磷脂包裹的层状双羟基复合无机物载药/基因纳米粒的构建及评价

Targeted delivery and gene delivery shows numerous potential applications and has drawn much attention in the fields of both clinical therapy and basic science research. Unfortunately, no satisfactory delivery system has been found yet. Owing to its advantages such as low immunogenecity, the absence of mutational insertion risks, and the ease of preparation and modification, non-viral vector is becoming an attractive alternative to viral vector. So far, numerous materials have been investigated as non-viral vectors for gene delivery. However, most of them suffered from low transfection efficiency, which suggested that other design principles should be employed to break the bottleneck. After drug/gene get into the cells, they pass through the lysosomes firstly where exists a large number of enzymes that can destroy the drug/gene. Therefore, lysosomal escaping is the key of improving the transfection efficiency. Layered double hydroxide nanoparticles can raise the osmotic pressure rapidly of the lysosomes, which leads to the destruction of the lysosomes and the release of drug/gene into cytoplasm. However, the application of LDH nanoparticles is limited by its poor in vivo stability. In order to overcome this disadvantage, the oppositely charged phospholipids are used to wrap the positively charged LDH nanoparticles. The core-shell structure can improve in vivo stability of the non-viral vector without affecting its high cell transfection efficiency. In addition, PLDH nanoparticles can be further modified to become a multi-funtional carrier. Our group has already successfully prepared PLDH nanoparticles, and its core-shell structure is ensured by TEM and XPS. These research laid a solid foundation for our further study. With low immunogenicity and and high transfection efficiency, PLDH nanoparticles have the potencial to be an excellent non-viral vector, which can greatly promote the development of gene therapy.

靶向治疗和基因治疗的关键技术是药物/基因传递载体的设计。非病毒载体载药量大、安全性高、易于修饰，但存在转染效率低的问题。基于此，本申请设计了一种新型核-壳式结构的非病毒载体，旨在提高载体的安全性又具有高效性。药物/基因进入细胞后，首先经过溶酶体，而溶酶体中存在大量可破坏药物/基因的酶系，因此逃离溶酶体是提高药效的关键。体外研究表明层状双金属氢氧化物（LDH）纳米粒进入溶酶体后可使渗透压迅速升高而破坏溶酶体，因而用于装载药物/基因时表现出较高的转染性；但其表面所带的正电荷导致其体内稳定性较差，无法用于体内。基于此问题，申请者设计了一种新型核-壳结构的脂包裹LDH（PLDH）纳米粒，以带相反电荷的磷脂膜为外壳，以荷正电的LDH为内核，同时对外膜进行PEG化和配体靶向修饰，不但提高了其体内稳定性，且保持高效的转染能力。本课题组经TEM、XPS等技术确证了所设计的核-壳结构的合理性及组分分布规律。

层状双金属氢氧化物（Layered double hydroxide，LDH）纳米粒是一种具备溶酶体逃逸功能的无机材料，具有良好的癌症治疗前景，但其体内稳定性差，静脉注射时可产生较大毒性，使其临床应用研究受阻。本研究利用静电自组装原理，制备了磷脂膜修饰的层状双金属氢氧化物（Phospholipid membrane modified layered double hydroxide，PLDH）纳米粒，旨在通过屏蔽LDH表面正电荷，增加其体内安全性，推动LDH纳米粒用于癌症治疗的研究进程。对PLDH的制备处方及工艺进行了筛选，并采用X射线光电子能谱仪、电感耦合等离子体光谱仪对PLDH纳米粒的元素组成进行了定量分析，使用透射电子显微镜、X射线光电子能谱仪对纳米粒的微观结构进行了确证。在此基础上，考察了磷脂膜修饰对LDH纳米粒放置稳定性、稀释稳定性、入胞途径及溶酶体逃逸功能的影响。结果发现，磷脂膜修饰可干扰LDH纳米粒与电解质、蛋白质等物质的相互作用，从而大大提升LDH纳米粒的血液稳定性；另一方面，磷脂膜由于改变了LDH纳米粒的表面性质，因此使其经网格蛋白入胞途径受到干扰，PLDH纳米粒主要通过巨胞饮作用被细胞摄取；然而，由于LDH与磷脂膜之间的静电吸引力破坏了磷脂膜的完整性，磷脂膜的修饰并不会显著削弱LDH的溶酶体逃逸功能。最后，评价了PLDH的细胞毒性，同时进行其体内药动学、药效学研究，发现磷脂膜修饰后的LDH纳米粒体内循环时间显著延长，因此而具有更强的抗肿瘤效果，且同时由于血液稳定性的提升，其体内安全性也得到显著改善，杜绝了LDH纳米粒因重复注射而出现的猝死现象。