Asp-AP/G95) contained a fraction of degraded products after guanidinylation. About the oligonucleotides, PS-Gapmer and PS-DNA elicited both greater hydrophobicity and stronger (poly)ion-pairing capacity compared with PO-Gapmer and PO-DNA, confirmed by reverse phase liquid chromatography and ion exchange liquid chromatography.Also, the duplex formation of oligonucleotides further reduced the SSOsome stability. The guanidinylated polypeptides facilitated the multimolecular PIC formation. And the stability of PICs was tolerable in the serum-containing medium.
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| 今後の研究の推進方策 |
The biological activity of ASOsomes prepared from P(Asp-AP/G80) and PS-Gapmer ASO will be investigated for cultured human lung carcinoma (A549) cells. Cellular uptake efficiency and gene knockdown efficacy will be evaluated. Also, RNase H encapsulated ASOsomes will be prepared and characterized, and then, compared with the empty ASOsome.
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