载阿霉素细胞膜纳米囊泡对黑色素瘤B16F10细胞的杀伤效应

doi:10.13481/j.1671-587x.20200502

吉林大学学报(医学版) ›› 2020, Vol. 46 ›› Issue (05): 905-910.doi: 10.13481/j.1671-587x.20200502

• 基础研究 • 上一篇

载阿霉素细胞膜纳米囊泡对黑色素瘤B16F10细胞的杀伤效应

杨泽斌, 王明月, 陈丽, 刘宁, 王浩, 崔梅英, 方楷漪, 夏薇, 关新刚

北华大学医学技术学院肿瘤靶向治疗重点实验室, 吉林吉林 132013

收稿日期:2020-02-11 发布日期:2020-10-23
通讯作者: 夏薇,教授,硕士研究生导师(Tel:0432-64608115,E-mail:xiawei4016@126.com);关新刚,研究员,硕士研究生导师(Tel:0432-64608115,E-mail:guanxg@ciac.ac.cn) E-mail:xiawei4016@126.com;guanxg@ciac.ac.cn
作者简介:杨泽斌(1996-),男,吉林省吉林市人,在读医学硕士,主要从事纳米抗肿瘤药物方面的研究。
基金资助:
国家自然科学基金资助课题（51503003）；吉林省科技厅科技发展计划项目资助课题（20180101213JC）；吉林省教育厅科学技术研究项目资助课题（JJKH20200033KJ）；吉林省人社厅人才开发资金项目资助课题（201858）；吉林省卫健委卫生计生青年科技骨干培养计划项目资助课题（2017Q040）；吉林省吉林市科技局科技创新发展计划项目资助课题（201831729）；北华大学青年科研创新团队项目资助课题（2017）；北华大学研究生创新计划项目资助课题（北华研创合字〔2019〕060）

Killing effect of doxorubicin-loaded cell membrane nanovesicles on melanoma B16F10 cells

YANG Zebin, WANG Mingyue, CHEN Li, LIU Ning, WANG Hao, CUI Meiying, FANG Kaiyi, XIA Wei, GUAN Xingang

Key Laboratory of Targeting Tumor Therapy, College of Medical Technology, Beihua University, Jilin 132013, China

Received:2020-02-11 Published:2020-10-23

摘要/Abstract

摘要： 目的：制备人胚胎肾细胞来源的细胞膜纳米囊泡（NVs），其内腔载阿霉素（DOX）得到新型纳米药物载NVs细胞膜DOX（NVs-DOX），探讨NVs-DOX的肿瘤细胞内吞作用及对黑色素瘤细胞的杀伤效应。方法：利用超速离心法提取HEK293细胞膜，脂质体挤出仪对细胞膜进行处理得到细胞膜NVs；应用电转化法将DOX担载于NVs的内腔，得到NVs-DOX，通过动态光散射检测NVs粒径。以不加囊泡细胞为对照组，细胞膜NVs为实验组，采用MTT法检测不同浓度（5、10、20、50和100 mg·L^-1）NVs对NIH3T3细胞的生物相容性。以小分子DOX（10 mg· L^-1）为对照组，不同浓度（0.001、0.005、0.010、0.050、0.100、0.500和1.000μmol·L^-1）NVs-DOX为实验组，采用荧光显微镜成像和流式细胞术检测NVs-DOX在黑色素瘤B16F10细胞中的荧光分布情况；细胞毒性实验，MTT法检测各组细胞存活率；活/死细胞染色法检测NVs-DOX（1 μmol·L^-1）处理后的B16F10细胞死亡情况。结果：利用细胞膜材料制备平均粒径为254.3 nm的NVs，电转化法得到了粒径约为289.6 nm的NVs-DOX。MTT法检测，不同浓度NVs组NIH3T3细胞存活率均>100%；荧光成像检测，药物孵育3 h后，NVs-DOX组细胞核内荧光强度略低于小分子DOX组；流式细胞术检测，NVs-DOX组的内吞效率（91.07%）略低于小分子DOX组（95.47%）；细胞毒性实验，NVs-DOX组与小分子DOX组均显示出对B16F10细胞浓度依赖性的杀伤效应，DOX浓度在0.1 μmol·L^-1时B16F10细胞存活率<20%，NVs-DOX组与小分子DOX B16F10细胞存活率比较差异无统计学意义（P>0.05）；活/死细胞染色，NVs-DOX组与小分子DOX组黑色素瘤B16F10细胞中死细胞占总细胞比率比较差异无统计学意义（P>0.05）。结论：成功制备了人胚肾细胞来源的细胞膜NVs及载DOX的新型纳米药物NVs-DOX，NVs-DOX具有较高的肿瘤细胞内吞效率及明显的黑色素瘤细胞的杀伤效应。

