靶分子/蛋白药物/载体天然机制一体化制备的纳米递送系统基础研究

Traditional delivery systems for protein drug are limited by some problems such as low drug loading efficiency, poor tumor targeting ability and susceptibility to environment. To address these problems, an integrated synthesis for protein drug and delivery platform is proposed by combining genomic engineering of yeast for protein drug expression and natural secretion mechanism of yeast extracellular vesicles for protein drug encapsulation. Specifically, the gene of tumor targeting membrane protein and protein drugs can be transformed into yeast by use of genetic engineering technique, which will spontaneous express in yeast cell. Meanwhile, taking advantage of the natural secretion mechanism of extracellular vesicles, the engineered membrane protein will be expressed onto the surface of extracellular vesicles for active tumor targeting, and the protein drug will be encapsulated into extracellular vesicles for anti-tumor. This system can one-step achieve of targeting molecule, protein drug and the nano carrier for highly efficient in vivo tumor targeting application. This project aims to investigate the characteristics of the integrated protein drug synthesis and delivery platform in vitro and in vivo at cellular and animal level, including test of drug stability, cellular uptake, secretion mechanism of extracellular vesicles, in vitro and in vivo anti-tumor efficiency, barrier permeability and other key scientific issues. A comprehensive examination of the pharmaceutical behavior of yeast extracellular vesicles drug delivery system and evaluation of the consistency of the system and traditional protein drug system will be performed. This project will provide a new strategy for combing protein drug delivery and the use of biological extracellular vesicles as a drug delivery system.

针对常规药物递送系统蛋白药物装载效率低、靶向性差及活性易受外界影响等问题，拟将基因工程与酵母微囊泡出芽分泌机制有机结合，构建靶分子/蛋白药物/载体一体化制备的药物递送系统。通过基因工程改造将酵母菌同时携带表达靶分子和蛋白药物的基因，利用酵母微囊泡出芽分泌机制，使微囊泡膜上含有特定膜蛋白（靶分子），具有肿瘤主动靶向作用，囊泡内含特定胞浆蛋白（蛋白药物），发挥抗肿瘤作用，实现靶分子/蛋白药物/载体一体化制备和蛋白药物体内肿瘤靶向高效转运目的。本课题拟在细胞和动物水平，围绕该药物递送系统体内外药学基本特征开展研究，如稳定性、细胞摄取、入胞机制、释放机制、体外活性以及体内肿瘤靶向性和分布、生物膜屏障渗透性和体内抗癌活性等问题，全面考察上述药物递送系统药学行为，评价利用基因工程与酵母分泌机制所得靶分子和蛋白药物与常规靶分子及蛋白药物体内一致性。为蛋白药物体内转运及生物囊泡作为递送系统应用提供新策略。

本课题以益生菌为基体，构建了4种益生菌仿生的药物递送系统，系统研究了其对口服药物递送系统的精准肠道结肠定位、肠道粘液层增强穿透、提高递送药物生物利用度、重塑肠道微生物种群等作用，进一步探索了益生菌仿生药物递送系统对结直肠癌、肠炎等胃肠道疾病的治疗作用。具体研究内容如下：1.针对口服药物递送系统难以实现精准的肠段定位及跨过肠粘液屏障困难的问题，本研究构建了具有近红外光响应的热泳纳米粒，利用纳米粒中二氧化硅的介孔结构高效装载化疗药物顺铂，最终使用金黄色葡萄球菌的外膜对上述自热泳纳米粒进行包裹，构建细菌仿生驱化定位的口服药物递送系统。该系统能够抵抗胃肠道环境对顺铂的影响，通过细菌仿生和结直肠癌肠段的IgG高效亲和，在结直肠癌肠段富集，近红外光照射后自主运动穿透肠粘液层，提高药物向结直肠癌的转运效率，显著提高抗肿瘤效果；2.益生菌具有良好的生物治疗作用，然而多数益生菌口服后难以抵抗胃肠道酸性及胆盐对其影响，导致口服后大量失活，更重要的是，在肠炎高ROS环境中，益生菌难以有效定植，影响疗效。本研究在益生菌上形成单宁酸自组装外膜，既能使益生菌抵抗酸性、胆盐等环境，又能自主的清除ROS，显著提高了益生菌的存活和定植，对肠炎治疗效果良好；3.针对益生菌在肠道定植中与肠粘液作用力低的问题，本研究采用肠粘液的主要成分黏蛋白为涂层修饰益生菌，研究表明，黏蛋白涂层提高了益生菌的抗逆特性，增强了与肠粘液的相互作用，显著提高了益生菌在结肠部位的滞留，进而进一步改善了其定植；4.结肠炎发生后，大量致病菌增殖，如何特异性的杀伤有害菌而促进益生菌的定植目前面临巨大挑战，本研究发现钨离子特异性杀伤大肠杆菌等病原菌，而对不依赖钼辅因子的益生菌不具备抑制作用，基于此构建了特异响应肠炎环境钙卫蛋白的钨酸钙纳米粒，同时采用酵母菌膜对其进行包裹，该系统定位至肠炎部位，响应钙卫蛋白释放钨离子，显著调控肠道菌群组成，短时间内提高益生菌水平，将病原菌压制至非致病水平，对肠炎治疗效果显著。本研究在该领域发表SCI论文共11篇，其中中科院一区文章11篇。依托本项目资助，培养了三名博士生和六名硕士生。