Department of Biomedical Engineering, University of Rochester Medical Center, University of Rochester, Rochester, NY 14627, USA.
Biomacromolecules. 2012 Nov 12;13(11):3841-9. doi: 10.1021/bm301294n. Epub 2012 Oct 11.
siRNA treatment has great promise to specifically control gene expression and select cell behaviors but has delivery challenges limiting its use. Particularly for applications in regenerative medicine, uniform and consistent delivery of siRNA to control gene expression and subsequent stem cell functions, such as differentiation, is paramount. Therefore, a diblock copolymer was examined for its ability to effectively deliver siRNA to mesenchymal stem cells (MSCs). The diblock copolymers, which are composed of cationic blocks for siRNA complexation, protection, and uptake and pH-responsive blocks for endosomal escape, were shown to facilitate nearly 100% MSC uptake of siRNA. This is vastly superior to a commercially available control, DharmaFECT, which resulted in only ~60% siRNA positive MSCs. Moreover, the diblock copolymer, at conditions that result in excellent knockdown (down to ~10% of control gene expression), was cytocompatible, causing no negative effects on MSC survivability. In contrast, DharmaFECT/siRNA treatment resulted in only ~60% survivability of MSCs. Longitudinal knockdown after siRNA treatment was examined and protein knockdown persists for ~6 days regardless of delivery system (diblock copolymer or DharmaFECT). Finally, MSC phenotype and differentiation capacity was examined after treatment with control siRNA. There was no statistically significant differences on cell surface markers of diblock copolymer/siRNA or DharmaFECT/siRNA-treated or cells measured 2 weeks after siRNA delivery compared to untreated cells. Upon differentiation with typical media/culture conditions to adipogenic, chondrogenic, and osteogenic lineages and examination of histological staining markers, there was no discernible differences between treated and untreated cells, regardless of delivery mechanism. Thus, diblock copolymers examined herein facilitated uniform siRNA treatment of MSCs, inducing siRNA-specific gene and protein knockdown without adversely affecting MSC survival or differentiation capacity and therefore show great promise for use within regenerative medicine applications.
siRNA 治疗在特异性控制基因表达和选择细胞行为方面具有巨大的潜力，但由于其传递方面的挑战，限制了其应用。特别是在再生医学应用中，均匀一致地传递 siRNA 以控制基因表达和随后的干细胞功能（如分化）至关重要。因此，研究了一种二嵌段共聚物将 siRNA 有效递送至间充质干细胞 (MSC) 的能力。该二嵌段共聚物由用于 siRNA 复合、保护和摄取的阳离子嵌段和用于内涵体逃逸的 pH 响应嵌段组成，可促进 MSC 对 siRNA 的几乎 100%摄取。这大大优于商业上可获得的对照物 DharmaFECT，后者仅导致约 60%的 siRNA 阳性 MSC。此外，在导致出色敲低（低至对照基因表达的10%）的条件下，二嵌段共聚物对 MSC 存活率无任何负面影响。相比之下，DharmaFECT/siRNA 处理仅导致60%的 MSC 存活率。在 siRNA 处理后进行了纵向敲低研究，无论使用哪种递送系统（二嵌段共聚物或 DharmaFECT），蛋白质敲低都会持续约 6 天。最后，研究了用对照 siRNA 处理后 MSC 的表型和分化能力。与未经处理的细胞相比，二嵌段共聚物/siRNA 或 DharmaFECT/siRNA 处理或 siRNA 递送 2 周后细胞的表面标志物上没有统计学上的显著差异。在用典型的培养基/培养条件向成脂、成软骨和成骨谱系分化并检查组织学染色标志物后，无论使用哪种递送机制，处理过的细胞与未经处理的细胞之间没有明显差异。因此，本文研究的二嵌段共聚物促进了 MSC 的均匀 siRNA 处理，诱导了 siRNA 特异性基因和蛋白敲低，而不会对 MSC 的存活或分化能力产生不利影响，因此在再生医学应用中具有广阔的应用前景。
