高载药率的多重靶向抗肿瘤纳米药物载体研究

Existing chemotherapeutic drugs are far from perfect with limitations such as lack of tumor selectivity and intrinsic or acquired drug resistance. At present, low drug loading efficiency of micro/nano drug carriers become a bottleneck of tumor targeted therapy as well. In this study, conjugates folate-chitosan (FA-CS) are synthesized by FA and chitosan to form tumor pHe responsive drug release nanogels. At the same time, PDTC is co-delivered with DOX using synthesized negative charged gold nanoparticles. Drug loaded gold nanoparticles is encapsulated into FA-CS nanogels through electrostatic interaction. Characterization is determined by NMR and UV-Vis spectrum. Size distribution is measured using a laser light scattering technique and transmission electron microscope (TEM) (JEM-2000EX) is also used to determine the size distribution and amorphous of nanogels. The amount of entrapped DOX and PDTC are determined by HPLC. The pH sensitive drug release profile is assayed by HPLC. To validate the choice of Hela folate free cell lines as a positive control overexpressing FR-α, the expression of FR-α is investigated by Western blot. To test the cytotoxicity of FA-CS and further evaluate the role of folate in the cellular uptake of drug loaded FA-CS nanogels, cancer cells are used for MTT assay, Fluorescence Microscopic and Flow cytometry study. The amounts of Au uptake are measured by ICP-MS in Hela, Hela folate free and A549 cells. We hope that using CS and FA-CS as delivery vehicles, the intracellular uptake of negatively charged drug loaded gold particle is enhanced to a greater extent. The design of this study is to achieve increasing drug loading efficiency of nanogel drug delivery vehicles, specific targeting, pH responsive drug release, and overcome DOX multidrug resistance. Our goal of this project is to obtain muliti-functional targeted anti tomr drug delivery system with high payload efficiency.

化疗药物的非特异性毒性及肿瘤细胞的耐药性等问题已成为当今肿瘤治疗亟待解决的问题。微纳米药物载体载药率普遍较低的现状也成为制约肿瘤靶向治疗的瓶颈。本课题拟采用叶酸和壳聚糖为原料，制备能主动靶向叶酸受体过多的肿瘤细胞，并能灵敏响应肿瘤pH值释药的纳米凝胶系统，同时合成高载药率负电荷纳米金，共转运抗肿瘤药物DOX及其增敏剂PDTC，并通过静电吸附将载药纳米金包裹于纳米凝胶内部。通过NMR、HPLC、透射电镜、体外荧光成像、流式细胞术、Western blot和ICP-MS等手段测定载药纳米凝胶理化性质、定量分析纳米金提高载药率的能力、及验证载体pH响应释药性能，增敏剂促进药物入胞的能力及主动靶向提高药物入胞率等相关生物学特性。本课题整体设计以同时实现提高载药率、主动靶向、智能响应释药，克服阿霉素多药耐药性为最终目的，以期制备出具有高载药率的多重靶向纳米抗肿瘤药物载体。

本课题设计针对目前纳米药物载体载药率难以提高、靶点单一及靶向效率低等缺点，旨在构建高载药率与多重靶向药物递送相结合的新型高效纳米药物转运体系。在完成项目标书设计内容的基础上，申请者进一步改进研究方案，构建出更高效靶向递送抗肿瘤药物的纳米药物载体。研究内容主要分为以下四个方面：.1. 构建壳聚糖、叶酸壳聚糖纳米凝胶包裹负电荷金纳米粒（AuCOOH@CS/FACS）为载体，实现化疗药物和生物制剂的同时靶向递送，具有高载药率，靶向递送及发挥药物协同作用的特点。AuCOOH@FACS体系可将95%以上的AuCOOH带入细胞。动物实验结果证实，AuCOOH@FACS给药组可显著抑制肿瘤生长，瘤体质量仅为对照组的1/40。 .2. 为进一步增强靶向性，减少载体的系统毒性，将靶向分子叶酸替换为合作实验室研发的新型FAT1分子靶向单克隆抗体，并将AuCOOH修饰配基（MUA）改为更具有生物相容性的（HS-PEG-COOH），在发挥金纳米粒载药率高的优势之余，增强靶向特异性。将FAT1分子特异性靶向单克隆抗体mAb198.3接合至AuCOOH表面，通过TEM，共聚焦显微镜及小动物活体呈像等手段，验证其肿瘤细胞及肿瘤组织靶向特异性。结果表明，mAb198.3不仅能将99%以上的AuCOOH带入colo-205细胞，且尾静脉给药后，裸鼠瘤体药物浓度为单纯抗体给药的15.5倍，为AuCOOH_isotype（同型对照）的28.4倍。.3. 在完成标书设计的基础上，为使体系能用于同时负载更多药物，将仅具有pH敏感特性的CS外壳换成可自降解的SiO2外壳，通过吸附和包裹两种不同的载药方式，制备不依赖于外部刺激即可实现多种药物逐级释放的序贯递送体系。该体系可通过表面修饰单抗进行主动靶向，并通过不同的药物装载机制及药物装载浓度调节药物释放的速率，使体系在不引入刺激响应化学键的基础上，能主动靶向，高效入胞及精确逐级释放不同种类的药物。完成该体系序贯递送研究后，继续将AuCOOH表面结合单抗与siRNA，与SiO2体系结合，制备出能同时递送三种不同药物的靶向纳米序贯递送体系。 . 标书设计的研究目标已基本实现，研究内容已发表于Biomaterials，Nanoscale，JECCR，Plos one等SCI杂志，共5篇，申请发明专利1项。