肿瘤靶向外泌体的工程化构建及共投递microRNA/吉非替尼抑制分子靶向治疗耐药研究

Searching for and developing vectors for efficient gene and drug delivery to treat cancers is a cutting edge, crossover research area. Being natural vesicles secreted by cells, exosomes can be excellent drug/gene vectors due to their unique features, such as ability to deliver their cargo microRNA through membrane fusion for intercellular communication, drug loading by lipid bimolecular layer and tumor tropism. However, there are several constraints that hinder the efficient clinical applications of exosomes, such as distinct performances resulting from variety of sources, separation and purification difficulty, complex introductions of targeting factors, and rapid in vivo clearance rate. In this application, we will integrate the advantages of exosomes mentioned above and construct engineering exosomes by chemically introducing tumor cell recognizing factor and cell membrane fusion factor, chemically or physically modifying the lipid composition and binding superparamagnetic nanoparticles to form exosome-superparamagnetic nanoclusters according to our previous reports. Such engineered exosomes will have the following features: magnetic targeted tumor function, tumor cell tropism and recognition ability. These exosomes can also deliver microRNA cargo through membrane fusion. MicroRNA/Gefitnib will be co-loaded into these exosomes and simultaneously delivered into tumor cell in vivo to overcome issue of drug resistance of molecular targeting therapy. The investigation will help develop new drug/gene vectors and provide a theory of overcoming clinic drug resistance of molecular targeted therapy.

寻找和开发性能优异的载体高效投递基因和药物治疗肿瘤是前沿交叉研究领域。细胞分泌的外泌体携带miRNA进行细胞间通讯，脂质双分子层结构利于负载药物，具有肿瘤趋向性并通过膜融合投递内含物，是优良的药物/基因同载体系。但存在不同来源外泌体性能各异、分离纯化困难、靶向因子引入过程复杂、体内清除速率快等问题。本课题拟以材料工程方法在外泌体表面键合肿瘤靶向识别因子，通过物理或化学方法引入细胞膜融合因子并改性脂质组成，集成外泌体的优势，结合前期以外泌体-超顺磁纳米团簇基础，构建具有磁靶向肿瘤功能、肿瘤细胞趋向和识别性能、可通过膜融合进入靶细胞投递基因和药物的工程化外泌体，以此外泌体同载miRNA和吉非替尼，解决分子靶向治疗的耐药问题，为开发高性能基因/药物载体探索新的路线，为克服分子靶向治疗耐药提供理论基础。

外泌体已成为新一代药物递送系统的理想候选者。具有良好生物安全性的外泌体的分离纯化及靶向因子的引入是其作为药物载体的关键。本项目基于超顺磁性纳米粒子团簇技术分离血液外泌体并应用于肿瘤靶向药物递送。进一步对分离方法进行了优化：对血清进行预透析，减少了游离配体的影响；引入PEG长链，使更多磁性粒子结合在外泌体上，从而获得“高安全性、良好的靶向能力、快速获取、高纯度”的外泌体。.血液转铁蛋白受体阳性（TfR+）外泌体是优选的药物载体，扩展其治疗应用有望推进外泌体的临床转化。本项目利用转铁蛋白和转铁蛋白受体间的pH响应性结合和解离特性，利用超顺磁性纳米粒子簇的方法精确分离血液TfR+外泌体，并实现它们从磁性粒子中的无损、温和释放。得到的血液TfR+外泌体具有良好的生物安全性，并能将化疗药物有效地递送至肿瘤，拓宽了基于外泌体的药物递送系统的应用。.开发高性能基因/药物共递送载体实现联合抗肿瘤治疗意义重大。外泌体天然地整合多种生物分子的能力使其具有作为共递送载体的潜力。本项目提出并设计工程化外泌体作为新型共递送纳米系统。结果显示，外泌体可以有效共载DOX和反义miRNA21；引入L17E肽增强了外泌体的细胞摄取和内涵体逃逸，提高了药物投递及基因转染效率；超顺磁性纳米粒子团簇结构使工程化外泌体具有优异的肿瘤靶向能力，肿瘤生长抑制效果显著，且无明显副作用。.进一步利用工程化外泌体同载反义miRNA21/吉非替尼，实现了吉非替尼耐药肺癌细胞的生长抑制。该研究证明了工程化外泌体作为共递送载体的可行性，为外泌体的临床应用提供了新思路。.本项目进一步延伸，分析了趋动肿瘤外泌体与肿瘤细胞强相互作用的因素，集成肿瘤外泌体优势对血液外泌体进行工程化改造。结果证明，调控外泌体的膜脂质组成可提高其肿瘤细胞摄取效率，进而提高药物递送效率。该研究为设计高效率的外泌体投递系统开辟了新途径。