非均匀温场下silibinin热敏脂质体-微泡复合体抑制肝肿瘤相关巨噬细胞的作用研究

Local thermal ablative techniques coagulates the tumor cells via inducing focal hyperthermia, and has emerged as one important theraputic method for liver cancer.However,the existence of residual tissue after ablation is one of problems demanding prompt solution.Our previous studies and literatures demonstrated that the inherent nonlinear structure of biological tissue easily lead to the nonuniform distribution of thermal field during ablation. Especially in the subthermal field, the tumor cells may not be necrotized and then develop into residual tissue in the support of the inflammatory microenvironment,which is made up of many kinds of inflammatory cells mainly involving tumor associated macrophages（TAMs）. Therefore,simply increasing the ablative energy was difficult to necrotize the residual viable cells completely. In that way, may the ablation combined with the drug be more effective, and by take advantage of the nonuniform thermal field,may the drug delivery target to residual tissues and inhibit TAMs, and finally reduce the residual viable tissue? Our previous studies and literatures confirmed that target microbubble destructon enhanced permeability of vascular endothelial cells locally. And within the subthermal field,the drug release of thermo-sensitive liposome will be triggered.These two conditions supplied probability for dual targeted drug delivery..Based on the previous results,in the current study,we plan to establish a new drug delivery system,the complex of thermosensitive liposome and microbubble.Anti-inflammatory agent silibinin, which is considered to have obvious effects on the inflammatory enviroment and TAMs, will be loaded on the complex. Using the effects of thermal field and microbubble destruction achieves the target,abundant and rapid drug delivery to the subthermal areas.The changes of biological characteristics of TAMs in liver cancer and the residual viable tissue within the subthermal field will be investigated. From above,we aim to establish a new strategy for thermal ablation on liver cancer,by using the thermosensitive liposome-microbubble as a critical drug carrier,combining the thermal effect,chemical effect and physical effect, and interferring the tumor microenviroment as the target.

热消融是利用高温灭活肿瘤的肝癌治疗重要方法，但消融后肿瘤残留存在是一大难题。我们前期研究和文献表明：生物固有的非线性结构易致热消融非均匀温场形成，其中亚高温场内细胞很难杀灭，且在肿瘤相关巨噬细胞（TAMs）为主的炎性细胞参与的微环境支持下，可发展为残留组织。因此单纯提高热能很难彻底消灭残留，那么是否能结合药物作用，利用非均匀温场，使之靶向残留区，干预TAMs，最终抑制残留细胞呢？我们前期研究及文献证实微泡爆破能增加局部血管内皮通透性，热敏脂质体能在亚高温场中释药，由此提供了双重靶向给药条件。本项目在前期基础上拟构建新型热敏脂质体微泡复合体，负载显著作用于炎性微环境及TAMs的抗炎药silibinin，通过药物靶向大量迅速释放，探索亚高温场内肝癌TAMs和残留肿瘤生物学变化，旨在建立以载药复合体为核心，以微环境干预为切入点，结合热、化学及物理效应的肝癌热消融治疗新策略，为减少残留提供新思路。

水飞蓟宾是具有抗炎抗氧化的保肝药物，也被认为能够显著改变肿瘤炎性微环境，从而抑制肿瘤生长。但目前水飞蓟宾水溶性和脂溶性小，生物利用度低，对组织渗透性较差。如能实现靶向递药，将有可能提高其吸收与利用率。超声造影微泡可在一定声强的局部声场中爆破，实现靶区血管内皮系统开放，促进药物进入组织间隙。热敏脂质体可在一定温度下释放药物，实现局部给药。本研究拟将这二者的特性相结合，研制新的热敏脂质体-微泡复合体载药体系，加强药物的组织渗透力和靶向性，实现局部释药控制。制备载水飞蓟宾热敏脂质体-微泡复合体并进行质量分析。并观察载药体对亚高温场中兔肝VX2肿瘤的作用。. 以薄膜旋蒸法制备载水飞蓟宾热敏脂质体，然后利用戊二醛偶联法将超声微泡Sonovue与载药脂质体相连接，通过紫外分光光度法检测热敏脂质体药物包封率及不同温度下释药特性。通过激光共聚焦显微镜检测载药脂质体与微泡的偶联率。结果发现，载水飞蓟宾热敏脂质体-微泡Sonovue复合体粒径2724 ± 559.9 nm， 药物包封率71.0 ± 8.3%， 41℃时药物平均释放率明显升高，达到55.6± 7.6%。偶联后，载药脂质体在微泡周围聚集，热敏脂质体与微泡偶联率为 90.0 ± 4.9%。. 将40只肝VX2荷瘤兔随机分为4组，每组10只动物： 微波亚高温辐照组(sub-hyperthermia microwave, SHM）、 水飞蓟宾载药微泡组（STLMC）、微波亚高温辐照联合水飞蓟宾载药微泡治疗组（SHM + STLMC），空白对照组（Blank control, BL）。分别给予不同干预。治疗后7 d及21 d，采用二维灰阶超声、超声造影成像评价术后各组肿瘤体积和最大径变化。透射电镜观察各组干预区超微结构变化。结果发现，SHM + STLMC组术后7 d及21 d肿瘤体积及最大径均明显小于同期其余各组（P < 0.05）；电镜下各组均出现染色质异常、细胞器受损的巨噬细胞，SHM + STLMC组受损巨噬细胞数目较其余各组明显增多（P < 0.05）。. 结论：载水飞蓟素热敏脂质体-微泡Sonovue复合体具有良好的偶联率，较高的药物包封率及控温释药特性，为药物递送提供一种新的载体。在亚高温场中STLMC可造成肿瘤肿瘤微环境中巨噬细胞形态破坏，抑制肿瘤生长。