Institute for Macromolecular Chemistry, University of Freiburg, 79104 Freiburg, Germany.
Institute for Macromolecular Chemistry, University of Freiburg, 79104 Freiburg, Germany; BIOSS-Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany.
Acta Biomater. 2019 Nov;99:397-411. doi: 10.1016/j.actbio.2019.09.006. Epub 2019 Sep 18.
In recent years evidence has been mounting for a role for mesenchymal stem cells (MSCs) in immunomodulation, anti-inflammatory processes, and paracrine signaling via secreted extracellular vesicles. In order to exploit these biological functions, systems to efficiently deliver genetic material into MSCs would therefore be highly desirable. In this study, efficient silencing of GFP expression by combining high N/P ratio siRNA and branched PEI (bPEI) polyplexes (siRNA-bPEI) polyplexes with glycosaminoglycans (GAGs), namely hyaluronic acid (HA), chondroitin sulfate (CS) and heparin sulfate (HS), and human serum albumin (HSA) is reported. These quaternary systems were characterized using surface charge, size and morphology and applied to MSCs, which represent a challenge due to their typically low transfection efficiency. The quaternary polyplexes promoted efficient charge shielding and release of siRNA in the cytoplasm with reduced toxicity. A high silencing efficiency of >90% (i.e., less than 10% remaining GFP expression) was achieved with noticeably reduced cellular toxicity, especially with siRNA-bPEI polyplexes modified with HA and HA + HSA. In general addition of GAGs led to more compact polyplexes. Endocytosis studies point to improved endosomal escape at high N/P ratios as a reason for high transfection efficiency and a role for hyaluronic acid in the uptake mechanism likely via CD44 interactions. Co-localization studies showed the polyplexes are stable in the cytosol over time, which correlates with a proper disassembly and subsequent silencing of GFP. Furthermore, GAG containing polyplexes were frequently co-localized with the nucleus. These findings in sum suggest that PEI/HSA/GAG based quaternary polyplexes are promising as transfection agents for MSCs. STATEMENT OF SIGNIFICANCE: Since mesenchymal stem cells (MSCs) are recruited to the site of tissue repair and play a role in immunomodulation, anti-inflammatory processes, and paracrine signaling, they present an excellent target for genetic engineering. However, delivery of genetic material into MSCs is challenging. In this study, >97% silencing of constitutive green fluorescent protein expression in human MSCs (hMSCs) using high N/P ratio polyplexes of branched-PEI-siRNA incorporating glycosaminoglycan as a charge neutralizer and human serum albumin as co-complexing agent is demonstrated. In addition to possessing good cytocompatibility and excellent cytosolic stability; polyplexes incorporating GAGs also showed altered endocytic uptake, with incorporation of hyaluronic acid promoting caveolae-mediated entry. Our system highlights the importance of physiologically derived macromolecules in delivery of genetic material into hMSCs.
近年来，间充质干细胞（MSCs）在免疫调节、抗炎过程和旁分泌信号传递中发挥作用的证据越来越多，这是通过分泌细胞外囊泡实现的。为了利用这些生物学功能，高效地将遗传物质递送到 MSCs 中的系统将是非常理想的。在这项研究中，通过结合高 N/P 比的 siRNA 和支化聚乙烯亚胺（bPEI）多聚物（siRNA-bPEI）多聚物与糖胺聚糖（GAGs），即透明质酸（HA）、硫酸软骨素（CS）和硫酸肝素（HS）以及人血清白蛋白（HSA），实现了 GFP 表达的高效沉默。这些四元系统的特性是表面电荷、大小和形态，应用于 MSCs，由于其通常较低的转染效率，因此具有挑战性。这些四元多聚物在细胞质中促进了有效的电荷屏蔽和 siRNA 的释放，同时降低了毒性。通过减少细胞毒性，实现了>90%的高效沉默效率（即 GFP 表达量减少到 10%以下），特别是用 HA 和 HA+ HSA 修饰的 siRNA-bPEI 多聚物。一般来说，添加 GAGs 会导致多聚物更加紧凑。内吞研究表明，在高 N/P 比下，内体逃逸的改善是高转染效率的原因之一，透明质酸在摄取机制中可能通过 CD44 相互作用发挥作用。共定位研究表明，多聚物在细胞质中随时间的推移保持稳定，这与 GFP 的适当解组装和随后的沉默有关。此外，含有 GAG 的多聚物经常与核共定位。这些发现表明，基于 PEI/HSA/GAG 的四元多聚物作为 MSCs 的转染剂具有很大的潜力。
由于间充质干细胞（MSCs）被招募到组织修复部位，并在免疫调节、抗炎过程和旁分泌信号传递中发挥作用，因此它们是基因工程的理想靶点。然而，将遗传物质递送到 MSCs 中是具有挑战性的。在这项研究中，使用高 N/P 比的支化聚乙烯亚胺-siRNA 多聚物，其中包含糖胺聚糖作为电荷中和剂，人血清白蛋白作为共复合剂，实现了人骨髓间充质干细胞（hMSCs）中组成型绿色荧光蛋白表达的>97%沉默。除了具有良好的细胞相容性和优异的细胞浆稳定性外；还表明，包含 GAG 的多聚物还表现出改变的内吞摄取，透明质酸的掺入促进了小窝介导的进入。我们的系统强调了生理衍生的大分子在将遗传物质递送到 hMSCs 中的重要性。
