载N2O微泡的声动力效应研究

Study on sonodynamic effects of N2O-microbubbles

目的探讨载N2O微泡的声敏性及其声动力效应。方法采用旋转成膜与机械振荡相结合的方法制备N2O微泡,检测其基本特征和细胞毒性。采用激光共聚焦显微镜观察N2O微泡与人乳腺癌MDA-MB-231细胞的结合情况。采用单线态氧荧光探针(SOSG)法、DCFH-DA法检测N2O微泡的声敏性。采用CCK-8法、流式细胞术和CAM/PI双染法检测N2O微泡的声动力治疗效果。结果制备的N2O微泡呈乳白色混悬液,形态规则,大小均匀,浓度为(2.65±0.41)×108/ml,粒径为(458.10±17.26)nm,电位为(–16.20±0.35)mV。N2O微泡与细胞共同孵育后,细胞膜周围和细胞质内出现大量微泡聚集。低强度聚焦超声(LIFU)辐照处理的N2O微泡溶液荧光强度值明显增加(P<0.05);吞噬N2O微泡的细胞经LIFU处理后,胞内可见明显增强的绿色荧光。LIFU辐照处理加入N2O微泡的细胞,细胞存活率明显降低(44.10%,P<0.05);LIFU结合N2O微泡处理后,细胞凋亡率明显增高(68.33%),并出现大量细胞死亡。结论成功制备的N2O微泡具有声敏性,其声动力效应可有效杀伤肿瘤细胞。

Objective To investigate the sonosensitivity of N2O-microbubbles(N2O-mbs)and its sonodynamic effect on tumor cells.Methods The N2O-mbs were prepared by improved mechanical oscillation method.The basic characteristics of N2Ombs were determined.The cytotoxicity of these microbubbles was evaluated by CCK-8 assay.The delivery efficiency of N2O-mbs to MDA-MB-231 cells was observed under a laser scanning confocal microscope.The sonosensitivity of N2O-mbs was evaluated by singlet oxygen sensor green(SOSG)assay and reactive oxygen species assay kit,which detected its extracellular and intracellular reactive oxygen species generation.In vitro sonodynamic properties of N2O-mbs on tumor cells were evaluated by CCK-8 assay,flow cytometry and CAM/PI double staining.Results These N2O-mbs presented with homogenized size distribution and milky white suspensions after water reconstitution.The average diameter of N2O-mbs was(458.10±17.26)nm and its zeta potential was(–16.20±0.35)mV.N2O-mbs exhibited a good biocompatibility and had an obvious delivery efficiency in cancer cells in vitro.These microbubbles could generate singlet oxygen(1O2)in aqueous solution and produce reactive oxygen species in the cells after irradiation with low intensity focused ultrasound(LIFU).Further,CCK-8 test results suggested N2O-mbs combined with LIFU action would cause a significant decrease in cell viability.Flow cytometry results showed a significant advanced apoptosis in cancer cells.A large number of dead cells were also observed using a laser scanning confocal microscope after N2O-mbs combined with LIFU treatment.Conclusion N2O-mbs has extraordinary sonosensitivity under the condition of microbubble encapsulation and could effectively kill tumor cells though sonodynamic effects in vitro.