微环境溶蚀性胶束抗耐药骨转移瘤的作用及其机制研究

Bone metastasis, a common serious complication in primary malignant cancer patients, usually leads to the refractory bone pain and pathological fracture. Bone metastasis often shows drug resistance to chemotherapy. The drug resistance of bone metastasis is caused by bone resorption microenvironment and mutation of bone metastatic tumor cell. The bone metastasis targeted micelle that only loaded chemotherapeutic drug showed weak effect on drug-resistant bone metastasis. Zoledronic acid exhibited strong inhibition activity on the bone resorption in in-vitro 3D model of docetaxel-resistant bone metastasis. In addition, zoledronic acid also showed strong inhibition on the expression of P-glycoprotein and Bcl-2 in docetaxel-resistant breast cancer cell. These results imply that zoledronic acid and docetaxel can exert an unique synergetic effect on drug-resistant bone metastasis. However, zoledronic acid exhibits strong binding affinity to bone matrix. Thus, for drug delivery system of drug-resistant bone metastasis, it is a key strategy to release zoledronic acid at bone resorption site and release both of zoledronic acid and docetaxel in docetaxel-resistant breast cancer cell. In this study, a docetaxel-loaded calcium phosphate hybrid micelle that targeted to bone resorption microenvironment and bone metastatic tumor cell will be developed by using zoledronic acid and conjugation of hyaluronic acid and polylactic acid as stabilizer. The in-vitro and in-vivo target characteristics of micelle will be thoroughly studied. Besides, the therapeutic effect and its mechanism of micelle on the drug-resistant bone metastasis will be further investigated. The study will supply a new strategy for the design of bone metastasis targeted drug delivery system and provide experimental evidence for the targeted therapy of drug-resistant bone metastasis.

骨转移瘤为原发恶性肿瘤常见严重并发症，对化疗有明显耐药性，常引起骨痛和骨折。骨转移瘤耐药由骨重吸收微环境和肿瘤细胞变异所致。本课题组发现只包载化疗药物的骨肿瘤靶向胶束抗耐药骨转移瘤的作用微弱，唑来膦酸（ZOL）可强效抑制耐多西他赛（DTX）骨转移瘤3D模型中的骨重吸收，并显著降低耐DTX乳腺癌细胞P-糖蛋白和抗凋亡蛋白Bcl-2的表达，提示ZOL与DTX可产生独特的协同抗耐药骨转移瘤作用，但骨基质对ZOL的吸附能力强，因此，既将ZOL释放于骨重吸收部位又使ZOL和DTX同时高效蓄积于耐药骨转移瘤细胞内，对耐药骨转移瘤的治疗非常关键。本研究拟以ZOL和双亲嵌段聚合物透明质酸-聚乳酸为稳定剂，与DTX共同构建靶向骨重吸收微环境和耐药骨转移瘤细胞的磷酸钙杂化胶束，研究胶束体内外靶向释药特性，阐明胶束抗耐药骨转移瘤作用及机制，为设计骨转移瘤靶向递药提供新策略，为耐药骨转移瘤靶向治疗提供实验依据。

骨组织能够为癌细胞生长提供优良的微环境，是癌细胞最好发的转移部位。70%的晚期乳腺癌患者以及85%的晚期前列腺癌患者会发生骨转移。骨转移癌容易形成全身多个部位骨转移病灶，局部手术或放疗无法完全清除所有瘤灶，化疗是治疗骨转移癌的重要手段。然而骨转移癌对化疗具有明显的耐药性，因此骨转移癌属于难治性疾病，临床迫切需要开发治疗耐药骨转移癌的新方法。本课题制备了共载唑来膦酸（ZOL）和多西他赛（DTX）磷酸钙杂化纳米粒（简称杂化纳米粒），研究了杂化纳米粒对骨转移癌的治疗作用及其机制。研究结果表明杂化纳米粒能够抑制骨微环境中耐药PC-3/DDR细胞和MDA-MB-231/DDR细胞的迁移及侵袭，其抑制作用明显强于同浓度DTX+ZOL。杂化纳米粒通过线粒体途径，促进PC-3/DDR细胞及MDA-MB-231/DDR细胞凋亡，通过抑制法尼基焦磷酸合成酶（FPPS），进一步增强对PC-3/DDR细胞及MDA-MB-231/DDR细胞凋亡的诱导作用。杂化纳米粒能够蓄积于裸鼠骨转移癌组织，对正常器官组织无明显毒性。与同剂量DTX+ZOL相比，杂化纳米粒能够显著抑制耐药骨转移癌的生长，明显提高骨转移癌部位骨密度。杂化纳米粒可降低耐药骨转移癌组织MMP-9、N-cadherin、CXCR4、CD44、RANKL、TGF-β、FPPS和Bcl-2蛋白表达，增加E-cadherin、OPG和Bax蛋白表达。结论：杂化纳米粒具有良好的骨转移癌靶向性。杂化纳米粒通过增加癌细胞之间的黏附，抑制耐药癌细胞侵袭；通过降低RANKL表达，增加OPG表达，抑制骨重吸收。最终杂化纳米粒通过DTX和ZOL的协同作用，双重途径阻断耐药骨转移癌细胞增殖与骨重吸收之间的恶性循环，提高了对耐药骨转移癌的疗效。该项研究为耐药骨转移癌的治疗探索了新方法，共载ZOL和DTX磷酸钙杂化纳米粒具有进一步研究开发的前景。