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首页> 外文期刊>Acta biomaterialia >Electrostatic immobilization of DNA polyplexes on small intestinal submucosa for tissue substrate-mediated transfection.
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Electrostatic immobilization of DNA polyplexes on small intestinal submucosa for tissue substrate-mediated transfection.

机译：小肠粘膜下层的DNA多链体的静电固定，用于组织底物介导的转染。

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摘要

Naturally occurring extracellular matrices (ECMs) such as small intestinal submucosa (SIS) have received significant attention for their therapeutic applications in tissue repair and regeneration. However, there have been no reports exploring the electrostatic properties of naturally occurring ECMs as a means to control transgene delivery. In the present study, we electrostatically adsorbed DNA polyplexes onto SIS for transfection upon cellular adhesion. To associate polyplexes with SIS, we first used a streaming potential method to characterize the surface charge of SIS and obtained a negative zeta potential at neutral pH, which can be attributed to the abundant glycosaminoglycan (GAG) content in SIS. We next prepared cationic polyethylenimine (PEI)/DNA polyplexes to associate with the negatively charged SIS for conjugation. Using the Cy(TM)3 dye-labeled control DNA as the reporter, we visualized the adsorption of PEI/DNA polyplexes at the SIS surface. Using luciferase, green fluorescent protein and beta-galactosidase as reporter proteins, we showed that the adsorbed PEI/DNA polyplexes were active and capable of carrying out transfection upon cellular adhesion, indicating that the electrostatic binding of polyplexes with SIS was reversible. In addition, the SIS-mediated transfection was contact-dependent: separation of SIS from the target cells via a 0.5mm porous polyester membrane significantly reduced the efficiency of transfection in comparison to a direct seeding of cells onto SIS. We conclude that electrostatic immobilization of PEI/DNA polyplexes on SIS is capable of initiating efficient transgene delivery, which can be a useful tool in developing localized gene transfer.
机译：天然存在的细胞外基质（ECM），例如小肠粘膜下层（SIS），因其在组织修复和再生中的治疗应用而受到广泛关注。然而，没有报道探索天然存在的ECM作为控制转基因传递的手段的静电特性。在本研究中，我们将DNA多链体静电吸附到SIS上，以便在细胞粘附后进行转染。要将多链体与SIS关联，我们首先使用流式电势方法表征SIS的表面电荷，并在中性pH值下获得负的ζ电势，这可以归因于SIS中丰富的糖胺聚糖（GAG）含量。接下来，我们制备了阳离子聚乙烯亚胺（PEI）/ DNA复合物，以与带负电的SIS结合进行缀合。使用CyTM3染料标记的对照DNA作为报告基因，我们观察到PEI / DNA多聚体在SIS表面的吸附。使用萤光素酶，绿色荧光蛋白和β-半乳糖苷酶作为报告蛋白，我们表明吸附的PEI / DNA多聚体是活跃的，并且能够在细胞粘附后进行转染，表明多聚体与SIS的静电结合是可逆的。此外，SIS介导的转染是接触依赖性的：通过将细胞从0.5mm多孔聚酯膜上分离出SIS，与直接将细胞直接接种到SIS上相比，显着降低了转染效率。我们得出结论，PEI / DNA多链体在SIS上的静电固定能够启动有效的转基因传递，这可能是开发局部基因转移的有用工具。

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