用于胞液递送的水溶性可降解两亲两性共聚物囊泡的优化及其递送机理研究

Nanocarriers are used to protect and deliver the drugs, which have their action sites in the cytosol or intracellular organelles, but are membrane impermeable and some are sensitive to hydrolysis. Unfortunately, we lack safe and efficient nanocarriers for cytosolic delivery. Among those nanocarriers, polymersomes prepared from amphiphilic block copolymers have attracted much attention because of their superb advantages: the special lumen structure resembling cells, high stability, biocompatibility, and easily adjustable natures, the ability to individually or simultaneously load hydrophilic, hydrophobic or amphiphilic compounds. Our preliminary study shows that amphiphilic and amphoteric lipopolysaccharide-amine (LPSA), synthesized by our lab, can self assemble into nanopolymersomes by direct dissolution in water, and LPSA nanopolymersomes (LNPs) are low cytotoxic and degradable. Their final degradation products are nontoxic, and can be metabolized and eliminated from the body. The further study demonstrates that LNPs can load enhanced green fluorescence protein plasmid (pEGFP), and more than 95% mesenchymal stem cells (MSCs) transfected by pEGFP-loaded LNPs express large target protein, indicating LNPs are promising as safe and efficient nanocarriers for cytosolic delivery. Based on these, this project aims to optimize LNPs, to study the structure and formation mechanism of LNPs, and the effects of mass ratios of three blocks in LPSA on properties of LNPs (such as surface charge, size, stability, drug loading and releasing, cytosolic delivering, and biodistribution). The cytosolic delivery mechanism will be further explored. We expect that this study will provide theoretical and practical basis for design and clinical applications of nanopolymersomes for cytosolic delivery.

许多药物的靶点位于细胞内，但自身无入胞能力，且有的不稳定易水解，需载体保护并运输至胞液内发挥疗效。目前缺乏安全高效胞液递送载体。聚合物纳米囊泡因具有类似细胞膜的特殊空腔结构、高稳定性、生物相容性、可调的物理化学性质、能同时或分别负载亲水、疏水或两亲药物而引起高度关注。我们前期研究发现：合成的两亲两性脂多糖胺嵌段共聚物可直接于水中自组装形成纳米囊泡，此囊泡低毒可降解、降解产物无毒可代谢；进一步研究发现囊泡能在水中高效负载pEGFP质粒，并使约95%的骨髓间质干细胞高效表达GFP蛋白，预示它有望成为高效低毒的胞液递送载体。本项目拟在此基础上，进一步优化囊泡，研究其结构和形成机理、聚合物中三嵌段质量比和囊泡性能（如表面电荷、粒径、稳定性、载释药能力、胞液递送能力及体内生物分布等）之间的关系，探讨胞液递送机制，为胞液递送囊泡体系的设计提供理论实践依据，并为临床提供易制备高效低毒胞液递送载体。

许多药物的靶点位于细胞内，但自身无入胞能力，且有的不稳定易水解，需载体保护并运输至胞液内发挥疗效，而目前临床上缺乏安全高效胞液递送载体。聚合物纳米囊泡具有类似细胞膜的特殊空腔结构、高稳定性、生物相容性、可调的物理化学性质、能同时或分别负载亲水、疏水或两亲药物等优点，因此特别适合用作药物载体。基于此，本项目设计制备了两亲两性三嵌段接枝共聚物脂多糖胺（LPSA）纳米囊泡，并对其理化/生物学性能进行优化，随后探索了LPSA自组装形成纳米囊泡（LNPs）的结构和机理，评价了LPSA的降解性和毒性等，研究了其胞液递送机理。最后用NPs分别负载5种基因药物（DNA/小干扰RNA）、抗癌药，通过体内外实验，评价其胞液递送能力及载药后疗效。结果表明，通过静电及亲疏水相互作用，LPSA能在水中自组装形成以胆固醇和PEI/氧化海藻酸聚电解质复合物为壁、以PEI为内外亲水冠的纳米囊泡，通过控制LPSA中三嵌段质量比可调控NPs的理化/生物学性能，获得高效——在多种细胞（包括干细胞rMSCs）中转染效率>95%，低毒——rMSCs存活率>85%，完全降解的纳米囊泡胞液递送载体。NPs具有强入胞能力，其胞吞具能量依赖性，且至少通过4种路径即巨胞饮、陷穴蛋白依赖的胞吞、网格蛋白依赖的胞吞、以及低密度脂蛋白介导的胞吞，其中前两者占主导作用。LNPs具有强内体逃逸能力，在胞内转运中可能直接从内体逃逸至胞质，避免了被溶酶体中的酶降解。囊泡递送DNA时，能使MSCs细胞表达治疗剂量的靶蛋白，在体内能显著促进靶组织如血管/骨组织生长；囊泡递送siRNA时，能沉默靶基因表达，在体内能显著抑制靶组织如血管等的生长；囊泡递送抗癌药盐酸阿霉素时，相同药物浓度下，载药囊泡与自由药物抑瘤效果相当但毒性大大减小，动物存活率显著提高。说明脂多糖胺纳米囊泡是一种高效低毒胞液递送载体，作为基因/药物载体，在临床上有广泛应用前景。