靶向阳离子脂质体递送miRNA对三阴性乳腺癌抑制作用的研究

doi:10.3877/cma.j.issn.1674-0793.2017.03.002

中华普通外科学文献(电子版) ›› 2017, Vol. 11 ›› Issue (03) : 148 -153. doi: 10.3877/cma.j.issn.1674-0793.2017.03.002

所属专题：文献；

论著

靶向阳离子脂质体递送miRNA对三阴性乳腺癌抑制作用的研究

徐枫¹, 刘婷¹, 赵亚男², 陆森¹, 陈园园¹, 叶园园¹, 刘培峰², 韩宝三¹^,^†(

)

^1. 200092　上海交通大学医学院附属新华医院普外科　普通外科实验室
^2. 200127　上海交通大学医学院附属仁济医院中心实验室

收稿日期:2017-04-06 出版日期:2017-06-01
通信作者: 韩宝三
基金资助:
国家自然科学基金资助项目(81172078); 宁波市社会发展重大择优科技攻关项目(2012C5013)

Delivery of tumor suppressive miRNA to triple negative breast cancer by targeting cationic liposome

Feng Xu¹, Ting Liu¹, Yanan Zhao², Sen Lu¹, Yuanyuan Chen¹, Yuanyuan Ye¹, Peifeng Liu², Baosan Han¹^,^†()

^1. Department of General Surgery, General Surgery Laboratory, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
^2. Medical Science Research Center, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200127, China

Received:2017-04-06 Published:2017-06-01
Corresponding author: Baosan Han
About author:
Corresponding author: Han Baosan, Email: hanbaosan@126.com

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引用本文：

徐枫, 刘婷, 赵亚男, 陆森, 陈园园, 叶园园, 刘培峰, 韩宝三. 靶向阳离子脂质体递送miRNA对三阴性乳腺癌抑制作用的研究[J]. 中华普通外科学文献(电子版), 2017, 11(03): 148-153.

Feng Xu, Ting Liu, Yanan Zhao, Sen Lu, Yuanyuan Chen, Yuanyuan Ye, Peifeng Liu, Baosan Han. Delivery of tumor suppressive miRNA to triple negative breast cancer by targeting cationic liposome[J]. Chinese Archives of General Surgery(Electronic Edition), 2017, 11(03): 148-153.

目的

探讨靶向阳离子脂质体（CLPs）递送miRNA对三阴性乳腺癌（TNBC）相关特异性抑瘤基因的作用。

方法

通过薄膜分散法评估理化性质对阳离子脂质成分进行优化，制备具有理想表征性质的新型透明质酸（HA）CLP，表征CLPs的粒径和zeta电位。对CLPs进行表面HA靶向修饰，并通过激光共聚焦观察和流式细胞术的荧光检测评估HA-CLPs进入细胞的能力。利用EdU细胞增殖检测和细胞划痕实验对负载miRNA的HA-CLPs对细胞产生的生物学效应进行研究。

结果

薄膜分散法制备的CLPs纳米颗粒分散性良好、形态均一，呈中空球形结构，CLPs平均粒径为（180.6±3.4）nm、平均zeta电位为（43.4±2.8）mV。经过HA修饰后的纳米颗粒及运载miR-205后的纳米颗粒平均粒径分别为（236.65±6.9）nm、（205.6±2.2）nm，平均电位分别为（29.1±5.4）mV、（11.2±1.1）mV。经过激光共聚焦检测和流式细胞术荧光检测，验证了HA-CLPs能够顺利将miR-205运送至细胞内而分布在细胞核附近，且HA的靶向作用显著。EdU实验和细胞划痕实验验证了运载入TNBC细胞内的miR-205和miR-34a对其增殖和迁移的抑制作用。

结论

制备的靶向阳离子脂质体对TNBC细胞具有较强的靶向特异性，且通过靶向递送miR-205和miR-34a对TNBC细胞的增殖和迁移起到了一定的抑制作用。

关键词: 脂质体, 阳离子, 透明质酸, 乳腺肿瘤

Objective

To investigate the effect of delivering specific tumor suppressor genes miRNA in triple negative breast cancer (TNBC).

Methods

Film dispersion method was utilized to prepare for the new-pattern hyaluronic acid (HA)-targeted cationic liposome (HA-CLPs). The physicochemical property, such as the representation of the size and zeta-potential of CLPs nanoparticles, was assessed and then the nanoparticles were modified with HA. Confocal and flow cytometry (FCM) was applied to assess targeting capacity, while EdU cell proliferation test and cell wound scratch assay to assess biological effects of HA-CLPs.

