Preparation and in vivo and vitro Evaluation of pH-Sensitive Liposomes Loading Arsenic Trioxide
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摘要: 目的 将三氧化二砷制备为pH敏感型脂质体，并进行体外抗胶质瘤活性与体内安全性评价。方法 采用反相微乳法制备疏水性修饰的锰砷共沉淀，后采用乳化蒸发法进行负载制备脂质体(Liposome/MnAs)。测定Liposome/MnAs的粒径、PDI以及Zeta电位；透射电镜观察Liposome/MnAs的形态；ICP-MS测定包封率；透析袋法考察其体外不同pH条件下的释药特性；考察酸性条件下体外MRI成像能力。共聚焦显微镜观察鼠源性脑胶质瘤GL261细胞对Liposome/C6的摄取情况和胞内分布情况；MTT法考察游离三氧化二砷及Liposome/MnAs对GL261的毒性；流式细胞仪检测游离三氧化二砷及Liposome/MnAs对GL261细胞的凋亡作用。结果 制备的锰砷共沉淀具有疏水性，可较好地被氯仿溶解；Liposome/MnAs呈规整的类球型，粒径约为(286.43±6.41)nm，As包封率为(48.32±5.95)%；可在pH5.4条件下迅速响应释放As3+及Mn2+，具有良好的体外MRI成像能力。GL261细胞对C6标记的脂质体的摄取情况较好，且主要分布在细胞质。Liposome/MnAs抑制GL261细胞生长的作用(IC50=2.99μmol/L)较游离三氧化二砷(IC50=4.74μmol/L)有所上升；细胞凋亡实验也表明Liposome/MnAs较游离三氧化二砷有更好地促进GL261细胞凋亡的作用，促凋亡率由13.73%提高至21.42%。给药周期内，Liposome/MnAs对小鼠体内主要脏器无明显毒性。结论 以反相微乳法制备的锰砷共沉淀具有良好的疏水性，被脂质体包载制备为Liposome/MnAs，具有pH响应释药的特性，能有效增强GL261细胞对脂质体摄取。可增强三氧化二砷对胶质瘤细胞GL261的毒性，且具有一定的体内安全性。Abstract: OBJECTIVE To prepare pH-sensitive liposomes with arsenic trioxide and evaluate the anti-glioma activity in vitro and safety in vivo.METHODS The hydrophobic modified MnAs coprecipitates were prepared by reverse phase microemulsion method, and liposomes loaded with MnAs coprecipitations (Liposome/MnAs) were prepared by emulsion evaporation method. The particle size, PDI and Zeta potential of Liposome/MnAs were measured. The morphology of Liposome/MnAs was observed by transmission electron microscopy. Encapsulation efficiency was determined by inductively coupled plasma mass spectrometer. Dialysis bag method was used to investigate the drug release characteristics under different pH conditions in vitro. The ability of in vitro MRI imaging was investigated under acidic conditions. The uptake and intracellular distribution of Liposome/C6 in mouse derived glioma cells GL261 were observed by confocal microscopy. The toxicity of free arsenic trioxide and Liposome/MnAs on GL261 was investigated by thiazolium blue assay. The apoptosis effect of free arsenic trioxide and Liposome/MnAs on GL261 cells was detected by flow cytometry. Liposomes were labeled with coumarin 6.RESULTS The prepared MnAs coprecipitations were hydrophobic and could be dissolved by chloroform well. Liposome/MnAs were spherical in shape, with a particle size of (286.43±6.41) nm and As encapsulation efficiency of (48.32±5.95)%. It could quickly respond to release As3+ and Mn2+ under the condition of pH 5.4, and had a good ability of in vitro MRI imaging Cell uptake assay showed that GL261 cells had a good uptake of C6-labeled liposomes, which were mainly distributed in the cytoplasm. The inhibitory effect of Liposome/MnAs on GL261 cell growth was higher than that of free arsenic trioxide, and the IC50 decreased from 4.74μmol/L to 2.99μmol/L. Apoptosis experiments also showed that Liposome/MnAs had a better effect on promoting the apoptosis of GL261 cells than free arsenic trioxide, and the proapoptotic rate was increased from 13.73% to 21.42%. Liposome/MnAs had no obvious toxicity to the main organs in mice within an administration cycle.CONCLUSION The MnAs coprecipitates prepared by reverse microemulsion method have good hydrophobicity and can be encapsulated to by liposome prepare Liposome/MnAs, which had pH-responsive drug release characteristics, could enhance ATO uptaked by glioma cell GL261. The Liposome/MnAs enhanced the toxicity of arsenic trioxide to GL261 cell and had certain safety in vivo.
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Key words:
- arsenic trioxide /
- pH sensitive liposomes /
- reversed microemulsion method /
- anti-glioma
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表 1 Liposome/MnAs及空白载体Liposome的粒径、PDI和Zeta电位
样品 粒径/nm PDI 电位/mV Liposome 196.7±1.75 0.23±0.03 -1.81±0.96 Liposome/MnAs 286.43±6.41 0.3±0.02 -14.03±2.03 
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