载雷西莫特微泡增效高强度聚焦超声消融及协同刺激免疫效应的实验研究  

Microbubbles carrying resiquimod enhance HIFU ablation and co-stimulate tumor immune response

作　　者：周颖 唐芮 吴念鸿 万莉 何红叶 操雨婷 熊夏林 李攀[1] Zhou Ying;Tang Rui;Wu Nianhong;Wan Li;He Hongye;Cao Yuting;Xiong Xialin;Li Pan(Department of Ultrasound,the Second Affiliated Hospital of Chongqing Medical University&Institute of Ultrasound Imaging of Chongqing Medical University,Chongqing 400010,China)

机构地区：[1]重庆医科大学附属第二医院超声科、重庆医科大学超声影像学研究所,重庆400010

出　　处：《中华超声影像学杂志》2023年第8期707-716,共10页Chinese Journal of Ultrasonography

基　　金：国家自然科学基金(81971633);重庆市自然科学基金(cstc2021jcyj-msxm X0220);重庆市卫健委中青年医学高端人才项目(2019GDRC006)。

摘　　要：目的制备载雷西莫特(R848)脂质微泡(R848-MBs), 观察其增强超声成像和增效高强度聚焦超声(HIFU)消融效果, 并评估其协同HIFU改善肿瘤免疫微环境的能力。方法通过薄膜水化-机械振荡法制备R848-MBs, 并检测其基本表征和安全性, 考察HIFU辐照R848-MBs体外控释药物作用及其体内药物代谢和生物分布情况;观察R848-MBs增强体内外超声成像效果;将6只EMT6荷瘤小鼠随机分为HIFU组和R848-MBs+HIFU组, 每组3只, 通过评估消融前后小鼠肿瘤区域的造影平均声强变化、肿瘤区域辐照后即刻超声图像灰度值变化和肿瘤TTC染色、肿瘤HE染色观察其凝固性坏死情况, 验证R848-MBs增效HIFU消融的效果;然后将45只荷瘤小鼠随机分为对照组、Free R848组、HIFU组、Blank-MBs+HIFU组、R848-MBs+HIFU组, 每组9只, 治疗后第3 d随机取各组中3只小鼠处死, 免疫荧光染色检测肿瘤细胞表面CRT的表达水平, 流式细胞术检测淋巴结、脾脏成熟DC比例和脾脏CD8+T细胞比例以评估R848-MBs协同HIFU改善肿瘤免疫微环境的能力, 通过测量剩余各组小鼠(n=6)的肿瘤体积、观察并绘制生存曲线以评估各组治疗效果。结果成功制备了安全性良好的载药脂质微泡R848-MBs, 微泡浓度为2.58×109/ml, 光镜和激光共聚焦显微镜下呈球形空泡, 粒径为 (1.72±0.11)μm, 表面电位为(-10.16±0.73)mV, 经HIFU(90 W, 3 s)辐照后药物累计释放率高达 (84.81±1.58)%, 体内血浆中R848浓度迅速下降, 治疗后24 h R848-MBs+HIFU组肿瘤组织中的药物浓度高于R848组 (P<0.01);体内外R848-MBs在造影模式下超声显像明显增强;同时, 载药微泡还能显著增效HIFU消融效果, R848MBs+HIFU组小鼠消融前后肿瘤区域的造影平均声强变化大于R848组 (P<0.05), HIFU联合R848-MBs组即刻超声灰度值变化(46.34±3.21)显著大于单纯HIFU组(10.67±1.53), 差异有统计学意义 (P<0.000 1), 肿瘤HE染色、TTC染色可观察到凝固性坏死;体内疗�Objective To prepare the resiquimod-loaded lipid microbubbles R848-MBs,evaluate their enhanced ultrasound imaging and high intensity focused ultrasound(HIFU)ablation effects,and explore their ability to improve tumor immune microenvironment synergize with HIFU.Methods R848-MBs were prepared by the thin film hydration-mechanical shock method;The basic characteristics and safety of R848-MBs were detected,the HIFU controlled-release characteristics were verified in vitro and the drug metabolism and biological distribution were investigated in vivo.The ability of enhancing ultrasound imaging was observed in vitro and in vivo.To investigate the enhanced HIFU ablation effect of R848-MBs,six EMT6 tumor-bearing mice were randomly divided into HIFU group and R848-MBs+HIFU group,three mice in each group,the changes in contrast average sound intensity before and after ablation in mouse tumor areas and the change of ultrasound image gray value in tumor area were evaluated,the tumor were resected to observe the coagulative necresis by TTC staining and HE staining.Forty-five tumor-bearing mice were randomly divided into control group,Free R848 group,HIFU group,Blank-MBs+HIFU group and R848-MBs+HIFU group,nine mice in each group.On the third day after treatment,3 mice in each group were randomly selected and killed,to evaluate the ability of R848-MBs to improve tumor immune microenvironment synergize with HIFU.The expression level of CRT on the surface of tumor cells were detected by immunofluorescence staining,the proportion of mature DC in lymph nodes,spleen,and CD8+T cells in spleen were detected by flow cytometry.The treatment effectiveness of each group(n=6)were evaluated by measuring tumor volume,observing and drawing survival curves.Results The R848-MBs lipid microbubbles with good safety were successfully prepared,with a concentration of 2.58×109/ml,as spherical bubbles under optical microscope and laser confocal microscopy,in a particle size of(1.72±0.11)μm,at a surface potential of(-10.16±0.73)mV.The cumulative dr

关 键 词：超声成像 高强度聚焦超声 雷西莫特 脂质微泡 免疫微环境 

分 类 号：R454.3[医药卫生—治疗学]