多功能"桑葚状"AQ4N∕噻替哌纳米载体及其乏氧选择、双药协同抗肿瘤作用

Hypoxia is a specific characteristic of solid tumor. In this project, a multi-functional mulberry like AQ4N∕thiotepa nanocarrier with the characteristics of hypoxia phosphorescence indication for drug release, hyaluronic acid tumor targeting, AQ4N hypoxia-selective reduction to AQ4, which can enhance anti-tumour of thiotepa significantly is designed .The nanocarrier is prepared as follows: (1) hydrophobic and hydrophilic cyanoacetic acid derivatives are obtained using the reaction of cyanoacetic acid with MePEG 2000 and 1-hexadecanol, respectively. The hypoxia sensitive polymer is prepared by the two of the cyanoacetic acid derivatives coordinating with hypoxia phosphorescence cyclometalated iridium(III) chloro-bridged dimmers and polymerization; (2) the polymeric micelles encapsulated thiotepa are fabricated with above polymer in solution using improved surface dispersion method; then positively charged chitosan is fabricated on the surface of the micelles; (3) AQ4N small nanoparticles is fabricated with hyaluronic acid by co-precipitation method, and final mulberry like AQ4N∕thiotepa nanocarriers are obtained by assemble AQ4N small nanoparticles onto the surface of the polymeric micelles encapsulated thiotepa with electron interaction. Their surface and chemical structures can be determined by IR , NMR spectra and so on. Their characteristics, such as targeting tumor cells, hypoxia-selective reduction of AQ4N, hypoxia phosphorescence indication etc, can be measured by the phosphorescence labeling，phosphorescence lifetimes, animal fluorescence/phosphorescence imaging, respectively. Their antitumor effects will be detected by cell tests in vitro and aminal tests in vivo. These results can conduct the nanocarrier design. This kind of multi-functional mulberry like AQ4N∕thiotepa nanocarrier with the characteristics of hypoxia phosphorescence indication, hyaluronic acid tumor targeting, AQ4N hypoxia-selective reduction to AQ4，which enhance anti-tumour of thiotepa significantly, thereby obtain higher thearpy effects for tumors with lower toxcity for normal tissues.

乏氧是实体肿瘤特有现象，本项目将乏氧磷光指示、透明质酸靶向、AQ4N乏氧还原及与噻替哌协同作用等策略集成在一起。将氰基乙酸分别与PEG 2000和十六醇结合形成亲水和疏水衍生物，将它们与铱二氯联桥化合物配位并聚合为乏氧磷光高分子化合物，利用其两亲性包裹噻替哌形成微胶束，再将其表面包裹一层阳离子壳聚糖。将透明质酸与AQ4N结合成为带负电纳米小球，利用电荷的相互作用将载有AQ4N的透明质酸纳米小球组装在微胶束表面形成桑葚状AQ4N∕噻替哌纳米载体。红外光谱、核磁共振等确定其化学组成和表面结构；磷光标记、活体成像等研究其肿瘤乏氧下磷光释药指示、选择靶向特性和双药协同治疗的作用机制；体内外实验研究其抗肿瘤效果并指导载体设计。这种多功能载体系统可在透明质酸介导下将双药同时靶向到肿瘤组织与细胞，在磷光指示下AQ4N释放并乏氧还原为活性物AQ4，通过其与噻替哌的双药协同作用，达到高效、低毒抗肿瘤的目的。

为提高肿瘤化疗药物的治疗效果、降低其毒副作用。将两种化疗药物负载在同一载体进行协同治疗是肿瘤化疗的重要策略。乏氧是实体肿瘤特有现象。AQ4N是一种乏氧靶向肿瘤化疗前药，在乏氧条件下可被还原成强效的拓扑异构酶II抑制剂AQ4抑制肿瘤，但是由于其水溶性太强而难以进入细胞及被纳米粒子负载。我们的前期研究发现AQ4N有强近红外荧光、可作为荧光监控剂。本项目将乏氧荧光指示、透明质酸肿瘤靶向、AQ4N乏氧还原及与棉酚协同作用等策略集成在一起。将Cu（II）配位AQ4N与棉酚形成无限配位配合物纳米粒子（ICPs），再在其外面包裹一层透明质酸（HA）形成多功能载体HA@AQ4N-Cu-Gossypol NPs，红外光谱、核磁共振等确定其化学组成和表面结构；荧光标记、活体成像等研究其肿瘤乏氧下荧光释药指示、选择靶向特性和双药协同治疗的作用机制；体内外实验研究其抗肿瘤效果并指导载体设计。结果表明HA@AQ4N-Cu(II)-gossypol NPs的药物包裹率为100%，总载药量高达77.41%。还同时具备EPR被动靶向、HA受体介导的靶向、pH响应性位点选择性三种主被动靶向能力。细胞实验表明，该纳米粒子具有极低的AQ4N和棉酚协同作用系数（0.097），表明AQ4N和棉酚具有很好的协同抗肿瘤能力。动物实验表明仅仅用常规剂量五十分之一的AQ4N、常规剂量二分之一的棉酚，HA@AQ4N-Cu(II)-Gossypol NPs的肿瘤抑制率仍高达87%，这一数值明显高于游离药物组或游离药物联用组。同时该多功能载体在体内的分布和药物释行为可通过AQ4N的近红外荧光实时自监控，这种反馈式药物释放和分布监控能力使得我们可以根据不同肿瘤类型和发展阶段调节给药剂量和给药时间，因而达到肿瘤个性化治疗的目的。该多功能载体系统具有高载药、高疗效和低毒副作用的特点，未来将有重要的应用前景。