多功能鸦胆子油分子配型组装纳米乳给药系统逆转肿瘤多药耐药的研究

In the study, multidrug resistance (MDR) in the chemotherapy is the breakthrough point. Brucea javanica oil capable of reversal MDR and anticancer was used as the oil phase of nanoemulsions (NEs), and the core-matched nanotechnology (CMT) was applied to prepare the multifunctional NEs loaded with functional molecular having different chemical and physical characters, targets and application. The NEs modified with tumor-penetrated peptide have dual-functions of therapy and diagnosis. Firstly, the hydrophobic paclitaxel (PTX) and hydrophilic fluorescent probe was loaded into Brucea javanica oil phase to prepare drug-carrier core-matched nanoemulsions (CMNEs) with high drug loading, good stability and controllable release. The CMNEs was endowed with tumor targeting and penetration by the modification of iRGD. Secondly, the target and stage of CMNEs reversing MDR was investigated from the molecular levels of protein expression, transcription and enzyme activity. And the tumor target and local pharmacokinetics of CMNEs was studied through the in vivo imaging system and microdialysis. Finally, from the level of cell-organ-tissue-animal, the mechanism of reversal MDR was clarified to provide the theory evidences and model functions for the multifunctional Traditional Chinese Medicine CMNEs.

本课题以肿瘤化疗的多药耐药(MDR)为切入点，以具有逆转MDR和抗肿瘤活性的鸦胆子油为纳米乳油相，采用分子配型组装纳米技术(core-matched nanotechnology, CMT)，将理化性质、作用靶点、用途各异的功能分子（药物、荧光探针等）载入油相，通过肿瘤穿膜肽修饰，形成具有治疗、诊断双重功能的纳米乳。首先，采用CMT将疏水性紫杉醇和水溶性荧光探针载入鸦胆子油，制备载药量高、稳定性好、释药可控的多功能分子配型组装纳米乳(CMNEs)，通过iRGD修饰赋予CMNEs肿瘤靶向性和肿瘤渗透性。其次，从分子水平，探讨鸦胆子油CMNEs逆转MDR的靶点和环节；借助活体成像技术和微透析技术，研究鸦胆子油CMNEs的肿瘤靶向性及肿瘤局部药动学。最终，从细胞-器官-组织-动物水平，阐明多功能鸦胆子油CMNEs逆转MDR的机制，为中药多功能CMNEs的构建提供理论基础和示范作用。

在肿瘤治疗方面，西医注重局部治疗，而中医更加重视整体调理。化疗能够直接杀伤或抑制瘤细胞，但受限于骨髓抑制，免疫低下等毒副作用和不良反应。因此，中药与化疗药联合使用在恶性肿瘤的综合治疗中可优势互补。本课题选择既能逆转肿瘤MDR，又具有抗肿瘤活性的中药鸦胆子油与化疗药紫杉醇联合用药，构建了多功能鸦胆子油分子配型组装纳米乳给药系统，从体外细胞、体内整体动物水平探讨了两者联合用药的抗肿瘤疗效及作用机制。.体外细胞试验结果显示PTX-OA/BJO CMNEs 在50-100nmol/L 的浓度范围内的细胞毒作用明显强于紫杉醇单独给药组（P<0.01）；PTX-OA/BJO CMNEs 组能使停滞在G2/M 期的细胞百分比提高至（63.34±0.96）%，与PTX-OA CMNEs组（37.82±1.32）%具有显著性差异（P<0.05）；PTX-OA/BJO CMNEs组和PTX-OA CMNEs组的凋亡细胞分别为（34.45±1.34）%和（28.40±0.85）%，具有显著性差异（P<0.05）。.体内动物试验表明鸦胆子油注射液、泰素组、PTX-OA/BJO CMNEs 组的抑瘤率分别为24.7%，39.6%，45.4%，PTX-OA/BJO CMNEs 组的抗肿瘤作用强于泰素组（P<0.05）；且在治疗过程中泰素组裸鼠体重和活跃度逐渐下降，说明泰素组存在的副作用较其他组大。HE 染色后高倍镜（×100）下观察PTX-OA/BJO CMNEs 组癌细胞密度减少比PTX-OA CMNEs 明显。TUNEL法检测细胞凋亡率顺序是：PTX-OA/BJO CMNEs>泰素>鸦胆子油注射液>生理盐水。PCNA 和拓扑异构酶II（Topo II）免疫组化阳性结果呈棕黄色，其表达量依次是生理盐水>鸦胆子油注射液>泰素>PTX-OA/BJO CMNEs。体内动物试验初步表明了体内组织和肿瘤部位的药物浓度和荧光强度在一定时间内比值基本一定，说明两者之间具有较好的相关性。.研究结果表明鸦胆子油与紫杉醇联用具有协同作用，鸦胆子油通过诱导肿瘤细胞凋亡、抑制肿瘤细胞DNA复制和细胞增殖等机制增强紫杉醇的抗肿瘤活性。本研究建立了多功能鸦胆子油CMNEs载体系统，为中药与化疗药联合用药发挥协同作用和诊断作用的复合纳米载体的构建提供了研究的思路与方法。