关键词: 细胞膜, 纳米囊泡, 阿霉素, 细胞内吞, 杀伤效应

Abstract: Objective: To prepare the cell membrane nanovesicles (NVs) derived from the human embryonic kidney cells and the doxorubicin (DOX)-loaded nanovesicles(NVs-DOX),and to investigate the cellular uptake of NVs-DOX and the killing effect on the melanoma cells. Methods: The cell membrane was prepared from HEK293 cells with ultracentrifugation method and the cell membrane was treated with Liposome extruder to obtain the cell membrane NVs;DOX was encapsulated into the inner cavity of NVs by electric shock to get the NVs-DOX. The size of NVs-DOX was determined by nanoparticle size analyzer. The cells without vescicles were used as control group,and the cell membrane NVs were used as experiment group.The biocompatibilities of NVs on the NIH3T3 cells under different concentrations(5,10,20,50 and 100 mg·L^-1)were detected by MTT method. The free DOX (10 mg·L^-1) was sued as control group,and different concentrations(0.001,0.005,0.010,0.050,0.100,0.500 and 1.000 μmol·L^-1) of NVs-DOX were used as experiment groups.Fluorescent imaging and flow cytometry were used to analyze the fluorescence distribution of NVs-DOX in melanoma B16F10 cells.In cytotoxicity experiment,the survival rates of cells in various groups were detected by MTT method;Live/dead cell staining method was used to detect the death of B16F10 cells after treatment with NVs-DOX (1 μmol·L^-1). Results: The NVs with a mean size of 254.3 nm were prepared with cell membrance materials and NVs-DOX with a mean diameter of 289.6 nm were also obtained with electric shock.The MTT results indicated that the survival rates of cells in different concentrations of NVs groups were higher than 100%.The fluorescence imaging results showed that after 3 h of drug incubation,the fluorescence intensity in the cell nucleus in NVs-DOX group was slightly lower than that in small molecule DOX group.The flow cytometry results showed that the internalization rate in NVs-DOX group(91.07%) was slightly lower than that in free DOX group(95.47%). The cytoxicity experiment results showed that both NVs-DOX group and small molecule DOX group displayed concentration-dependent killing effects on the B16F10 cells.When the concentration of DOX was 0.1 μmol·L^-1,the survival rate of B16F10 cells was lower than 20%. There was no significant difference in the survival rate of B16F10 cells between NVs-DOX group and free DOX group(P>0.05).The Live/dead cell staining results showed that there was no significant difference in the ratio of dead cells to total cells in the melanoma B16F10 cells between NVs-DOX group and free DOX group(P<0.05). Conclusion: The cell membrane NVs derived from human embryonic kidney cells and new DOX-loaded drug NVs-DOX are successfully prepared. NVs-DOX have good cellular uptake in the B16F10 cells and significantly killing effect on the melanoma cells.

Key words: cell membrane, nanovesicle, doxorubicin, cellular uptake, killing effect

中图分类号:

杨泽斌, 王明月, 陈丽, 刘宁, 王浩, 崔梅英, 方楷漪, 夏薇, 关新刚. 载阿霉素细胞膜纳米囊泡对黑色素瘤B16F10细胞的杀伤效应[J]. 吉林大学学报(医学版), 2020, 46(05): 905-910.

YANG Zebin, WANG Mingyue, CHEN Li, LIU Ning, WANG Hao, CUI Meiying, FANG Kaiyi, XIA Wei, GUAN Xingang. Killing effect of doxorubicin-loaded cell membrane nanovesicles on melanoma B16F10 cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(05): 905-910.

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载阿霉素细胞膜纳米囊泡对黑色素瘤B16F10细胞的杀伤效应

Killing effect of doxorubicin-loaded cell membrane nanovesicles on melanoma B16F10 cells

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