脑靶向硫化银量子点的构建及体外跨血脑屏障作用

doi:10.3969/j.issn.2095-9400.2020.03.008

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摘要目的：设计脑胶质瘤靶向的Ag2S量子点及Ag2S-PEG量子点近红外二区成像探针，并比较修饰脑靶向肽Angiopep-2（ANG）前、后量子点通过体外血脑屏障及靶向脑胶质瘤细胞的能力。方法：Ag2S量子点及Ag2S-PEG量子点和脑靶向肽ANG经EDC和NHS介导，发生氨基和羧基的缩合反应进行连接。对Ag2S、Ag2S-ANG、Ag2S-PEG、Ag2S-PEG-ANG量子点的结构及粒径进行琼脂糖电泳、动态光散射及透射电镜等表征，并评价其对U87 MG和bEnd.3细胞的细胞毒性以及穿透体外血脑屏障的能力。结果：修饰ANG肽后的Ag2S-ANG、Ag2S-PEG-ANG量子点水合粒径较Ag2S、Ag2S-PEG增大，差异有统计学意义（P＜0.05）；表面Zeta电位正电性增强；在琼脂糖电泳中迁移距离变短。MTT实验发现，Ag2S、Ag2S-ANG、Ag2S-PEG、Ag2S-PEG-ANG四种量子点在100 μg/mL以下对U87 MG和bEnd.3细胞的存活率没有影响。在体外血脑屏障模型中，Ag2S-ANG、Ag2S-PEG和Ag2S-PEG-ANG均能够穿过上层的bEnd.3细胞到达下层培养基中，而Ag2S-ANG被下层的U87 MG细胞摄取的量有较大提升，约是Ag2S量子点的6倍，差异有统计学意义（P＜0.01）。结论：本研究成功构建了2种脑靶向硫化银量子点，其中Ag2S-ANG能够跨体外血脑屏障系统，靶向脑胶质瘤细胞，为进一步的体内脑靶向成像奠定了基础。

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	徐毅
	闫美玲
	马继飞
	赵芳菲
	孙艳红
	王丽华
	高基民

关键词 ：硫化银量子点, 脑靶向, Angiopep-2, 血脑屏障

Abstract：Objective: To design the brain targeted Ag2S and Ag2S-PEG quantum dots and evaluate the property of the modified quantum dots in crossing the in vitro blood-brain barrier. Methods: Ag2S was conjugated with Angiopep-2 (Ag2S-ANG) and Ag2S-PEG was conjugated with Angiopep-2 (Ag2S-PEG-ANG) through the condensation reaction of amino and carboxyl groups mediated by EDC and NHS. The structure and size of Ag2S, Ag2S-ANG, Ag2S-PEG, Ag2S-PEG-ANG were characterized by agarose electrophoresis, dynamic light scattering transmission, electron microscope (TEM). The property of four kinds of quantum dots across the blood-brain barrier was evaluated in vitro by using the in vitro blood-brain barrier model. The cytotoxicity of four kinds of materials on U87 MG and bEnd.3 cells was observed. Results: Ag2S-ANG and Ag2S-PEG-ANG had a shorter migration distance compared with Ag2S and Ag2S-PEG in the agarose gel electrophoresis. The hydrate particle size of Ag2S-ANG and Ag2S-PEG-ANG was respectively increased compared with Ag2S and Ag2S-PEG. And their Zeta potential exhibited electropositive reinforcement. From the TEM results, we founded that the partical size of Ag2S-ANG and Ag2S-PEG-ANG was bigger than Ag2S and Ag2S-PEG (P<0.05). Ag2S, Ag2S-ANG, Ag2S-PEG, Ag2S-PEG-ANG QDs had no obvious cytotoxicity when the concentration was lower than 100 μg/mL. In vitro blood-brain barrier cell model, Ag2S-ANG, Ag2S-PEG and Ag2S-PEG-ANG quantum dots could cross the bEnd.3 cells in the upper layer, especially when the uptake of Ag2S-ANG was 6 times that of Ag2S by U87 MG cells in the lower layer (P<0.01). Conclusion: Brain targeted Ag2S-ANG and Ag2S-PEG-ANG are synthesized successfully. Ag2S-ANG QDs exhibit excellent ability to cross the blood-brain barrier system in vitro and target glioma cells, which lays the foundations for further study of in vivo brain targeting imaging.

Key words： Ag2S quantum dot brain-targeting angiopep-2 blood brain barrier

基金资助:国家自然科学基金资助项目（11575278，21675167，U1632125）；国家重点研发计划项目（2016YFA040 0902）；放射医学与辐射防护国家重点实验室开放性项目（GZK1201813）。

通讯作者: 高基民，教授，Email：jimingao64@163.com

作者简介: 徐毅（1993-），男，湖北黄冈人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/10.3969/j.issn.2095-9400.2020.03.008 或 https://xb.wmu.edu.cn/CN/Y2020/V50/I3/206

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