巨噬细胞靶向性PLGA载药微球的制备及其在抗结核药物靶向递送中的应用研究

Pulmonary tuberculosis(TB) is still a major infectious disease that threats human health. Systemic administration of anti-TB drugs was usually adopted for TB treatment in clinic, which was, however, inefficient in controlled release and targeted delivery of drugs.The PLGA will be employed as the base material in the project, and we will firstly optimize the preparation of PLGA micropheres for rifampicin delivery. Meanwhile, for the surface antigen of alveolar macrophages of F4/80, its dominant epitope will be predicted using BioSun Version software. Our institute owned the independent intellectual property rights of the software.Then, the "integrated dominant epitope antigen " will be prepared by means of genetic engineering. Next, the "integrated dominant epitope antigen "is used to immunize animals for preparation of high-quality F4/80 antibodies. The functional Fab’portion of the F4/80 antibodies is obtained by enzymatic hydrolysis and mild reduction with mercaptoethanol. Then, chitosan with maleimide groups at N end is used to covalently bind sulfhydryl of Fab’fragments, resulting in chitosan-Fab’ conjugates. The microphage-targeted anti-TB drug delivery pharmaceutical preparation will be obtained by coating drug-carrying PLGA micropheres with the chitosan-Fab’ conjugates. Further, the targeting and anti-TB efficiency of the targeted PLGA micropheres will be systematicaly evaluated in BCG infected microphages. The study will provide a novel and efficient strategy of anti-TB drug delivery and is promising in improving the therapeutic index of anti-TB drus, which is of great significance.

肺结核依然是威胁人类健康的重大传染病。临床上常采用的全身系统性给药治疗方式在药物控释、靶向性等方面存在明显不足。本项目拟以PLGA为基体材料，首先优化用于抗结核药物利福平递送的最佳PLGA微球体系；同时，针对肺巨噬细胞表面抗原F4/80，利用我单位自主知识产权的BioSun Version软件预测其优势抗原表位，并通过基因工程手段制备“优势表位集成抗原”，用于免疫动物制备高性能F4/80抗体；进一步，通过酶解和β-巯基乙醇还原获得F4/80抗体功能区Fab’，利用N端携带马来酰亚胺基团的壳聚糖与Fab’的巯基结合获得壳聚糖-Fab’聚合物，用于PLGA微球的表面包被修饰，从而获得巨噬细胞靶向性抗结核药物递送制剂。之后，在BCG感染巨噬细胞中，系统评价这一靶向性PLGA微球的药物递送及抗结核菌效能。本项目将为抗结核药物递送提供一种新型、有效策略，有望显著提高抗结核药物治疗指数，具有重要意义。

肺结核是威胁人类的重大传染病。临床上常采用的全身系统性给药治疗方式存在明显不足。通过改善给药方式，使药物靶向性进入荷菌细胞，有望显著提高治疗效果并降低负效应，具有重要研究意义。本项目深入开展了PLGA载药微球制备新方法、新型抗巨噬细胞标志物F4/80多克隆抗体制备的研究工作，在此基础上探讨了巨噬细胞靶向性PLGA载药微球的制备及抗结核菌实验。执行过程中，改进建立了两种新型的PLGA微球制备方法，大大简便了制备过程，同时提高了微球的均匀性，并获国家发明专利；开展了PLGA微球的生物相容性、携带抗结核药物效能实验，获得了体外缓释效果好、药物携带量较高的微球载体；通过体外向巨噬细胞进行抗结核药物递送实验表明，PLGA载药微球能够较为高效地将利福平递送进入Raw264.7巨噬细胞；为了制备抗F4/80多克隆抗体，通过Biosun软件对抗原免疫表位进行了预测，对这一蛋白的细胞外全序列进行了克隆表达，同时根据生物信息学对免疫表位的预测结果对全长蛋白进行了分段克隆表达，通过多克隆抗体分析了免疫表位在蛋白中的分布情况，揭示了抗体识别的区域主要位于蛋白的N端，为后续抗体制备奠定了基础；在此基础上，获得了针对F4/80蛋白N端免疫表位的抗体，与商业化同类抗体相比，对巨噬细胞具有显著较高的敏感性，在巨噬细胞检测等相关诊断应用中具有重要价值。随后，将这种抗体用于PLGA药物载体的修饰，制备了巨噬细胞靶向性的药物载体，给药实现显示显著提高了药物载体与巨噬细胞的结合能力与抗结核药物运输能力；利用BCG感染的巨噬细胞模型验证显示，这种靶向性的载药颗粒显著提高了抗结核效能。此外，课题组还通过大量临床样本筛选建立了一种新型诊断方法，显著提高了肺结核免疫诊断效能，具有临床应用前景。本课题资助下已发表论文4篇，其中SCI论文2篇，投稿SCI论文2篇，获得国家发明专利2项，申请国家发明专利1项。