多层聚丙交酯共聚物/壳聚糖电纺膜定位负载microRNA的血管再生活性材料

Cardiovascular diseases seriously threaten human's health and create an important demand for small-diameter (<6mm) vascular grafts. Due to the limitation of autografts and allografts, development of tissue engineering vascular grafts is a promising solution. Although many relevant studies on small-diameter vascular scaffolds have been done, it is still difficult to keep long-term patency, because restenosis usually happens after implantation. One of the most important factors causing restenosis is intimal hyperplasia by the excessive proliferation of vascular smooth muscle cells (SMCs). Besides, rapid endothelialization is also important. Therefore, it is necessary to promote endothelialization and inhibit SMC hyperproliferation during the regeneration of blood vessels. In this project, we propose a novel method to prepare bioactive small-diameter vascular grafts by loading microRNAs to regulate vascular regeneration. It was reported that angiogenic growth factors such as vascular endothelial growth factor (VEGF) modulate vascular endothelial cell (EC) proliferation, migration and adhesion. MicroRNA-126 (miR-126) could inhibit production of endogenous VEGF repressors such as Spred-1 within ECs and promote VEGF signaling. On the other hand, SMC excessive proliferation is modulated by many cytokines, and one of them is platelet-derived growth factor-bb (PDGF). It was reported that miR-145 could inhibit PDGF-induced SMC proliferation and migration through targeting KLF5. In addition, the peptide sequences Arg-Glu-Asp-Val (REDV) and Val-Ala-Pro-Gly (VAPG) are known to bind preferentially to ECs and SMCs, respectively. Thus, multi-layered (2 or 3) electrospun membranes or tubes consisted of poly(ethylene glycol)-b-poly(L-lactide-co-ε-caprolactone) and other related copolymers will be developed, while chitosan nanoparticles loading microRNAs (miR-126 and miR-145) will be encapsulated in inner and middle layers (or outer of double-layers) of the multilayered electrospun fibers, respectively for spatio-temporal control of EC and SMC growth. Also, chitosan nanoparticles, the inner and middle layers will be surface-functionalized by targeting peptides (REDV and VAPG) to promote binding to ECs and SMCs, respectively. The microRNA encapsulation and transfection efficiency will be controlled by the chitosan nanoparticle physiochemical properties and the morphology of electrospun membranes. The spatio-temporal modulating mechanism of EC and SMC growth, the encapsulation approaches of chitosan nanoparticles within electrospun membranes and the release behaviors under biomechanical stimulation will be focused on. In vivo replacement experiments in an animal model using the small-diameter vascular grafts will be carried out to evaluate the in situ regeneration of the vascular tissue. The investigation of bioactive vascular grafts based on microRNAs will provide new strategies and fundamental theories for the small-diameter vascular reconstruction.

心血管疾病是人类常见的重大疾患之一，直径<6mm的小口径人工血管材料虽已研究多年，但移植后仍会出现再狭窄等问题，这主要与血管平滑肌细胞(SMCs)过度增殖有关。本项目拟从调控血管再生的microRNA出发，构建新型血管再生活性材料。以壳聚糖偶联选择性多肽(REDV或VAPG)为载体包载microRNA成纳米粒子，并负载于以聚乙二醇-b-聚(丙交酯-co-己内酯)等为原料、由同轴电纺等方法制备的多层超细纤维膜(管)中。使内层和中层分别定位负载miR-126和miR-145，以定时定位调控血管内皮细胞(ECs)和SMCs，促进血管再生初期快速内皮化、中期稳定性和长期维持血管通畅的能力。重点研究壳聚糖载体及多层电纺膜对miRNA的负载方法、生物力学环境下的释放行为和控制因素，以及体外和体内定位、定时调控ECs和SMCs及相互作用的影响机制。为构建新型原位诱导血管再生材料提供新思路和理论基础。

血栓、内膜增生和钙化等不良症状是制约小口径血管再生的主要因素。本项目将微小核酸(miRNAs)与电纺纤维膜相结合，制备了新型生物活性小口径人工血管材料。在电纺纤维中载入miRNA-126和/或miRNA-145，调控血管内皮细胞(VECs)和血管平滑肌细胞(VSMCs)的生理功能，以促进快速内皮化，并抑制VSMCs过度增殖。.制备了三甲基化壳聚糖偶联选择性小肽REDV和VAPG的载体TMC-g-PEG-REDV (TPR)、TMC-g-PEG-VAPG (TPV)和葡聚糖-g-聚赖氨酸偶联VAPG (DPV)，分别用于复合miRNA-126和miRNA-145，载入电纺纤维膜，分别定向调控VECs和VSMCs。以聚乙二醇-b-聚(L-丙交酯-co-ε-己内酯) (PELCL)、聚(L-丙交酯-co-乙交酯) (PLGA)和聚(ε-己内酯) (PCL)等为主要原料，由乳液电纺方法制备了双层或三层超细纤维膜和小口径人工血管，内层和外层(或中层)分别负载miRNA-126和miRNA-145，进行了体外释放、细胞培养和体内动物试验研究。.以负载TPR/miRNA-126的PELCL与PCL/明胶制备了双层电纺纤维膜，表明miRNA-126可保持活性；以PELCL为内层，在外层PLGA中负载DPV/miRNA-145制备的双层人工血管，可使VSMCs正常表达收缩表型。以PELCL/PEG电纺纤维负载TPR/miRNA-126为内层、微米直径PLGA电纺纤维负载TPV/miRNA-145为外层制备的双层人工血管材料，具有较快释放miRNA-126和较慢释放miRNA-145的作用，可促进血管再生。以PELCL/PELCL-REDV为内层负载TPR/miRNA-126、中层为PELCL负载TPV/miRNA-145、外层为PCL制备的三层电纺纤维膜人工血管，进一步表明上述两种miRNAs分别对VECs和VSMCs具有调控作用，可促进内皮化，抑制内膜增生，同时还可调控巨噬细胞表型，减少血管再生过程中的炎症和钙化现象。因此，负载miRNA-126和/或miRNA-145的生物活性多层电纺纤维膜可为小口径人工血管的制备提供新方法。.在此项目资助下，已在Acta Biomater.、J. Mater. Chem. B等期刊发表研究论文11篇，及1篇综述，申请国家发明专利9项，授权1项。

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