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首页> 外文会议>World biomaterials congress >Silica-collagen nanocomposites for local and sustained release of Interleukin 10 (IL-10) to treat cutaneous chronic wounds
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Silica-collagen nanocomposites for local and sustained release of Interleukin 10 (IL-10) to treat cutaneous chronic wounds

机译：硅胶原纳米复合材料，用于局部和持续释放白介素10（IL-10），用于治疗皮肤慢性伤口

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Cutaneous chronic wounds are characterized by the absence of healing six weeks after the injury. The classic treatment is the debridement of the wound bed followed by a compression method. When this technique is not effective enough, the application of wound dressings is required. Several hulman trials with protein therapy have failed because of the rapid diffusion and short half-life of the cytokines in the wound. Therefore gene therapy represents an interesting alternative. Local delivery of nucleic acids through a hydrogel extends the application of gene therapy for the treatment of chronic wounds. However, the inactivation and fast release of encapsulated DNA strongly compromise their efficacy. Nanoscale delivery systems such as silica nanoparticles (SiNPs) hold great promise for gene therapy owing to their high cellular uptake, DNA protection and possible cell targeting. In this study, nanocomposites consisting of plasmid DNA-PEI-SiNP complexes, collagen hydrogel and 3T3 mouse fibroblasts have been evaluated as gene delivery systems to obtain a local and sustained release of biomolecules. Plasmid DNA encoding for Interleukin 10 (IL-10) was chosen as this cytokine is able to modulate inflammation of chronic wounds. SiNPs were modified with PEI (polyethyleneimine) of different molecular weights (1.8 kDa, 10 kDa and 25 kDa) by electrostatic absorption. SiNP-PEI complexes were associated with plasmids encoding for Luciferase or human IL-10 at a weight ratio of 30:1. Then, complexes were encapsulated within collagen hydrogel with 3T3 mouse fibroblasts. Cell transfection was assessed by measuring the luciferase activity or the IL-10 production. Cell viability was evaluated by Alamar Blue Assay. Subsequently, the effect of nanocomposites to modulate inflammation was evaluated on activated mouse RAW 264.7 macrophages. The ability of IL-10 doses released from nanocomposites to downregulate the TNF-α expression in macrophages was analyzed over one week. Successful cell transfection within the collagen/silica nanocomposites was detected by sustained release of luciferase over one week. The best transfection efficiency was obtained with PE1 10KDa (Figure 1). When compared with the soluble form of PEI 25 KDa, the transfection was lower. However, the incubation with increasing quantities of soluble PEI or complexed with SINP revealed a cytotoxicity of PEI 25 KDa and 10 KDa from 10 μg/mL (Figure 1). In contrast, PEI was not toxic until the 100 μg/mL when associated with silica nanoparticles. Interestingly, SiNP-PEI complexes were confined within the collagen network as no transfection was possible out of the nanocomposites. Finally, SiNP-PEI complexes were confined within the collagen network as no transfection was possible out of the nanocomposites. Tested on activated macrophages, nanocomposites permitted the sustained release of IL-10 (500 pg/mL) which could inhibit the expression of TNF-α by 50% in macrophages (Figure 2). These results show that nanocomposites obtained by entrapping SiNP-PEI-DNA together with cells inside a collagen hydrogel can work as a reservoir for local and controlled gene delivery. In addition, it is possible to create cell factories producing high doses of IL-10 which modulate inflammation. These biomaterials are promising for the local treatment of cutaneous chronic wounds.
机译：皮肤慢性伤口的特征是受伤后六周没有愈合。经典的治疗方法是对伤口床进行清创术，然后采用加压方法。当该技术不够有效时，就需要使用伤口敷料。由于伤口中细胞因子的快速扩散和半衰期短，一些蛋白质疗法的霍尔曼试验失败了。因此，基因治疗代表了一种有趣的选择。通过水凝胶的核酸局部递送扩展了基因疗法在治疗慢性伤口中的应用。但是，被包封的DNA的失活和快速释放强烈损害了它们的功效。诸如二氧化硅纳米颗粒（SiNPs）之类的纳米级递送系统因其对细胞的高摄取，DNA保护和可能的细胞靶向而具有广阔的基因治疗前景。在这项研究中，由质粒DNA-PEI-SiNP复合物，胶原蛋白水凝胶和3T3小鼠成纤维细胞组成的纳米复合物已被评估为基因传递系统，以获取生物分子的局部和持续释放。选择编码白介素10（IL-10）的质粒DNA，因为该细胞因子能够调节慢性伤口的炎症。通过静电吸收，用不同分子量（1.8 kDa，10 kDa和25 kDa）的PEI（聚乙烯亚胺）修饰SiNP。 SiNP-PEI复合物与编码萤光素酶或人IL-10的质粒以30：1的重量比结合。然后，将复合物与3T3小鼠成纤维细胞一起包封在胶原蛋白水凝胶中。通过测量荧光素酶活性或IL-10产生来评估细胞转染。通过Alamar Blue Assay评估细胞活力。随后，在活化的小鼠RAW 264.7巨噬细胞上评估了纳米复合材料调节炎症的作用。在一周内分析了纳米复合物释放的IL-10剂量下调巨噬细胞中TNF-α表达的能力。通过持续一周以上的萤光素酶的持续释放，可以检测到胶原蛋白/二氧化硅纳米复合材料内成功的细胞转染。 PE1 10KDa可获得最佳转染效率（图1）。与PEI 25 KDa的可溶形式相比，转染率较低。但是，与增加量的可溶性PEI或与SINP配合使用的孵育显示，PEI从10μg/ mL起具有25 KDa和10 KDa的PEI的细胞毒性（图1）。相反，PEI与二氧化硅纳米颗粒缔合后，直到100μg/ mL才具有毒性。有趣的是，SiNP-PEI复合物被限制在胶原蛋白网络内，因为不可能从纳米复合物中转染。最后，SiNP-PEI复合物被限制在胶原蛋白网络内，因为不可能从纳米复合物中转染。在活化的巨噬细胞上进行测试，纳米复合材料允许IL-10（500 pg / mL）的持续释放，从而可以在巨噬细胞中抑制TNF-α的表达达50％（图2）。这些结果表明，通过将SiNP-PEI-DNA与胶原蛋白水凝胶中的细胞包埋而获得的纳米复合材料可以充当局部和受控基因传递的储存库。另外，有可能建立产生高剂量调节炎症的IL-10的细胞工厂。这些生物材料有望用于皮肤慢性伤口的局部治疗。

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