肿瘤微环境响应的Zn/Cu@CM纳米粒子制备及研究  

Preparation and Study of Zn/Cu@CM Nanoparticles in Response to Tumor Microenvironment

作　　者：刘文涛[1] 李晓婧 朱丽丽 唐成武 权静[1,3] LIU Wentao;LI Xiaojing;ZHU Lili;TANG Chengwu;QUAN Jing(Shanghai Engineering Research Center for Nanobiomaterials and Regenerative Medicine,School of Biological and Medical Engineering,Donghua University,Shanghai 201620,China;Huzhou Key Laboratory of Translational Medicine,The First Affiliated Hospital of Huzhou University,Huzhou 313000,China;Guangxi Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor,Nanning 530022,China)

机构地区：[1]东华大学生物与医学工程学院、上海纳米生物材料与再生医学工程研究中心,上海201620 [2]湖州师范学院附属第一医院、湖州市转化医学重点实验室,湖州313000 [3]广西区域性高发肿瘤早期防治研究重点实验室,南宁530022

出　　处：《中国生物工程杂志》2025年第1期13-23,共11页China Biotechnology

基　　金：湖州市转化医学重点实验室重点开放课题(HZZHYX-2024-02);区域性高发肿瘤早期防治研究教育部重点实验室开放课题(GKEKF202401)资助项目。

摘　　要：目的:构建主动靶向的金属酶纳米平台Zn/Cu@CM,用于实现高效杀伤肿瘤细胞。方法:通过透射电子显微镜(transmission electron microscope,TEM)、粒径和电位、X射线衍射仪(X-ray diffractometer,XRD)、X射线光电子能谱(X-ray photoelectron spectroscopy,XPS)和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PACE)等对制备的Zn/Cu NPs和Zn/Cu@CM进行表征;通过亚甲基蓝(methylene blue,MB)来探究Zn/Cu NPs催化生成羟基自由基(hydroxyl radical,·OH)的能力;通过CCK-8实验验证Zn/Cu NPs的细胞杀伤性;通过细胞活性氧染色实验进一步探究催化生成·OH的能力;最后通过流式细胞术和细胞靶向实验探究细胞凋亡情况和主动靶向性。结果:制备出大小均一的球形Zn/Cu@CM纳米粒子,粒径在20~200 nm大小左右;亚甲基蓝实验证明Zn/Cu NPs在肿瘤微环境(tumor microenvironment,TME)下能够高效催化生成·OH;CCK-8实验表明,质量浓度为200μg/mL的纳米粒子与正常小鼠成纤维细胞(L929)共培养后,细胞存活率仍然保持在82%以上,而与乳腺癌细胞(4T1)共培养后,细胞存活率不足25%;活性氧探针2',7'-二氯二氢荧光素二乙酸酯(2',7'-dichlorodihydrofluorescein diacetate,DCFH-DA)染色实验证明,Zn/Cu@CM在肿瘤细胞内产生大量ROS以杀伤肿瘤细胞;流式细胞术表明,当Zn/Cu@CM质量浓度为150μg/mL时,细胞坏死和细胞凋亡分别占61.3%、0.349%,表明该复合纳米粒子可以高效产生ROS而导致细胞坏死,进而杀伤肿瘤细胞;细胞靶向实验证实Zn/Cu@CM具有良好的靶向性。结论:成功构建了主动靶向的Zn/Cu@CM复合纳米粒子,通过一系列表征和细胞实验证明其具有高效杀伤肿瘤细胞的能力,可为多功能纳米复合材料的抗肿瘤应用提供有价值的参考,并且为进一步双重杀伤肿瘤提供新思路。Objective:To construct an actively targeted metalloenzyme nanoplatform Zn/Cu@CM for the efficient killing of tumor cells.Methods:The prepared Zn/Cu NPs and Zn/Cu@CM were characterized using TEM,particle size and zeta potential,XRD,XPS,and SDS-PAGE.The catalytic generation of hydroxyl radicals(·OH)by Zn/Cu NPs was investigated using methylene blue(MB)as a probe.The cytotoxicity of Zn/Cu NPs was verified by CCK-8 assays.The capacity for catalytic generation of·OH was further explored through cellular reactive oxygen species(ROS)staining experiments.Finally,apoptosis and active targeting were investigated using flow cytometry and cellular targeting experiments.Results:Uniform spherical Zn/Cu@CM nanoparticles with sizes ranging from 20 to 200 nm were successfully synthesized.Methylene blue experiments demonstrated that Zn/Cu NPs could efficiently catalyze the generation of·OH in the tumor microenvironment(TME).CCK-8 assays indicated that after co-culture with normal mouse fibroblast cells(L929),the cell viability remained above 82%at a nanoparticle concentration of 200μg/mL,whereas it was less than 25%when co-cultured with breast cancer cells(4T1).ROS staining experiments confirmed that Zn/Cu@CM produced a significant amount of ROS inside tumor cells to kill them.Flow cytometry revealed that at the Zn/Cu@CM concentration of 150μg/mL,cell necrosis and apoptosis accounted for 61.3%and 0.349%,respectively,indicating that the composite nanoparticles could efficiently generate ROS leading to cell necrosis and thus kill tumor cells.Cellular targeting experiments confirmed the good targeting ability of Zn/[e-mail]:The actively targeted Zn/Cu@CM composite nanoparticles were successfully constructed,and a series of characterization and cellular experiments proved their ability to efficiently kill tumor cells.This provides a valuable reference for the application of multifunctional nanocomposite materials in antitumor therapy and offers new insights for further dual-targeted tumor killing.

关 键 词：肿瘤微环境响应 纳米粒子 化学动力学 靶向 类FENTON反应 

分 类 号：Q819[生物学—生物工程]