靶向载药纳米微囊激发免疫协同化疗诊治三阴性乳腺癌的实验研究

Triple-negative breast cancer is a special subtype of breast cancer. Patients of triple-negative breast cancer could not benefit from endocrine therapy and targeted therapy for lacking effectively biomarker. Its therapeutic efficacy has not been satisfactory yet. Programmed death-ligand 1 (PD-L1) is a trans-membrane protein expressed on breast cancer. It shows higher expression in triple-negative breast cancer than that in other molecular subtypes of breast cancer. Immunotherapy is the main focus in many anticancer researches. Studies demonstrated that PD-L1 could be an important marker for prognostic stratification and for planning immune checkpoint inhibitors therapies in patients with cancer. The combination of chemotherapy and immunotherapy is one comprehensive treatment for breast cancer with wide application prospect. However, precise synergistic effects of immuno-chemotherpy therapy is difficult to realize because traditional treatment techniques cannot targeted breast cancer tissue at the molecular level, and the existing imaging tools cannot timely monitor the progress of treatments. How to achieve complete and effective visible therapy of breast cancer，and to monitor the treatment procedure in real-time, and to assess the effectiveness after therapy is an urgently problem that need to be resolved. In this study, we want to prepare and characterize an organic biodegradable muti-porous-structure poly (lactic-co-glycolic acid)- monomethoxypoly (ethylene glycol) (mPEG-PLGA) NCs encapsulating PTX, and then grafted with anti-PD-L1. The targeted-PTX-nano-carriers were prepared for “muti-holes” special structure through double emulsion method. With this multifunctional nanocapsules, we can target triple-negative breast cancer tissue at the molecular level. The nanocapsules can trigger immune response and stepwise release of doxorubicin，which can improve treatment effect and reduce drug toxicity and tumor drug resistance. It can avoid or postpone the metastasis and recurrence of the breast cancer，and kill the underlying tiny metastases. The multifunctional nanocapsules will prove a new way for the integration of molecule targeted adjuvant therapy and therapeutic effect evaluation of triple-negative breast cancer.

三阴性乳腺癌（TNBC）是乳腺癌的一种特殊亚型，由于缺乏有效生物标志物，TNBC患者无法从内分泌和靶向治疗中获益, 目前TNBC的治疗仍不理想。PD-L1是免疫抑制受体-配体，在乳腺癌特别是TNBC中呈明显高表达。研究证实PD-L1可作为乳腺癌免疫治疗的有效靶点。免疫治疗和化疗联合是当前研究热点。但目前联合治疗模式多为非靶向性，无法从分子水平定位癌组织，且现有的成像手段无法及时监测，因此很难实现精准的特异性可视化靶向治疗。本研究首次将mPEG-PLGA表面修饰抗PD-L1抗体,内包载紫杉醇,复乳法制备“多空穴”纳米微囊。可靶向定位TNBC组织，激发机体免疫的同时靶向调控药物释放,实现免疫/化疗协同增敏治疗, 以提高治疗效果，降低药物的毒副作用和耐药的产生。其激发的免疫反应对潜在转移灶也有杀伤作用，可防止肿瘤转移和复发。该纳⽶微囊将会为实现TNBC精准有效的可视化诊治提供新思路。

三阴性乳腺癌（TNBC）是乳腺癌的一种特殊亚型，目前TNBC的治疗仍不理想。免疫治疗和化疗联合治疗乳腺癌是当前研究热点。但目前联合治疗模式多为非靶向性，无法从分子水平定位癌组织，且现有的成像手段无法及时监测，因此很难实现精准的特异性可视化靶向治疗。如何实现三阴性乳腺癌精准有效的特异性靶向治疗是当前研究热点。本研究采用单乳化溶剂挥发法首次将PEG-PLGA表面修饰抗PD-L1抗体,内包载DOX和SPIOs纳米粒子,制备多功能纳米微囊,并对其进行表征；CCK-8法检测纳米微囊的细胞毒性；激光共聚焦下观察靶向PD-L1的纳米微囊的体外靶向性；成像仪器检测纳米微囊的体外成像效果。流式细胞技术检测经纳米微囊作用后细胞的凋亡率。Western blot分析不同处理组细胞Bcl-2和capase-8蛋白表达来评价纳米材料对肿瘤细胞凋亡的影响。通过检测血液生化指标和HE染色标本来评估非载药纳米粒子对小鼠体内重要脏器的毒副作用。尾静脉注射10mg/mL的靶向纳米粒子200μL，评估纳米粒子的体内成像效果。探究纳米微囊体内治疗效果时实验分四组：Control、DOX、DOX-SPIOs-PLGA-PEG和DOX-SPIOs-PLGA-PEG-antiPD-L1组。治疗结束后，取每组肿瘤组织做H&E染色和TUNEL检测观察疗效。通过组织免疫荧光和流式细胞技术对纳米微囊能否激活免疫做进一步的探索。本研究成功制备了可靶向定位TNBC组织的DOX-SPIOs-PLGA-PEG-antiPD-L1纳米微囊。体内外实验显示该纳米材料无明显毒性，且有良好的靶向性，可实现US/MRI双模态成像。尾静脉注射靶向纳米材料2h后MRI成像效果最佳。靶向联合治疗组在小鼠肿瘤模型上取得较好的治疗效果，可在一定程度上激发机体免疫，实现免疫/化疗协同增敏治疗。该纳⽶微囊将会为实现TNBC精准有效的可视化靶向诊治提供新思路。