两相释药降低乳腺癌细胞耐药靶向给药系统的研究

Modern drug delivery system demands high therapeutic efficacy and low toxicity which depends on efficient intracellular transportation of therapeutics to specific organisms, cells, even targeted organelles such as cytosol, nucleus, mitochondria, lysosome and endoplasmic reticulum. This project was designed to bypass problems of the custom drug carriers: simple type, single structure, limited function, using the inorganic / organic multilayer composition. We adopt new technology to give the classic biodegradable polymer (FDA approved) new functions. Develop a new technology of core-shell cRGD targeted multifunctional and biodegradable nanoparticles drug delivery system, using phospholipid-PEG and chemotherapeutic agent (Mitoxantrone,MIT) as core(control release phase), CaP hybrid porous nanospheres and reverse drug resistance (Verapamil ,Ver) as shell (release phase).The CaP/GPC-mPEG hybrid porous nanospheres have been prepared through a facile polymer assisted route at a relatively low temperature. The hybrid CaP/GPC-mPEG NSs obtained were 30-60 nm in diameter and exhibited a porous structure. The mechanism of release drug: First release phase of drug partially release, accumulating in drug resistance cancer cells in order to against P-gp. Second control release of chemotherapeutic agent then begin to release in core. Synergistic effects enhance the reversal of multidrug resistance effects. The interaction of biphasic nanoparticles with MCF-7 and MCF-7/Adr:adhesion,uptake,retention and transportation following temporal and different time ,this can help us to undstand how the composite,structre and physicochemical properties of biphasic nanoparticle effect on the endocytosis ,exocytosis and transportation. The purpose of using drug resistance cancer cell line in this project is not only to study the mechanism interaction of core-shell cRGD targeted multifunctional biodegradable nanoparticles and resistance cancer cells and effect of reverse multidrug resistance of tumor cells, but also to provide a new mode for the industrialization and preclinical trials of targeted nanoparticles delivery drugs system.

现代给药系统要求药物在充分发挥疗效的同时毒副作用小, 这就需要药物能被特异转运至靶组织、靶细胞, 甚至是特定的细胞器, 如细胞质基质、细胞核、线粒体、溶酶体、内质网等。本项目采用无机/有机多层组合和功能化设计的思路，突破传统载体材料种类单一、结构单一和功能单一的局限性，采用新技术，赋予传统经典材料（FDA批准用于人体）新功能，发展以磷脂-PEG聚合物和化疗药（米托蒽醌MIT）为核（控释相）、磷酸钙和逆转耐药抑制剂（维拉帕米VER）为壳（即释相）的生物可降解多功能纳米载体，构建靶向基团RGD修饰的复合靶向纳米缓释给药系统。本项目拟以耐药细胞株为模型，研究两相释药纳米粒与耐药细胞的结合/粘附、摄取、胞内滞留及转运等过程，揭示两相释药纳米粒的组成、结构和理化特性对其入胞 、出胞过程及胞内行为的影响。研究结果将为靶向纳米系统的产业转化提供实验数据，为纳米靶向递送缓释系统应用于临床提供理论支持。

本项目设计了一种高分子纳米药物载体，PEG-PS(phosphatidylserine- Monomethoxy (polyethylene glycol) ) /CaP纳米粒，其粒径为20nm左右，易于通过EPR效应到达肿瘤部位。制备了以磷脂-PEG聚合物和化疗药（米托蒽醌MIT）为核（控释相）、磷酸钙和逆转耐药抑制剂（维拉帕米VER）为壳（即释相）的生物可降解多功能纳米载体。PEG-PS/CaP纳米粒对MIT和VER的包封率均达99%以上。对米托蒽醌的载药量为6.67%，对维拉帕米的载药量为6.69%。. 生物相容性评价是考察生物材料应用于临床的基础。本研究对PEG-PS/CaP纳米粒进行了生物相容性的初步评价。实验结果表明，PEG-PS/CaP纳米粒不改变血液的溶血和凝血性质。对正常肝细胞没有毒性。不引起免疫因子的释放和免疫反应。PEG-PS/CaP纳米粒在斑马鱼体内循环时间较长，能够被代谢，并且不引起斑马鱼体细胞凋亡。实验结果证实，PEG-PS/CaP纳米粒生物相容性良好。.本研究以耐药细胞株MCF7/MDR(耐药乳腺癌肿瘤细胞)为模型，研究了两相释药纳米粒与耐药细胞的结合/粘附、摄取、胞内滞留及转运等过程，结果显示，MCF7/MDR细胞对纳米粒中药物的摄取量高于游离药物。这说明在纳米粒的作用下，耐药细胞对MIT的外排作用得到了抑制。两相释药纳米粒通过巨胞饮和膜穴样凹陷途径进入耐药肿瘤细胞形成内涵体，并且消耗一定量的ATP （ATP的消耗辅助逆转耐药）。纳米粒外壳的维拉帕米在内涵体的酸性环境中首先释放，阻断P-糖蛋白功能。两相释药纳米粒能携带所载药物从溶酶体逃逸，溶酶体崩解，米托蒽醌释放。释放进细胞质的米托蒽醌进入细胞核，阻断DNA的合成。.本研究验证了载药纳米粒对肝肿瘤耐药细胞的逆转效果。首先，在细胞水平上，对逆转耐药效果进行了探讨，结果表示，两相释药纳米粒能够促进肝肿瘤耐药细胞的死亡和凋亡，具有逆转耐药的效果。然后，本文进一步建立了荷瘤裸鼠模型，对纳米粒的肿瘤靶向性以及整体治疗效果进行动物层面的研究。研究结果证实，纳米粒在体内循环时间较长，能通过EPR效应有效被动靶向至肿瘤组织，并且深入到肿瘤组织内部。同时，两相释药纳米粒能够减缓荷瘤裸鼠体重的降低速率，降低肿瘤组织的增长速度，增强肿瘤组织的坏死程度，从而达到逆转耐药的效果。