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/α CD8) 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.

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）