载药型纳米层片状HAp/PLGA骨组织工程支架的构建、双药可控释放及其功能响应研究

Bone tumor and the corresponding bone defect are one of the most difficult diseases to tackle. The drug-loaded tissue engineering scaffold is believed a promising method for the treatment of this kind of disease. Based upon the results obtained from the last project supported by the Natural Science Foundation of China, a novel dual-drug-loaded tissue engineering scaffold is proposed. The scaffold will take full advantages of the characteristics of nano-lamellar hydroxyapatite (L-HAp) by simultaneously carrying a gene drug (small interfere RNA, siRNA) and a chemical drug (doxorubicin, DOX). The siRNA will be intercalated into the gallery of L-HAp and the DOX will be immobilized on the external surface of L-HAp and the dual-drug loaded L-HAp will be incorporated into the electrospun nanofibers of poly(lactic-co-glycolic acid) (PLGA). The strong interaction between L-HAp and tumor cells will be employed to allow the fragile siRNA to break the cell membrane, which is almost impossible for conventional drug carrier. Furthermore, the fact that drug in the gallery of L-HAp can be released at moderate pH will be tactfully used to trigger release of siRNA in intracellular target site and the endosome bursts caused by dissolved calcium and phosphate ions result in endosome escape and timely releases the siRNA and DOX into suitable site (cytoplasm). In addition to the preparation of drug-loaded carrier, the project will focus on the investigation on the carrying, delivery, protection, and release of dual drugs and their affects on the bone regeneration and anticancer behavior of the drug-loaded scaffold. The findings of the project will find a new way to design a drug-loaded scaffold with ‘high-dose’ drug loading, oriented delivery, effective protection, and timely and accurately release. Moreover, this project will provide scientific criterion and technological support for the preparation of the drug-loaded scaffold with desirable bone regeneration and effective anticancer properties.

骨肿瘤及伴生的骨缺损治疗是临床所面临的重大难题之一，载药型组织工程支架的出现为该类疾病的治疗带来了曙光。本项目在总结上一个国家基金项目的基础上，拟充分发挥纳米层片状羟基磷灰石（L-HAp）的特性，率先提出构建载双药（基因药siRNA插层于L-HAp层间，化学药阿霉素负载于层外，静电纺丝将载双药L-HAp掺入PLGA纳米纤维中）L-HAp/PLGA骨组织工程支架的新思想。本项目拟充分利用L-HAp与肿瘤细胞亲和力极强的特点突破大分子siRNA难以穿越细胞膜的瓶颈，并巧妙地利用L-HAp层间药物释放具备pH响应以及HAp降解产物使内涵体破裂等特性实现其适时与适地的可控释放。在完成载药支架制备科学的基础上，重点研究载药支架的双药装载、递送与释放行为及其对骨修复和抗肿瘤功能的调控机制，为双药的高量携带、递送、保护、可控释放提供新的途径，为兼具骨修复与抗肿瘤功能支架的制备提供新的科学依据与技术支撑。

骨肿瘤及伴生的骨缺损治疗是临床所面临的重大难题之一，载药型组织工程支架的出现为该类疾病的治疗带来了曙光。本项目选择了阿霉素（DOX）和siRNA分别为模型化学药和基因药，以广泛应用于载体领域的聚乳酸-羟基乙酸共聚物（PLGA）为纳米纤维的基体材料。为充分发挥纳米层片状羟基磷灰石（LHAp）的特性，本项目通过插层法使siRNA装载于LHAp的层间；通过LHAp表面与DOX的键合作用将其固定于LHAp的外表面，最后通过静电纺丝技术将携带DOX和siRNA的LHAp掺入PLGA纳米纤维中，由此制备了一种新型的载药型骨组织工程支架。这种新型载药支架具备众多独特的优势：首先，由于采用静电纺丝法制备，这种载药型骨组织工程支架可很好地模拟天然细胞外基质，并含有高生物活性的LHAp。其次，由于DOX和siRNA分别负载于LHAp的外表面和层间，通过LHAp几何结构和药物负载量等参数的控制实现了两类不同药物的可控释放。本项目首先采用模板技术制备了LHAp，并采用插层技术将siRNA插层于LHAp的层间，随后，将DOX负载到LHAp的外表面，最终利用静电纺丝法制备了PLGA/LHAp/DOX/siRNA载药支架。结果显示，LHAp对siRNA具有抗酶解保护。体外药物释放结果显示，载双药支架可以实现对DOX和siRNA的可控释放。抗肿瘤实验证实，该支架具有优异的抗肿瘤功能。动物体内实验证实，该支架在体内同样具备抗肿瘤和促进成骨的双重功能。本项目揭示了载体几何学特性与载药量及药物释放的内在联系以及双药协同抗肿瘤的机制，其结果可为临床提供一种兼具骨修复与抗肿瘤功能的新型载药型骨组织工程支架。