骨科钛植入物表面基于结核宿主细胞响应释放与自噬调控的水凝胶药物递送系统研究

Anti-tuberculosis chemotherapy is the mainstay for treatment of spinal tuberculosis which could lead to severe vertebrae destruction, instrumentation is usually indicated to reconstruct the spinal stability. In addition to explore new types of anti-tuberculosis drugs and drug delivery systems, the host directed therapy based on autophagy regulation for Mtb infected macrophage has become one of researching hotspots in the meantime. Multiple studies have focused on bionic bone scaffolds for anti-tuberculosis drug delivery in recent years. However, the strategy of drug release coating fabricated on titanium substrates for anti-tuberculosis and autophagy regulation has not been investigated. Based on the microenviroment of spinal tuberculosis and previous technical system, “Macrophage targeting, smart pH responsive and INH/RMP/RAPA combined delivery” PLGA nanoparticles would be fabricated to PDA-PEG-PLGA coating on Ti substrate in this study. We assume that, the novel DDS could achieve macrophage targeted and controlled release of PLGA nanoparticles to tuberculous focus. Based on the multi-model nano drug delivery technology for antimicrobial and host-directed therapy, the DDS could increase the intra-cellular concentrations of INH and RMP. Moreover, the targeting delivery of RAPA to macrophage based on “mTOR dependent autophagy induction” may have the synergetic effect with antimicrobial drugs delivery. The proof-of-concept study would be performed through characterization of coating materials, drug release profiles, Mtb inhibition test and evaluation of autophage induction effect. This study could provide theoretical and technical supports for the fabrication of an optimal DDS for spinal tuberculosis.

脊柱结核严重破坏骨质结构，在全身抗结核化疗基础上，常需使用内植物重建脊柱稳定性。随着对结核病发病进程深入研究，除了继续发展新型抗结核药物或药物递送方式外，针对结核宿主细胞-巨噬细胞的自噬免疫调节也成为研究重点。目前已有多项针对脊柱结核局部药物递送的研究，但尚无在钛内植物表面负载抗结核或自噬调节药物的策略出现。基于病灶微环境与前期工作，本研究拟设计巨噬细胞靶向、pH响应、联合载药PLGA纳米粒子，将其复合于钛表面PEG水凝胶层中，以期实现载药纳米粒子在病灶内控释与巨噬细胞精确递送效应。基于多模态纳米药物递送平台的抗微生物和免疫调节作用，一方面提高巨噬细胞内异烟肼和利福平的浓度；另一方面基于mTOR依赖途径，通过雷帕霉素靶向递送以诱导巨噬细胞自噬，最终实现两种策略对胞内寄生菌的协同杀灭作用。通过材料表征、释药行为评价、抑菌试验、自噬诱导评价等系列试验，为脊柱结核局部药物治疗提供新思路与新技术。

结核病诊疗技术不断进步，但彻底控制该传染病仍十分困难。即便患者完成抗结核治疗，之后仍面临复发风险，尤其是免疫缺陷患者或合并症较多的患者。导致结核分枝杆菌不能彻底杀灭或休眠的主要原因是细菌侵入体内后被巨噬细胞吞噬，一方面可通过细胞凋亡、自噬等途径被消化清除，另一方面可通过抑制吞噬溶酶体融合而逃避免疫杀伤，进而形成结核性肉芽肿，既可控制感染，也为结核菌生存创造了适宜微环境。另外，多数抗结核药物细胞膜渗透性差、体内半衰期短，无法有效到达巨噬细胞内部并持续作用。本研究目的是设计一种能够识别结核分枝杆菌并在其表面实现自组装的多肽纳米材料，其包含三个功能单元：颗粒单元、纤维单元和识别单元。其中颗粒单元为芳香族双芘（Bis-Pyrene，BP），由于它的强疏水作用，可使多肽链在极性溶剂中自组装形成纳米颗粒，此外，BP的聚集诱导发光（Aggregation induced emission，AIE）效应引起的荧光信号可实现检测全身给药纳米材料的位置。纤维单元为KLVFF序列，是β-折叠纤维结构的肽骨架。识别单元为HPTGSHWKC序列，其可靶向结合卡介苗（Bacillus Calmette-Guérin，BCG）。通过自组装成纠缠的纤维网络，捕获结核分枝杆菌并阻止其入侵宿主巨噬细胞。实验方法是首先合成了具有颗粒单元、纤维单元和识别单元的多肽纳米材料。通过透射电子显微镜（Transmission Electron Microscope，TEM）和荧光光谱（Fluorescence，FL）观察其形貌表征。纳米材料与BCG共孵育，通过激光共聚焦显微镜（Confocal laser scanning microscope，CLSM）、扫描电子显微镜（Scanning Electron Microscope，SEM）观察纳米材料对BCG的靶向功能及在BCG表面的自组装行为。纳米材料、BCG和巨噬细胞共孵育，观察纳米材料对巨噬细胞吞噬BCG的影响。实验结果显示由BP诱导的纳米材料在水溶液中可自组装形成纳米颗粒，4小时之后可进一步形成纤维结构。纳米材料与BCG孵育后能够成功识别、捕获BCG，并在其表面自组装成纳米纤维，且能有效抑制BCG入侵巨噬细胞。我们得出结论，多肽纳米材料能成功抵御BCG进入巨噬细胞，进而抑制结核肉芽肿形成，使化疗药物更有效、更彻底杀灭结核分枝杆菌成为可能。