载替莫唑胺纳米粒联合低强度聚焦超声对人胶质瘤细胞的促凋亡作用

Apoptosis-promoting effect of temozolomide loaded nanoparticles combined with low intensity focused ultrasound on human glioma cells

目的制备由聚乳酸-羟基乙酸共聚物(PLGA)包载全氟戊烷(PFP)与化疗药替莫唑胺(TMZ)的液气相变型纳米粒(TMZ/PFP/PLGA NPs),联合低强度聚焦超声(LIFU)辐照,探讨其体外超声显影能力及对人胶质瘤细胞的体外干预效果。方法采用复乳法制备TMZ/PFP/PLGA NPs,检测其基本理化性质及药物包载能力,观察纳米粒体外超声显影效果;CCK-8法检测纳米粒的体外细胞毒性及与LIFU协同干预对胶质瘤细胞存活率的影响;Western blot检测协同干预后细胞内凋亡相关蛋白Bcl-2、bax及caspase-3的表达水平变化。结果在透射电镜下,TMZ/PFP/PLGA NPs呈形态规则的类圆形核壳结构,粒径(137.9±63.31)nm,对TMZ的包封率为(83.01±5.57)%,载药量(3.19±0.22)%;纳米粒与U251细胞共孵育24 h后细胞存活率仍可保持在70%以上,在同时施加LIFU辐照的协同作用下,可使细胞凋亡加速,细胞存活率明显下降;Western blot检测结果显示,协同干预可明显下调细胞内凋亡相关蛋白Bcl-2的表达,同时明显上调bax蛋白与caspase-3蛋白的表达(均P<0.05)。结论TMZ/PFP/PLGA NPs本身具有良好的基本理化性质,在高效包载化疗药物替莫唑胺的同时,具有较低的体外细胞毒性,在LIFU辐照下的协同干预可明显加速U251胶质瘤细胞凋亡,具有良好的应用前景。

Objective To prepare liquid-gas phase modified nanoparticles(TMZ/PFP/PLGA NPs)of perfluoropentane(PFP)and temozolomide(TMZ)encapsulated by polylactic-glycolic acid copolymer(PLGA),combined with low intensity focused ultrasound(LIFU)irradiation,and to investigate its ultrasound imaging ability and intervention effect on human glioma cells in vitro.Methods TMZ/PFP/PLGA NPs were prepared by compound emulsion method.The basic physical and chemical properties and drug loading ability of TMZ/PFP/PLGA NPs were detected.CCK-8 assay was used to detect the cytotoxicity of nanoparticles in vitro and the effect of synergistic intervention with LIFU on the survival rate of glioma cells.The expression levels of apoptosis related proteins Bcl-2,Bax and caspase-3 were detected by Western blot.Results Under transmission electron microscope,TMZ/PFP/PLGA NPs showed a circular core-shell structure with regular morphology,particle size was(137.9±63.31)nm,encapsulation efficiency of TMZ was(83.01±5.57)%,drug loading was(3.19±0.22)%.The survival rate of U251 cells was still above 70%after 24 hours of co-incubation with nanoparticles.Under the synergistic effect of LIFU irradiation,the apoptosis of U251 cells was accelerated and the survival rate of U251 cells was significantly decreased.The results of Western blot showed that the synergic intervention could significantly down-regulate the expression of apoptosis related protein Bcl-2,and significantly up-regulate the expression of Bax protein and caspase-3 protein(all P<0.05).Conclusions TMZ/PFP/PLGA NPs have good basic physical and chemical properties.TMZ/PFP/PLGA NPs have low cytotoxicity in vitro while efficiently loading chemotherapeutic drug timozolomide.Synergistic intervention under LIFU irradiation can significantly accelerate the apoptosis of U251 glioma cells,which has a good application prospect.