Results

Nanoparticles of CLPs were of good dispersibility and shaped as homogeneous form of hollow globe. The average size of CLPs was (180.6±3.4) nm and the average zeta-potential was (43.4±2.8) mV. The average size of HA-targeted nanoparticles and miRNA-loaded HA-targeted nanoparticles was (236.65±6.9) nm and (205.6±2.2) nm. The average zeta-potential of HA-targeted nanoparticles and miRNA-loaded HA-targeted nanoparticles was (29.1±5.4) mV and (11.2±1.1) mV. Confocal and FCM verified the targeting capacity of HA-CLPs to deliver miR-205 around the MDA-MB-231 cell nucleus observably. EdU cell proliferation test and cell wound scratch assay proved that miR-205 and miR-34a sent into MDA-MB-231 cell had the ability to inhibit cell proliferation and migration.

Conclusion

Targeted cationic liposome has specific targeting ability towards MDA-MB-231 cell, and the delivery of miR-205 and miR-34a plays a part in the proliferation and migration inhibition of MDA-MB-231.

Key words: Liposomes, Cations, Hyaluronic acid, Breast neoplasms

表1 不同CLPs的理化性质表征（t检验）

组别（纳米粒子）	粒径（nm，±s）^a	zeta电位（mV，±s）^a	多分散系数/PDI
CLPs	180.6±3.4	43.4±2.8	0.298
CLPs-miR-205	200.8±2.5	38.1±1.7	0.259
HA-CLPs	236.65±6.9	29.1±5.4	0.35
HA-CLPs-miR-205	205.6±2.2	11.2±1.1	0.314
t₁,t₂,t₃,t₄,t₅,t₆	8.292，12.620，10.691，8.461，2.497，7.426	2.802，4.072，18.544，2.754，3.011，5.426	-
P₁,P₂,P₃,P₄,P₅,P₆	<0.01,<0.01,<0.01,<0.01,>0.05,<0.01	<0.05,<0.05,<0.01,>0.05,<0.01,<0.01	-

表1 不同CLPs的理化性质表征（t检验）

组别（纳米粒子）	粒径（nm，±s）^a	zeta电位（mV，±s）^a	多分散系数/PDI
CLPs	180.6±3.4	43.4±2.8	0.298
CLPs-miR-205	200.8±2.5	38.1±1.7	0.259
HA-CLPs	236.65±6.9	29.1±5.4	0.35
HA-CLPs-miR-205	205.6±2.2	11.2±1.1	0.314
t₁,t₂,t₃,t₄,t₅,t₆	8.292，12.620，10.691，8.461，2.497，7.426	2.802，4.072，18.544，2.754，3.011，5.426	-
P₁,P₂,P₃,P₄,P₅,P₆	<0.01,<0.01,<0.01,<0.01,>0.05,<0.01	<0.05,<0.05,<0.01,>0.05,<0.01,<0.01	-

图1 透射电镜下CLPs纳米颗粒的形态观察

图2 HA-CLPs-miR对MDA-MB-231细胞毒性检测结果Ctr为不加任何处理的空白对照组；CLPs为加入正常浓度的CLPs组；HA-CLPs1为加入高浓度HA-CLPs组；HA-CLPs2为加入正常浓度HA-CLPs组

图3 激光共聚焦显微镜对于两种CLPs纳米颗粒转运miR能力的观察Ctr为空白对照组；CLPs为经CLPs转运的非靶向转运miR-205组；HA-CLPs为经HA-CLPs转运的靶向转运miR-205组

图4 流式细胞法定量检测两种CLPs纳米颗粒转运miR的能力　A为MCF-7细胞；B为MDA-MB-231细胞；C为两种CLPs纳米颗粒转运miR后各组平均荧光强度的对比；HA-CLPs+HA为受试前3 h经HA靶向阻断后的HA-CLPs靶向转运miR-205组

图5 EdU实验检测MDA-MB-231细胞的增殖能力hoechst为经hoechst染色后细胞核；EdU为同一镜下经EdU染色后具有增殖能力细胞核；Merge为两种不同染色的融合图像；Ctr为空白对照组；miR-205为通过HA-CLPs转运miR-205组；miR-34a为通过HA-CLPs转运miR-34a组；mix为通过HA-CLPs转运等量的miR-205和miR-34a的共递送组

图6 细胞划痕实验检测不同miR组分对MDA-MB-231细胞迁移的影响　A为加入受试溶剂后0 h；B为加入受试溶剂后24 h时；Ctr为空白对照组；HA-CLPs-miR205为通过HA-CLPs转运miR-205组；HA-CLPs-miR34a为通过HA-CLPs转运miR-34a组；HA-CLPs-miRmix为通过HA-CLPs转运等量的miR-205和miR-34a的共递送组

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