负载双介孔硅与P65 siRNA/布洛芬复合纳米粒子的PELA电纺纤维膜 防治肌腱粘连的作用及机制研究

Tendon adhesion is a common complication after injury, which seriously affects the limb function. The treatment is troublesome, and the efficacy of many methods is still not precise. We confirmed earlier that P65 small interfering (si) RNA plays the role of anti-adhesion by inhibiting cyclooxygenase (COX) 2, regulating cell growth, but easy to inactivation. Mesoporous silica can supports siRNA activity, but the effect of P65 siRNA on barely COX2 limits its efficacy. Ibuprofen can simultaneously inhibit COX1 and COX2 to prevent tendon adhesion. However, over-inhibition of COX1 can promote hematoma which aggravates adhesion after organization. Using mesoporous silica to load siRNA and ibuprofen with an optimized ratio is expected to achieve a desirable effect. We intend to use the large and small holes of dual-mesoporous silicon for the load of siRNA and ibuprofen, respectively, to make the composite particles, and produce an anti-adhesion membrane with high activity, sustained-release properties by electrospinning the particles with polylactic acid-polyethylene glycol (PELA). Afterwards, we will test the effectiveness of the system to maintain activity and achieve sustained release in vitro and in vivo, and confirm a better efficacy of double-drug combination for tendon adhesion prevention. We will also study the interaction between the two drugs, and discuss the molecular mechanism of combined drug treatment for the prevention and treatment of adhesion by comparing the expression of COXs, nuclear factor-κB and transforming growth factor -β pathways related factors, cell growth and extracellular matrix synthesis in different drug loading groups in order to provide a new way of thinking for tendon adhesion treatment.

肌腱粘连是外伤后常见并发症，它严重影响患肢功能。其治疗棘手，诸多方法疗效仍不确切。我们前期证实：P65小干扰（si）RNA通过抑制环氧化酶（COX）2、调控细胞生长，发挥防粘连作用，但易失活。介孔硅载体可保持其活性，但P65 siRNA仅作用COX2限制了其疗效。布洛芬可同时抑制COX1、COX2防治粘连。但COX1过度抑制可促进血肿，机化后加重粘连。利用双介孔硅以优化比例联载siRNA与布洛芬有望取得理想的疗效。我们拟利用双介孔硅大、小孔分别搭载siRNA与布洛芬制备复合粒子并与聚乳酸-聚乙二醇（PELA）电纺，制成高活性、缓释的防粘连膜，并在体内外验证该体系保持活性、实现缓释的有效性，明确双药联载是否具有更好功效，考察两药相互作用。通过比较不同载药情况下COX、核因子-κB与转化生长因子-β通路相关因子表达、细胞生长和外基质合成的差异，探讨联药治疗的分子机制，为粘连防治提供新的思路。

肌腱粘连是临床常见疾病。由于缺乏有效的防粘连措施，粘连松解术后往往陷入“粘连—松解—再粘连”的恶性循环，为患者家庭及社会带来巨大负担。本研究旨在构建一种新型载药介孔硅生物材料，并在体内外考察其防治肌腱粘连的有效性，通过对比不同载药方式探究其分子机制。为提高材料在临床的可操作性，该研究将原设计中的电纺膜改为水凝胶。水凝胶无需外科手术切开即可微创植入，操作更为便捷。本研究成功合成双孔介孔硅，并实现硅纳米粒子对siRNA的负载（介孔硅-siRNA）。研究中进一步完成水凝胶对介孔硅-siRNA的包覆，并在体内环境中进行初步验证，为双载药体系的构建及其疗效研究提供实验基础。项目组后续将按原技术路线在此基础上继续研究，完成双载药纳米粒子的水凝胶的制备，并在体内外实验中验证其防治肌腱粘连的疗效并考察其机制。此外，在该项目支持下，课题组探究了脂肪干细胞（ASC）细胞片对肌腱修复再生的疗效。通过生长分化因子5 (GDF-5) 体外诱导ASC成肌腱分化，并在体内外实验中考察诱导后ASC细胞片复合纳米纱材料促进肌腱修复再生的可行性与有效性，并探究其相关分子机制。同时，项目组还设计出一种对活性氧和近红外刺激具有双响应性释放、聚多巴胺表面修饰的载药新型杂化纳米粒子(dual-responsive hybrid micelle , DHMP/M)，并检测其稳定性，载药能力及生物相容性；考察其双响应性药物释放，及体内外对ROS 清除效果。以上两项研究为肌腱疾病的治疗提供新的治疗思路及方法。