骨靶向性抗结核缓释微球的制备及体内外评价

Bone tuberculosis is a common extra-pulmonary tuberculosis. It is difficult to cure for its long course, high disability and recurrence rate. Due to the necrosis, encapsulation and calcification of lesion tissue, it is uneasy to reach the effective concentration by conventional drugs and usual methods, and the adequate dosage and duration of the drug combination increase the toxicity and side effects. In order to solve the above problems, Rifapentine polylactic acid sustained-release microspheres had been successfully prepared and patented in the early stage. It has been proved that the minimum therapeutic dose preventing the recurrence in the lesion is 15mg, and the minimum inhibitory concentration lasting for 60 days. In this study, bone targeting molecules, such as Bisphosphonates, Oligopeptides, Tetracyclines and Poly malonates linkaged with Polylactic acid, encapsulate Rifapentine to develop a new non-oral anti-tuberculosis microsphere with the characters of bone targeting and sustained release. The drug targeting, the therapeutic effect and the lowest therapeutic dosage are evaluated by the targeting efficiency, pharmacokinetics and drug distribution experiments in vivo and in vitro. It is expected that the bone targeting Rifampentine sustained-release microspheres can enhance the drug concentration in the lesion while decrease the drug concentration in non-bone tissue, reduce the side effects and drug resistance, lengthen the interval time between dosing to improve patients’compliance and lower the recurrence rate, and increase the cure rate of bone tuberculosis.

骨结核病是常见肺外结核病，致残率和复发率高、病程长且难以治愈。因病灶组织坏死、包裹、钙化，常规药物及给药途径很难在骨组织中达到有效治疗浓度，而足疗程的联合用药又增加了毒副作用。为解决上述问题，前期已制备利福喷丁聚乳酸缓释微球（获专利）和兔脊柱结核模型，并证实在病灶局部预防复发的最低有效治疗剂量为15mg，维持最低抑菌浓度达60天。本研究在前期基础上，选择具有骨靶向性的分子：双膦酸盐、寡肽、四环素、聚丙二酸类与聚乳酸键合形成新膜材包裹利福喷丁，制备具有骨靶向和缓释双重作用的非口服抗结核微球。结合体内外靶向性实验、药代动力学及药物组织分布实验分析、评估药物靶向性及治疗效果、探索药物最低有效治疗剂量。期望此具有骨靶向性利福喷丁缓释微球能在提高病灶局部药物浓度的同时，降低非骨组织药物浓度以减少毒副作用和耐药性，进而延长给药时间间隔，增加病人依从性，减少复发并提高骨结核病的治愈率。

目的：基于利福喷丁（RPT）、乳化技术、聚乳酸-羟基乙酸（PLGA）类高分子聚合物及骨靶向的羟基磷灰石/磷酸三钙和四环素，构建可供局部植骨/术中应用的利福喷丁递药系统（RPT/Bone-graft DDS）和可供外周给药靶向到骨/术前或术后应用的利福喷丁递药系统（RPT/Bone-target DDS），并通过体内外实验对所制备的递药系统进行评价。方法：采用SPG膜乳化技术制备聚乳酸微球包载利福喷丁，与羟基磷灰石/磷酸三钙支架复合构建RPT/Bone-graft DDS，并利用紫外及红外光谱的检测方法确定共聚物的成分；再采用超声乳化技术包载利福喷丁制备RPT/Bone-target DDS，并测量其粒径、表面电位、多分散系数，观察其表观特征。分别评估PLGA利福喷丁微球及骨靶向纳米粒的包封率、载药量、体外释放及体外抑菌情况。分离、培养大鼠rBMSCs并对其进行鉴定，检测利福喷丁微球、纳米粒及支架的细胞毒性。建立大鼠股骨外侧髁骨缺损模型，植入RPT/Bone-graft DDS，检测RPT/Bone-graft DDS在大鼠股骨髁内的释放情况；采用小动物活体成像仪检测RPT/Bone-target DDS小鼠给药后的骨靶向性能。结果：（1）利福喷丁微球粒径约为36.63 μm，RPT/Bone-target DDS表面带负电，平均粒径为48.83 nm，在紫外检测条件下于477 nm及336 nm处有最大吸收峰，两处波长均可计算标准曲线方程用于浓度检测，且336 nm处具有更低的检测下限；测得利福喷丁微球的包封率为70.28%，载药量为36.12%，体外释放初始阶段呈爆发释放，第40天累积释放78.3%；体外抑菌实验表明于局部植骨系统中释放出的利福喷丁其抑菌能力与利福喷丁原药一致。（2）RPT/Bone-target DDS的载药量为8.1%，包封率为76.3%，体外释放于6小时内呈现爆发释放，随后64小时缓慢释放；最低抑菌药物浓度为0.094 μg/mL，低于游离药物；体外骨靶向骨亲和能力为60.80%，高于非骨靶向递药系统。结论：RPT/Bone-graft DDS及RPT/Bone-target DDS可以缓慢释放利福喷丁，有良好的缓释性能和生物相容性，具有明显的骨靶向。