癌症仍然是全球范围内致死率最高的疾病之一，尽管如放射疗法，光热疗法和免疫疗法等癌症治疗手段不断出现，传统化疗仍然是临床上癌症治疗的主要手段。系统毒副作用大，缺乏肿瘤特异性等缺点严重限制了传统化疗药物在癌症临床治疗中的应用。纳米技术在药物递送领域的应用和发展有效解决了传统化疗药物面临的诸多问题，但是药物在血液循环过程中的提前释放以及纳米载体进入肿瘤细胞后的药物释放效率低下使得纳米药物递送系统在临床上的深入应用受阻。 本文构建了多种不同的肿瘤微环境响应性纳米载药体系用于化疗药物的靶向运输，内容和结果如下所述： 1、我们分别合成了ROS响应性前药聚合物mPEG2k-TK-DOX(PTD)和ROS和pH双响应性前药聚合物mPEG2k-TK-hyd-DOX(PTHD)。前药聚合物的化学结构通过¹H NMR进行了表征。PTD和PTHD前药聚合物均能够在水溶液中自组装形成纳米胶束，粒径均在200 nm左右并且胶束的PDI较小，说明胶束的粒径均一且分布较窄。PTHD胶束在PBS和含有10％胎牛血清的PBS溶液中均具有良好的稳定性。体外释放实验表明在模拟肿瘤ROS微环境条件下PTD前药胶束能够缓慢释放DOX且随着ROS浓度的增加DOX的释放量增大，这一结果表明PTD前药胶束具有ROS响应性。PTHD前药胶束具有pH和ROS双响应性，这保证了PTHD在进入肿瘤细胞后能够更加快速高效地释放DOX。细胞毒性方面，PTD前药胶束能够选择性地杀伤肿瘤细胞，而对正常组织细胞没有毒性。 PTHD前药胶束对不同类型的肿瘤细胞的细胞毒性均要大于PTD前药胶束的细胞毒性。 2、我们成功合成了PTHD／TPGS和PTHD／TPP-TPGS混合胶束。混合胶束的粒径分布在100-200nm，TEM结果表明两种混合胶束粒子都具有较为规则的球形结构。胶束粒子无论是在PBS还是含有10％胎牛血清的PBS溶液中都有很好的胶体稳定性。体外细胞毒性结果表明PTHD／TPP-TPGS和PTHD／TPGS混合胶束中的两种组分之间具有协同作用，都能够一定程度上逆转MCF-7／ADR细胞的耐药性。CLSM结果显示PTHD／TPP-TPGS混合胶束处理组细胞内红色荧光绝大部分与线粒体的绿色荧光重叠，说明PTHD／TPP-TPGS混合胶束具有线粒体靶向性。PTHD／TPP-TPGS混合胶束能够显著提高MCF-7／ADR细胞内的ROS水平。ROS水平提高一方面能够加速混合胶束释放DOX，另一方面使细胞遭受氧化应激诱导细胞发生凋亡。三维细胞球体实验表明，PTHD／TPP-TPGS混合胶束具有较好的细胞球浸润能力并且能够破坏三维细胞球的完整性并抑制三维球体的生长。 3、我们首次将D-α-生育酚琥珀酸酯(α-TOS)与聚乙二醇单甲醚(mPEG2k)通过具有ROS响应性的酮缩硫醇连接臂(Thioketal linker)键连制备了前药复合物mPEG2k-TK-TOS(PTK)。PTK在水溶液中能够自组装成粒径约14 nm的纳米胶束。我们利用薄膜水化法制备了负载DOX和α-TOS载药纳米胶束(PTKTD)。 PTKTD胶束在肿瘤细胞内的药物释放行为具有ROS响应并且药物释放存在正向反馈调节。体外细胞毒性实验表明PTKTD载药胶束能够有效克服MCF-7／ADR细胞的多药耐药性，极大地增强了DOX对耐药细胞的细胞毒性。雌性BALB／c荷瘤裸鼠的体内抗肿瘤实验结果显示PTKTD载药胶束干预后的小鼠体重没有明显改变，而且H&E染色结果表明PTKTD载药胶束对心脏，肝脏和脾脏等主要组织器官没有明显的损伤作用，这些结果说明PTKTD载药胶束对机体没有明显的毒副作用。瘤体生长曲线表明PTKTD载药胶束能够显著抑制MCF-7／ADR实体瘤的生长。H&E和TUNEL结果显示PTKTD载药胶束干预后的肿瘤组织切片有大量的细胞坏死现象，这同样说明PTKTD载药胶束对MCF-7／ADR实体瘤有很强的杀伤作用。 关键词：肿瘤微环境；刺激响应性；胶束；多药耐药；药物控制释放

 Cancer is still one of the leading causes of death all over the world. Although emerging anticancer strategies such as radiotherapy, photothermal therapy and immunotherapy have been utilized in clinic to treat cancer, conventional chemotherapy is still an indispensable technique for cancer treatment. The application of traditional chemotherapeutic drugs in clinic was severely limited for their systemic side effects and lack of tumour specificity. The development and application of nanotechnology in the field of drug delivery have solved many problems faced by traditional chemotherapeutic drugs. However, the premature drug release during circulation and insufficient drug release inside the tumour cells still hinders the application of drug delivery systems in clinic. In this paper, a variety of different tumor microenvironment-responsive drug delivery systems were constructed for targeted drug delivery. The contents and results are as follows: 1.We successfully synthesized ROS responsive polymer prodrug (mPEG2k-TK-DOX, PTD) and ROS and pH dual responsive polymer prodrug (mPEG2k-TK-hyd-DOX, PTHD), respectively. The chemical structures of these polymers were characterized by ¹H NMR. PTD and PTHD can self-assemble into micelles in aqueous solution with particle size about 200 nm and small PDI. PTHD micelles were stable both in PBS and PBS containing 