イメージングシステムを利用した低分子量キトサン-siRNA複合体の結合能とRNA干渉評価

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Chitosan is one of the attractive non-viral carriers for gene delivery including siRNA. However, common chitosan, which has a relatively high molecular weight, is insoluble in water, which might make it difficult to apply clinically. In this study, we investigated the efficacy of low-molecular-weight chitosan (LMWC), which is soluble in water, as a carrier for siRNA delivery. To evaluate the binding affinity and RNA interference (RNAi) of LMWC/siRNA complexes, a multi-well imaging system (IVIS^[○!R]) was adapted. CT26 cells stably expressing firefly luciferase (CT26/Luc cells) were established to evaluate RNAi. Evaluation of RNAi using lipofectamine^<TM> 2000 was carried out by employing a luminometer with cell lysis and IVIS^[○!R] without cell lysis. The results were closely correlated, suggesting the advantages of the multi-well imaging system regarding screening, the visualization of results, and nondestructive evaluation. Fluorescence generated by ethidium bromide intercalated in the double strand of siRNA was markedly quenched at a higher ratio of LMWC to siRNA (N/P) and lower pH. Evaluation of the particle size and zeta potential of LMWC/siRNA complexes also indicated the higher binding affinity of LMWC with siRNA. At N/P=300 and pH 6.5, which satisfied the high-level binding affinity of LMWC with siRNA, significantly lower luminescence was detected in CT26/Luc cells treated with LMWC/siRNA compared with those treated with LMWC alone, suggesting the presence of RNAi. These results suggested that LMWC may be an effective carrier for siRNA delivery, and that the multi-well imaging system may be a powerful tool to evaluate the binding affinity and RNAi.
社団法人日本薬学会の論文
2009-04-01

イメージングシステムを利用した低分子量キトサン-siRNA複合体の結合能とRNA干渉評価

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