双重基质金属酶响应性智能材料递送miRNA再生椎间盘

The chronic diseases such as intervertebral disc degeneration (IDD) frequently start from a long-term and gradual pathological changes inside tissues. Excessive matrix metalloproteinases (MMPs) contributes to adverse tissue remodeling during this process. Sustainable and on-demand microRNA delivery to inhibit the process is presumably a promising treatment strategy. Herein, toward this aim, we explore to develop matrix metalloproteinase (MMP)-responsive hydrogels as the reservoir for on-demand release of the miRNA polyplexes to realize a long-term treatment considering the significantly overexpressed MMPs during IDD. Specifically, the MMP-cleavable cationic block copolymer, poly(ethylene glycol)-GPLGVRG-poly{N´-[N-(2-aminoethyl)-2-aminoehtyl] aspartamide}-cholesteryl (PGPC), was designed to complex miRNA as a nanocarrier via formation of polyplex micelles, which were further encapsulated into the MMP-responsive PEG hydrogels inside discs with an injectable manner. The novel smart polyplex micelles in responsive hydrogels (miRNA/PGPC@HG) showed two-stage delivery of miRNA into nucleus pulposus cells for sustainable and on-demand silence of MMP. Polymer/miRNA polyplexes micelles were first released from PEG hydrogels under the cleavage of MMP and distributed in the target tissues; and subsequently, detachment of PEG shells from polyplexes micelles by MMP contributes to improved cellular uptake and intracellular delivery of miRNAs. This two-stage bioresponsive local delivery of miRNA is verified to functionally inhibit fibrosis and reverse IDD in a sustained and on-demand fashion, which possesses great potentials for the treatment of IDD.

基质金属蛋白酶（MMPs）表达水平的动态变化是椎间盘退变的关键因素。miRNA为椎间盘再生提供了新的手段，但缺乏作用持久、剂量准确的给药手段。为此，我们设计了双重MMPs响应性智能生物材料：利用嵌段共聚物PEG-GPLGVRG-PAsp(DET)-Chole（PGPC）作为载体，通过水凝胶包裹，其中PGPC和水凝胶均含有可被MMPs特异性剪切的肽段GPLGVRG。该材料可感受环境中的MMPs浓度，智能反馈性释放miRNA:当MMPs浓度大于设定的阈值，水凝胶可逐渐降解释放PGPC和miRNA复合物；随后PGPC被MMPs剪切去掉PEG壳层递送miRNA至细胞内，调节MMPs的表达直至恢复正常水平。我们前期工作基础表明该体系具有靶向性好，两阶段按需释放等特点。本项目将在此基础上进一步探讨通过智能生物材料控释miRNA的规律和分子机理，为椎间盘退变治疗提供新思路。

背景：椎间盘退行性变(IDD)常导致慢性下腰痛，甚至致残，已成为目前社会的巨大健康问题和医疗负担。随着各种生物治疗技术的发展，生长因子治疗、细胞治疗以及基因治疗在内的各种其他治疗方法为我们提供了修复椎间盘的新思路。既往研究表明，IDD与基质金属蛋白酶(MMPs)的表达增加密切相关，过度表达的MMPs导致纤维化侵袭，显著降低了基质的完整性，促进其降解，从而加速了椎间盘的退变并抑制了再生。因此，持续抑制基质降解和纤维化可能是治疗椎间盘退变的有效策略。而目前诸多对于miRNA影响椎间盘退变的研究发现，多种miRNA主要通过作用于细胞凋亡、炎症信号反应、细胞外基质(extracellular matrix，ECM)成分等环节参与影响椎间盘退变。于是课题组拟设计一种新型响应递送系统，通过响应外部MMPs水平的变化智能提送治疗作用的miRNA到退变椎间盘靶点，实现对椎间盘退变的精准治疗。.主要研究内容：课题组拟设计一种新型MMPs响应型 miRNA智能递送系统，通过外部响应炎症MMPs信号，分阶段地精准递送具有治疗作用的miRNA小分子，对椎间盘退变起到治疗作用。.重要结果及关键数据：课题组合成了有巯基的 CGPLGVRGC肽段及八臂-PEG-马来酰亚胺混合的miRNA/PGPC/水凝胶MMPs响应型 miRNA智能递送系统，并验证了其缓释miRNA的效果，在体外及体内证实该缓释载体的有效性及准确性。通过针对mirBase/Targetscan的生物信息学分析，再结合双荧光报告基因测定，证实了miR-29a通过结合MMP-2的mRNA 3'非编码区以直接调节MMP-2的蛋白表达。通过PGPC装载miRNA-29a转染含过量转化生长因子-β1（TGF-β1）刺激纤维化的髓核细胞，验证下游椎间盘退变相关基因（II型胶原，蛋白多糖）以及纤维化相关基因（I型胶原，α-SMA）的表达，证实了该系统能灵敏响应外部MMPs水平并精准递送miR-29a从而一定程度抑制椎间盘纤维化。.科学意义：本项目为椎间盘退变的基因治疗提供一个高效安全的非病毒载体PGPC聚离子复合物，为目前临床基因治疗开拓了新的思路，为治疗椎间盘退变提供新的选择与解决办法。