10% fetal bovine serum. In vitro release experiments showed that PTD prodrug micelles could release DOX slowly under the simulated tumor ROS microenvironment. The release profile of DOX is ROS concentration-dependent. PTHD micelles are both pH and ROS responsive, which ensures that PTHD micelles can release DOX more rapidly and efficiently after entering tumour cells. As for in vitro cytotoxicity, PTD showed selective killing ability of tumor cells, while not affecting the proliferation of normal tissue cells. The cytotoxicity of PTHD prodrug micelles on different types of tumor cells was higher than that of PTD prodrug micelles. 2.PTHD/TPGS and PTHD/TPP-TPGS mixed micelles were successfully synthesized. The particle size distribution of the mixed micelle was 100-200 nm, and the TEM results showed that both micelles had regular spherical structure. Mixed micelles showed good colloidal stability both in PBS and PBS containing 10% fetal bovine serum. In vitro cytotoxicity results showed that there was a synergistic effect between the PTHD and TPGS or TPP-TPGS. Both mixed micellar systems could reverse the drug resistance of MCF-7 /ADR cells to some extent. CLSM results showed that most of the red fluorescence overlapped with the green fluorescence in cells treated with PTHD/TPP-TPGS, indicating the mitochondrial targeting ability of PTHD/TPP-TPGS mixed micelles. The PTHD/TPP-TPGS mixed micelles can significantly improve the ROS level in MCF-7 /ADR cells. The increased ROS can accelerate DOX release from mixed micelles and can induce cell apoptosis for oxidative stress. Three-dimensional cell culture experiment showed that PTHD/TPP-TPGS mixed micelles had better infiltration ability. PTHD/TPP-TPGS mixed micelles could break down the integrity of 3D cell sphere and inhibit the growth of cell sphere. 3.The mPEG2k-TK-TOS (PTK) was firstly constructed by combining mPEG2k with α-TOS via ROS responsive thioketal linker. PTK can self-assemble into nano-micelle with particle size of 14 nm in aqueous solution. We prepared DOX and α-TOS loaded micelles (PTKTD) by thin-film hydration method. The in vitro cytotoxicity experiments showed that PTKTD micelles could effectively overcome the multidrug resistance of MCF-7 /ADR cells and greatly enhance the cytotoxicity of DOX to drug resistant cells. During the treatment period, no obvious body weight changes were found in PTKTD micelle groups. No noticeable tissue damage was detected in PTKTD micelle group for this test dosage, reflecting the negligible systemic toxicity and better safety profiles of PTKTD micelles. The PTKTD micelles showed preferable antitumor efficacy compared with other groups and the tumours almost stopped growing. H&E and TUNEL results showed a large amount of cell necrosis in tumor tissue sections after PTKTD micelles intervention, which indicated the strong killing effect of PTKTD micelles against MCF-7 /ADR solid tumors. Key Words: Tumor microenvironment; Stimulus responsiveness; Micelles; Multidrug resistance; Controlled drug release