Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore.
Biomaterials. 2011 Sep;32(25):5915-23. doi: 10.1016/j.biomaterials.2011.04.065. Epub 2011 May 18.
Nanofiber scaffold-mediated delivery of small-interfering RNA (siRNA) holds great potential in regenerative medicine by providing biomimicking topographical signals and enhanced gene silencing effects to seeded cells. While the delivery of naked siRNA was demonstrated previously using poly (ε-caprolactone) (PCL) nanofibers, the resulting siRNA release kinetics and gene knockdown efficiencies were sub-optimal. In this study, we investigated the feasibility of encapsulating siRNA and transfection reagent (TKO) complexes within nanofibers comprising of a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP, diameter ∼ 400 nm). Sustained release of bioactive naked siRNA and siRNA/TKO complexes were obtained for at least 28 days. By copolymerizing EEP with caprolactone, siRNA release was significantly enhanced (total siRNA that was released by day 49 was ∼ 89.3-97.2% as compared to previously reported 3% by plain PCL nanofiber delivery). Using GAPDH as the model protein, bioactivity analyses by supernatant transfection revealed the partial retention of bioactivity of naked siRNA and siRNA/TKO complexes for at least 30 days. In particular, GAPDH siRNA/TKO supernatant alone induced significant gene silencing (∼40%), indicating the feasibility of co-encapsulating siRNA and transfection reagent within a single scaffold construct for sustained delivery. Direct culture of cells on siRNA incorporated scaffolds for scaffold-mediated gene transfection revealed significant gene knockdown even in the absence of transfection reagent (21.3% knockdown efficiency by scaffolds incorporating naked siRNA only). By encapsulating siRNA/TKO complexes, more significant gene knockdown was obtained (30.9% knockdown efficiency as compared to previously reported 18% by plain PCL scaffold-mediated transfection). Taken together, the results demonstrated the feasibility of co-encapsulating siRNA-transfection reagent complexes within a single nanofiber construct for sustained siRNA delivery and enhanced gene knockdown efficiency. The study also highlights the potential of PCLEEP as a platform for tailoring siRNA release kinetics for long-term gene silencing applications.
纳米纤维支架介导的小干扰 RNA（siRNA）传递在再生医学中有很大的潜力，它为接种细胞提供了仿生拓扑信号和增强的基因沉默效果。虽然之前已经证明了使用聚己内酯（PCL）纳米纤维传递裸 siRNA 的可行性，但结果表明 siRNA 的释放动力学和基因敲低效率并不理想。在这项研究中，我们研究了将 siRNA 和转染试剂（TKO）复合物包封在由己内酯和乙基乙烯磷酸酯共聚物（PCLEEP，直径约 400nm）组成的纳米纤维内的可行性。至少 28 天内获得了生物活性裸 siRNA 和 siRNA/TKO 复合物的持续释放。通过将 EEP 与己内酯共聚，siRNA 的释放得到了显著增强（到第 49 天释放的总 siRNA 约为 89.3-97.2%，而之前报道的纯 PCL 纳米纤维递送为 3%）。使用 GAPDH 作为模型蛋白，通过上清液转染进行的生物活性分析表明，裸 siRNA 和 siRNA/TKO 复合物的生物活性至少保留了 30 天。特别是，单独的 GAPDH siRNA/TKO 上清液诱导了显著的基因沉默（约 40%），表明在单个支架构建体中共同包封 siRNA 和转染试剂用于持续递送的可行性。直接在包含 siRNA 的支架上培养细胞进行支架介导的基因转染，即使没有转染试剂，也能获得显著的基因敲低（仅包含裸 siRNA 的支架的敲低效率为 21.3%）。通过包封 siRNA/TKO 复合物，获得了更显著的基因敲低（与之前报道的纯 PCL 支架介导转染的 18%相比，效率为 30.9%）。总的来说，结果表明在单个纳米纤维构建体中共同包封 siRNA-转染试剂复合物用于持续 siRNA 传递和增强基因敲低效率的可行性。该研究还强调了 PCLEEP 作为一种平台的潜力，用于调整 siRNA 释放动力学以实现长期基因沉默应用。
