Department of Controlled Drug Delivery, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
Department of Developmental BioEngineering, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands; Department of Tissue Regeneration, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
J Control Release. 2015 May 10;205:181-9. doi: 10.1016/j.jconrel.2015.01.034. Epub 2015 Jan 28.
Two linear poly(amido amine)s, pCABOL and pCHIS, prepared by polyaddition of cystamine bisacrylamide (C) with 4-aminobutanol (ABOL) or histamine (HIS), were explored to form alternating multilayer thin films with DNA to obtain functionalized materials with transfection capacity in 2D and 3D. Therefore, COS-7 cells were cultured on top of multilayer films formed by layer-by-layer dipcoating of these polymers with GFP-encoded pDNA, and the effect of the number of layers and cell seeding density on the transfection efficiency was evaluated. Multilayer films with pCABOL were found to be superior to pCHIS in facilitating transfection, which was attributed to higher incorporation of pDNA and release of the transfection agent. High amounts of transfected cells were obtained on pCABOL films, correlating proportionally over a wide range with seeding density. Optimal transfection efficiency was obtained with pCABOL films composed of 10 bilayers. Further increase in the number of bilayers only marginally increased transfection efficiency. Using the optimal multilayer and cell seeding conditions, pCABOL multilayers were fabricated on poly(ε-caprolactone) (PCL), heparinized PCL (PCL-HEP), and poly(lactic acid) (PLA) disks as examples of common biomedical supports. The multilayers were found to completely mask the properties of the original substrates, with significant improvement in cell adhesion, which is especially pronounced for PCL and PLA disks. With all these substrates, transfection efficiency was found to be in the range of 25-50% transfected cells. The pCABOL/pDNA multilayer films can also conveniently add transfection capability to 3D scaffolds. Significant improvement in cell adhesion was observed after multilayer coating of 3D-plotted fibers of PCL (with and without an additional covalent heparin layer), especially for the PCL scaffold without heparin layer and transfection was observed on both 3D PCL and PCL-HEP scaffolds. These results show that layer-by-layer dip-coating of pCABOL with functional DNA is an easy and inexpensive method to introduce transfection capability to biomaterials of any nature and shape, which can be beneficially used in various biomedical and tissue engineering applications.
两种线性聚（酰胺胺），pCABOL 和 pCHIS，通过胱胺双丙烯酰胺（C）与 4-氨基丁醇（ABOL）或组氨酸（HIS）的加成聚合制备，与 DNA 交替形成多层薄膜，以获得具有转染能力的二维和三维功能化材料。因此，将 COS-7 细胞培养在这些聚合物与 GFP 编码的 pDNA 层层浸泡形成的多层薄膜顶部，并评估层数和细胞接种密度对转染效率的影响。发现 pCABOL 的多层膜比 pCHIS 更有利于转染，这归因于更高的 pDNA 掺入和转染剂的释放。在 pCABOL 膜上获得了大量转染的细胞，与接种密度成正比，范围很广。用 10 个双层组成的 pCABOL 膜获得了最佳的转染效率。进一步增加双层的数量仅略微增加转染效率。使用最佳的多层和细胞接种条件，将 pCABOL 多层膜构建在聚（己内酯）（PCL）、肝素化 PCL（PCL-HEP）和聚乳酸（PLA）盘上，作为常见生物医学支架的实例。发现这些多层膜完全掩盖了原始基底的性质，细胞粘附得到了显著改善，对于 PCL 和 PLA 盘尤其明显。在所有这些基底上，转染效率在 25-50%转染细胞的范围内。pCABOL/pDNA 多层膜还可以方便地为 3D 支架添加转染能力。在 PCL（带有和不带有额外的共价肝素层）的 3D 绘图纤维上进行多层涂层后，观察到细胞粘附的显著改善，特别是对于没有肝素层的 PCL 支架，并且在 3D PCL 和 PCL-HEP 支架上都观察到转染。这些结果表明，用功能 DNA 进行 pCABOL 的层层浸涂是一种简单且经济的方法，可以将转染能力引入任何性质和形状的生物材料中，这在各种生物医学和组织工程应用中都将是有益的。
