靶向性CPI-444载药纳米微粒的制备及其对T细胞活性和抗肿瘤效应的影响  

Preparation of targeting CPI-444-loaded nanoparticles and investigation of its effects on T cell activity and anti-tumor response

作　　者：陈明水[1] 李洁羽[1] 王玲[1] 周智锋[1] 张麟腾 CHEN Mingshui;LI Jieyu;WANG Ling;ZHOU Zhifeng;ZHANG Linteng(Clinical Oncology School of Fujian Medical University,Fujian Cancer Hospital,Fuzhou 350014,Fujian,China)

机构地区：[1]福建医科大学肿瘤临床医学院,福建省肿瘤医院,福建福州350014

出　　处：《中国肿瘤生物治疗杂志》2024年第8期777-785,共9页Chinese Journal of Cancer Biotherapy

基　　金：福建省医学创新课题(No.2020CXA012);福建省自然基金联合资金(No.2023J011272);福建省肿瘤医院院内基金(No.2023YN10)。

摘　　要：目的:制备并表征包载CPI-444并偶联CD8抗体的纳米微粒(CNP/αCD8),探讨其对CD8^(+)T细胞活化、增殖和抗肿瘤作用的影响。方法:采用复乳溶剂蒸发法和EDC/NHS法制备包载腺苷受体A2A(A2AR)特异性拮抗剂CPI-444(C)或香豆素6(C6)荧光素的纳米微粒并分别在其表面偶联CD8抗体,制得CNP/αCD8和C6NP/αCD8。扫描电镜和NanoPlus粒度测定仪表征纳米微粒形态和粒径,液相色谱与串联质谱联用(LC-MS/MS)法和离心法测定纳米微粒的载药量和药物释放情况,荧光显微镜和流式细胞仪检测CD8^(+)T细胞内化C6NP/αCD8的情况,流式细胞仪、ELISA和LDH法检测CNP/αCD8对CD8^(+)T细胞增殖、活化、细胞毒活性和杀瘤能力的影响。结果:CNP/αCD8纳米微粒为圆形、粒径约150 nm,能有效包载CPI-444和偶联CD8抗体,药物包封率和CD8抗体偶联效率分别约为60%和53.4%;CNP/αCD8纳米微粒具有良好稳定性,能被CD8+T细胞内化,抑制A2AR分子表达。生物学功能实验显示,CNP/αCD8增强CD8^(+)T细胞的增殖能力、促进T细胞活化、分泌细胞因子及产生颗粒酶B和穿孔素,并增强CD8^(+)T细胞杀伤肿瘤细胞的能力。结论:CNP/αCD8纳米微粒能显著增强CD8^(+)T细胞免疫效应功能,其增强CD8^(+)T细胞功能可能是通过抑制A2AR分子的表达起作用。Objective:To prepare and characterize CD8 antibody-conjugated CPI-444(C)-loaded nanoparticles(CNP/αCD8)and investigate their effects on CD8^(+)T cell activation,proliferation,and anti-tumor activity.Methods:Nanoparticles encapsulating the adenosine A2A receptor(A2AR)antagonist CPI-444(C)or the fluorescent dye Coumarin-6(C6)were prepared using the double emulsion solvent evaporation method and EDC/NHS chemistry for antibody conjugation,resulting in CNP/αCD8 and C6NP/αCD8.The morphology and size of the nanoparticles were characterized by scanning electron microscopy and NanoPlus particle size analyzer.Drug loading and release profiles were determined using liquid chromatography-tandem mass spectrometry(LC-MS/MS)and centrifugation.The internalization of C6NP/αCD8 by CD8^(+)T cells were examined by flow cytometry and fluorescence microscopy.The effects of CNP/αCD8 on the proliferation,activation,cytotoxicity,and tumor killing ability of CD8^(+)T cells were examined by flow cytometry,ELISA,and lactate dehydrogenase(LDH)assay.Results:The CNP/αCD8 nanoparticles were spherical with an average diameter of about 150 nm,effectively encapsulating CPI-444 and conjugating CD8 antibodies,with a drug encapsulation efficiency and a CD8 antibody conjugation efficiency of approximately 60%and 53.4%,respectively.The nanoparticles exhibited good stability and were efficiently internalized by CD8^(+)T cells,inhibiting A2AR expression.Biological function assays showed that CNP/αCD8 enhanced CD8^(+)T cell proliferation,promoted T cell activation,cytokine secretion,granzyme B,and perforin production,and improved the tumorkilling ability of CD8^(+)T cells.Conclusion:CNP/αCD8 nanoparticles can significantly enhance the immune functions of CD8^(+)T cells,likely by inhibiting A2AR expression.

关 键 词：纳米微粒 腺苷受体A2A 过继细胞疗法 

分 类 号：R730.3[医药卫生—肿瘤] R979.1[医药卫生—临床医学]