多重靶向型诊疗一体化聚合物纳米载体的构筑及其靶向诊疗研究

Functional nano drug carriers are nowadays an indispensable and important means for the diagnosis and therapy of cancer. However, achieving precise diagnosis and targeted therapy of tumour using nano carriers and improving the effect of integrated diagnosis and therapy remain a challenging topic. In this study, a nano carrier with multi-targeting and integrated diagnosis and treatment was prepared utilizing the self-assembly of a 3-miktoarm star polymer which includes chemotherapy drugs and is terminated by fluorescence probe, targeting molecule and photosensitizer. The mechanism of multi-targeting combining fluorescent probe diagnosis mediated positively charged membrane-targeting and nucleus-targeting with traditional molecule-targeting was established. The mechanism of diagnosis of tumour by fluorescent probe and combination therapy of photosensitizers and chemotherapy drugs was systematically investigated. The evaluation methodology of the combined effect of fluorescent probe with photosensitizers and chemotherapy drugs was explored. The models of cell experiment and animal experiment were constructed, and the mechanism of multi-tumour-targeting, tumour diagnosis and combination therapy of chemotherapy and photodynamic therapy was investigated at cellular and molecular level. The expectation is to construct nano carrier model with integration of diagnosis and treatment combining efficient targeting and therapy to provide new thoughts and new methods for the investigation of integrated diagnosis and treatment of tumour.

如今，在癌症的诊断与治疗中功能性纳米药物载体已成为一种不可或缺的重要手段。然而如何利用纳米载体同时实现对肿瘤的精确诊断和靶向治疗，提高诊疗一体化效果仍然是一个具有挑战性的课题。本研究利用荧光探、靶向分子和光敏剂封端的三杂臂星形聚合物自组装包载化疗药物制备得到具有多重靶向/诊疗一体化的纳米载体。建立了荧光探针诊断介导的正电荷膜靶向和细胞核靶向与传统分子靶向联合的多重靶向机制；系统研究了荧光探针成像诊断和光敏剂-化疗药物联合治疗的机理；探寻荧光探针诊断剂与光敏剂和化疗药物共同作用的评价方法；建立细胞和动物实验模型，在细胞和分子水平上研究了纳米药物载体的肿瘤多重靶向、肿瘤诊断和化疗-光动力治疗联合治疗的机理。以期建立高效靶向和高效治疗的诊疗一体化载药模型，为肿瘤的诊疗一体化研究提供新思路与新方法。

近年来，肿瘤的荧光诊断段与联合治疗已成为生物医学领域的研究热点。如何利用肿瘤细胞微环境设计合成纳米载体实现对肿瘤的诊断与联合治疗，以及如何设计合适的诊断分子对与肿瘤相关的活性分子进行示踪与诊断是目前亟待解决的问题。首先本项目通过巧妙合计，将光敏剂、靶向分子和具有电荷翻转功能的基团引入到星形聚合物中，制备得到一系列三杂臂星形聚合物（TPP）PCL-PEGMA(CA)-PEG-FA，实验结果表明该三杂臂星形聚合物在水中自组装包载抗癌后形成了以（TPP）PCL为核，PEGMA(CA)-PEG-FA为壳的纳米胶束。且弱酸性条件下通过席夫碱键水解CA在聚合物中解离下来，产生的-NH2质子化，促使纳米胶束发生电荷翻转，且包载DOX后电荷的翻转特性和TPP的光敏特性仍能保持。细胞毒性实验和细胞成像实验表明该载药纳米胶束具有正电荷和叶酸分子双重靶向性，具有化疗-光动力治疗联合治疗效果。活体成像实验表明该载药纳米胶束能够快速靶向肿瘤，具有双重靶向和诊疗一体应用前景，为肿瘤的诊疗一体化研究提供新思路与新方法。接着，受实验中光敏剂光照产生活性氧的启发，我们研究开发了多种用于以检测与肿瘤相关的活性氧（如:次氯酸、超氧自由基阴离子（O2·-）的荧光探针。光谱测试、细胞成像和斑马鱼成像结果表明探针NS-HClO可以专一性检测内源性和外源性次氯酸；探针NS-HClO可以快速、专一性的响应内源性和外源性超氧自由基阴离子（O2·-）；而探针NBDS可以快速专一性检测生物体内的半胱氨酸。通过本项目的研究我们研发了一种以光敏剂和电荷翻转基团修饰的功能化星形聚合物构筑诊疗一体化纳米载体的新方法，为多功能线纳米药物载体的制备提供了一种新思路；同时通过对检测与肿瘤相关的活性氧的荧光探针的开发，为肿瘤等疾病的诊断研究提供了新途径。