声敏型脂质纳米粒联合低功率聚焦超声对HepG2细胞增殖的影响及其体外CT成像的实验研究

Effect of sound sensitive nano-liposomes combined with low intensity focused ultrasound on proliferation of HepG2 cells and its CT imaging in vitro

目的制备一种载血卟啉单甲醚(hematoporphyrin monomethyl ether,HMME)包裹全氟溴辛烷(1-Bromoheptadecafluorooctane,PFOB)及阿霉素(doxorubicin,DOX)的声敏型脂质纳米粒(sound sensitive nano-liposomes,SNLs),探究其联合低功率聚焦超声(low intensity focused ultrasound,LIFU)体外释药、产生活性氧的能力以及对HepG2细胞增殖的影响,和其体外CT成像的能力。方法采用薄膜水化法制备SNL;检测其一般性质;高效液相色谱法检测其体外释药;荧光分光光度计和激光共聚焦显微镜观察其产生活性氧的能力; CCK-8实验检测其对HepG2细胞增殖的影响,CT观察其体外CT成像。结果成功制备SNL,粒径为(282. 53±6. 95) nm; LIFU处理8h内SNL释放阿霉素达(83. 45±2. 97)%,释药速度随LIFU强度提高而加快;以DPBF(1,3-二苯基异苯并呋喃)为活性氧探针,产生活性氧的相对量随SNL中血卟啉单甲醚增加及LIFU强度增高而增多;以DCFH-DA(2’,7’-二氯荧光黄双乙酸盐)为活性氧探针,激光共聚焦观察显示SNL+LIFU组较SNL组绿色荧光强; CCK-8检测结果显示SNL+LIFU组的细胞存活率明显较DOX组,载血卟啉单甲醚包裹全氟溴辛烷的脂质纳米粒(hematoporphyrin monomethyl ether nano-liposomes,HNL)+LIFU组和SNL组低(P <0. 05),可见SNL+LIFU明显增强了对HepG2细胞生长的抑制作用; CT成像结果显示SNL的CT值较对照组和空白组高,且CT值随PFOB浓度增大而增高。结论成功制备的声敏型脂质纳米粒的声动力效果呈HMME浓度及LIFU强度依赖性,药物释放速度受LIFU控制,可联合化疗及声动力治疗增强HepG2细胞生长的抑制效果,同时具有良好的CT成像效果。

Objective To prepare a sound sensitive nano-liposomes(SNLs) loaded with hematoporphyrin monomethyl ether(HMME),1-bromoheptadecafluorooctane(PFOB) and doxorubicin(DOX),and to investigate their abilities in releasing drugs,generating reactive oxygen species(ROS) and inhibiting HepG2 cells proliferation under low intensity focused ultrasound(LIFU) and explore its CT imaging in vitro. Methods SNLs were prepared by membrane hydration,and their basic features were tested. After LIFU,the drug-release of SNLs was detected by high performance liquid chromatography(HPLC). The ability of SNLs to produce ROS was detected via fluorescence spectrophotometry and observed via laser confocal microscopy. The proliferation of HepG2 cells was detected by CCK-8 assay and CT imaging was observed by Philips Ingenuity CT scanner. Results SNLs were successfully prepared with their particle size of 282. 53 ± 6. 95 nm. Under LIFU irradiation within 8 h,the nano-liposomes released(83. 45 ± 2. 97) %DOX,and the releasing speed was increased with the increase of LIFU intensity. With DPBF as ROS probe,the relative amount of produced ROS was increased as the dose of HMME or the intensity of LIFU rose. When DCFH-DA was used as ROS probe,the green fluorescence in the HepG2 cells treated with SNLs + LIFU was brighter than those treated with SNLs alone. The results of CCK-8 assay indicated that SNL + LIFU treatment decreased the cell viability than the cells treated with DOX or SNLs alone,and HMME nano-liposomes(HNL) + LIFU(P < 0. 05),indicating that SNL + LIFU treatment enhancing the inhibition on HepG2 cells proliferation. The CT value in SNL was higher than it in control group or blank group,and the value was increased as the increase of PFOB dose. Conclusion The sonodynamic effect of our prepared SNLs is in a HMME-dose and LIFU-intensity dependent manner. LIFU can prompt drug release in the nano-liposomes.SNLs + LIFU combined with sonodynamic therapy and chemotherapy enhances the inhibition on HepG2 cells proliferation. Besides,SNL can be used in CT imaging in vitro.