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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (6): 1-11    DOI: 10.13523/j.cb.2201020
研究报告     
工程化外泌体介导巨噬细胞清除肿瘤外泌体*
王璐1,陈梦丽1,何芳1,2,项建1,尹斌成1,2,3,**(),叶邦策1,2
1.华东理工大学生物反应器国家重点实验室 上海 200237
2.浙江工业大学药学院 长三角绿色制药协同创新中心 杭州 310032
3.石河子大学化学化工学院 石河子 832003
Clearance of Tumor Exosomes by Engineered Exosomes-assisted Phagocytosis of Macrophages
WANG Lu1,CHEN Meng-li1,HE Fang1,2,XIANG Jian1,YIN Bin-cheng1,2,3,**(),YE Bang-ce1,2
1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
2. Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310032, China
3. School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003 China
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摘要：

目的:通过膜表面修饰改造技术构建工程化外泌体(engineered exosomes,enExos),并以此介导巨噬细胞特异性清除膜表面富含表皮生长因子受体(epidermal growth factor receptor,EGFR)的肿瘤外泌体。方法:利用表面展示技术获得膜表面展示趋化因子(chemokine 8,CXCL8)的外泌体,同时在其磷脂双分子层上修饰EGFR核酸适配体制备工程化外泌体;纳米颗粒跟踪和纳米粒度电位分析enExos的尺寸、电位;CCK-8试剂盒检测细胞活力;透射电子显微镜观察enExos与高表达EGFR的肿瘤外泌体的特异性结合;荧光成像技术及流式细胞术分析探究enExos靶向趋化巨噬细胞吞噬高表达EGFR的肿瘤外泌体。结果:成功构建膜表面展示EGFR与CXCL8的工程化外泌体,enExos可以特异性识别并捕获高表达EGFR的肿瘤外泌体,同时利用其趋化因子CXCL8特异性靶向巨噬细胞膜表面趋化因子受体CXCR1/CXCR2,刺激巨噬细胞对肿瘤外泌体的捕获及清除。结论:工程化外泌体促进了特定肿瘤外泌体的清除,为后续深入研究工程化外泌体抑制癌症转移的作用奠定基础,并期望为癌症转移治疗提供新的研究方向。
关键词： 工程化外泌体;  肿瘤外泌体;  表皮生长因子受体;  白细胞介素-8;  吞噬    
Abstract:

Objective: This paper aims to construct engineered exosomes (enExos) by membrane surface modification technology, and use enExos to mediate the removal of epidermal growth factor receptor (EGFR)-rich tumor exosomes on the membrane surface by macrophages. Methods: The exosomes displaying the chemokine 8 (CXCL8) on the membrane surface were obtained by surface display technology. Meanwhile, the EGFR nucleic acid aptamer was modified on the CXCL8-expressing exosomes to prepare enExos. The size and potential of enExos were analyzed by nanoparticle tracking analysis and nanoparticle size potentiometer. Cell viability was measured by CCK-8 assay. The specific binding of enExos to EGFR-expressing tumor exosomes was observed by transmission electron microscopy. Finally, the phagocytosis of tumor exosomes with high EGFR expression by enExos-targeted chemotactic macrophages was detected by fluorescence imaging and flow cytometry. Results: The enExos with EGFR and CXCL8 displayed on the membrane surface were successfully constructed. enExos can specifically recognize and capture tumor exosomes with high EGFR expression, and at the same time use their chemokine CXCL8 to specifically target the macrophage membrane. Surface chemokine receptors CXCR1/CXCR2 stimulate the capture and clearance of tumor exosomes by macrophages. Conclusion: enExos promote the clearance of specific tumor exosomes, laying a foundation for the subsequent in-depth study of their role in inhibiting cancer metastasis, and they are expected to provide new research directions for cancer metastasis therapy.
Key words: Engineered exosomes(enExos)    Tumor exosomes    Epidermal growth factor receptor    Interleukin-8    Phagocytosis
收稿日期: 2022-01-15 出版日期: 2022-07-07
ZTFLH:  Q813  
基金资助: *国家自然科学基金(21822402);国家自然科学基金(22134003);浙江省自然科学基金联合基金(LHDMZ22H300008)
通讯作者: 尹斌成     E-mail: binchengyin@ecust.edu.cn
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王璐
陈梦丽
何芳
项建
尹斌成
叶邦策

引用本文:

王璐,陈梦丽,何芳,项建,尹斌成,叶邦策. 工程化外泌体介导巨噬细胞清除肿瘤外泌体*[J]. 中国生物工程杂志, 2022, 42(6): 1-11.

WANG Lu,CHEN Meng-li,HE Fang,XIANG Jian,YIN Bin-cheng,YE Bang-ce. Clearance of Tumor Exosomes by Engineered Exosomes-assisted Phagocytosis of Macrophages. China Biotechnology, 2022, 42(6): 1-11.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2201020    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I6/1

图1  工程化外泌体(enExos)的设计及消除肿瘤外泌体的工作原理示意图
名称 序列(5'-3')
EGFR/AP-FAM Cholesterol-TTTTTTTTTTTTTACCAGTGCGATGCTCAGTGCCGTTT CTTCTCTTTCGCTTTTTTTGCTTTTGAGCATGCTGAC
GCATTCGGTTGAC-FAM
EGFR/AP Cholesterol-TTTTTTTTTTTTTACCAGTGCGATGCTCAGTGCCGTTTCTTCTCTTTCGCTTTTTTTGCTTTTGAGCATGCTGACG
CATTCGGTTGAC
B BHQ1-GTCAACCGAATGCGTCAGCATGCTCAAA
表1  本研究中使用的核酸适配体序列
图2  CXCL8-Exos的制备与表征
图3  外泌体的工程化及表征
图4  enExos的细胞相容性分析
图5  enExos靶向识别巨噬细胞
图6  EGFR适配体的功能验证
图7  体外enExos对肿瘤外泌体的识别与结合
图8  enExos介导巨噬细胞吞噬肿瘤细胞
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