磁性长循环热敏脂质体—endoglin靶向微泡载药复合体构建及其对亚高温环境肝肿瘤基质细胞的作用研究

The principle of thermal ablation for necrotizing hepatic tumor depends on high temperature in local areas. However, because of heterogeneity of biological structures,sub-high temperature fields exist during the procedure of thermal ablation,which may result in the residual tumors and recurrence. How to reduce the residual tumors in sub-high temperature fields is considered as one critical problem for improving the ablative effects .Our previous study and other literatures verified that the development of residual tumor cells was supported by the tumor microenvironment, in which stroma cells were the important component. Is it feasible to interfere with the growth of residual tumor by destroying the microenvironment ? Our previous researches disclosed destruction of microbubbles could help the thermosensitive liposome loaded with anti-inflammatory drug to be delivered into sub-high temperature areas. The biological behavior of tumor associated macrophages were inhibited. However, the effects of single drug, the targeting of ordinary microbubble and the short existence duration of the drug carriers were limited. . Based on the previous results, we aim to build a new drug delivery system, which will combine magnetic long-circulating thermosensitive liposome(MLCTL) with endoglin-targeted microbubbles.This drug complex carrier will load ganoderma appanatum polysaccharides(GAPs) in liposome and COX-2 inhibitors in microbubbles. Anti-endoglin antibody on microbubbles may recognize the tumor vessels specifically, and then the microbubbles were destroyed under high power ultrasound beam which could increase the permeability of vascular endothelial cells in local areas and send the COX-2 inhibitors into the ablated areas. GAPs were released by MLCTL under both sub-high temperature field and magnetic field of ablation. Both GAPs and COX-2 inhibitors will act on the tumor stroma cells. Through observing the change of biological features of stroma cells, including tumor associated fibroblast cells and tumor associated macrophages, the role of the new carriers on the tumor microenviroment was determined, which may supply a new method for improving ablation effects.

热消融技术通过高温灭活细胞。但因生物结构的非均一性，热消融中存在亚高温分布，可导致组织灭活不全。如何减少亚高温带来的肿瘤残留成为提高消融疗效的关键问题之一。我们前期研究及文献证实：肿瘤微环境为残留肿瘤细胞生长提供“土壤”，其中基质细胞是重要组份。是否能通过破坏微环境而干扰残留肿瘤生长？我们前期发现通过超声微泡的定向爆破，将携抗炎药物热敏脂质体递送至靶区，能抑制肿瘤相关巨噬细胞。但单一药物作用有限，普通微泡靶向性差，且载药体循环时间短。本项目在前期基础上拟构建新型载药复合体，将载树舌多糖的磁性长循环热敏脂质体与携COX2抑制剂的endoglin靶向微泡相联合，通过特异抗体使微泡识别肿瘤微血管，利用微泡被动定向爆破及磁性热敏脂质体磁场聚集、温场释药等多重靶向作用，将针对基质细胞的药物高效输送至亚高温环境，干预细胞活性，重塑肿瘤微环境，为减少亚高温带来的热消融术后肿瘤残留寻求新思路。

树舌多糖具有良好抗肿瘤活性和增强机体免疫功能等作用可以阻止肿瘤生长发展，但是目前仍存在口服用药利用度低的问题。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。本项目制备载树舌多糖磁性长循环热敏脂质体-微泡复合体，利用微泡被动定向爆破，在亚高温场释药，将针对基质细胞的药物高效输送至亚高温环境，并观察载药体对亚高温场中兔肝VX2肿瘤的作用，减少亚高温带来的热消融术后肿瘤残留。通过对比薄膜分散法、乳化法溶剂挥发法、复乳化-溶剂挥发法三种方法制备树舌多糖长循环热敏脂质体的粒径、zeta电位、包封率和载药量，确立了复乳化-溶剂挥发法制备树舌多糖长循环热敏脂质体的处方和工艺，通过苯酚硫酸法、紫外分光光度法检测长循环热敏脂质体药物包封率及不同温度下释药特性。所制备的脂质体粒径163.2±17.1nm, 包封率为82.4%，药量为7.2%，在28天内脂质体稳定性良好，37℃时基本不释放，42℃时累计释药量到达90%以上。通过薄膜分散法制备全氟丙烷微泡，粒径为630±18nm，用生物素-亲和素将脂质体和微泡偶联后，载树舌多糖长循环热敏脂质体-全氟丙烷微泡的粒径1510±100 nm。建立VX2兔肝肿瘤模型，肝VX2荷瘤兔随机分为微波亚高温辐照组(SHM）、 树舌多糖载药微泡组（GLMC）、微波亚高温辐照联合树舌多糖载药微泡治疗组（SHM + GLMC），空白对照组（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，免疫组化和HE染色进行病理分析。结论：确立复乳化-溶剂挥发法制备树舌多糖长循环热敏脂质体的处方和工艺，所制备的脂质体具有良好的热敏特性，可为进一步递药系统研究提供依据。载树舌多糖脂质体-微泡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并可在亚高温场中对肿瘤肿瘤微环境中巨噬细胞形态破坏，抑制肿瘤生长。