载咖啡酸叶酸壳聚糖纳米粒联合甲氨蝶呤作为一种新型以腺苷 A2A 受体下游级联为靶点的 HepG2 免疫治疗药物。

BACKGROUND: Methotrexate (MTX) is a common chemotherapeutic drug that inhibits DNA synthesis and induces apoptosis. Treatment with MTX increased CD73 expression, which leads to higher levels of extracellular adenosine. Adenosine levels are also high in the tumor microenvironment through Cancer cells metabolism. That promotes the survival of cancer cells and contributes to tumor immune evasion through the Adenosine 2a Receptor. A2A receptor antagonists are an emerging class of agents that treat cancers by enhancing immunotherapy, both as monotherapy and in combination with other therapeutic agents. Caffeine is an adenosine receptor antagonist. Herein, we demonstrate the ability of a novel well prepared and characterized nano formula CAF-FA-CS-NPs (D4) for A2aR blockade when combination with MTX to improve its antitumor efficacy by enhancing the immune system and eliminating immune suppression. METHODS: CAF-FA-CS-NPs (D4) were prepared and characterized for particle size, loading efficiency, and release profile. Molecular docking was used to validate the binding affinity of caffeine and folic acid to A2A receptor. The effects of the nano formula were evaluated on human liver cancer cells (HepG2), breast cancer cells (MCF-7), and MDA-MB-231, as well as normal human cells (WI-38). Different combination ratios of MTX and D4 were studied to identify the optimal combination for further genetic studies. RESULTS: Molecular docking results validated that caffeine and folic acid have binding affinity to A2A receptor. The CS-NPs were successfully prepared using ionic gelation method, with caffeine and folic acid being loaded and conjugated to the nanoparticles through electrostatic interactions. The CAF loading capacity in D4 was 77.9 ± 4.37% with an encapsulation efficiency of 98.5 ± 0.37. The particle size was optimized through ratio variations. The resulting nanoparticles were fully characterized. The results showed that (D4) had antioxidant activity and cytotoxicity against different cancer cells. The combination of D4 with MTX (IC50 D4 + 0.5 IC50 MTX) resulted in the downregulation of Bcl-2, FOXP3, CD39, and CD73 gene expression levels and upregulation of Bax and A2AR gene expression levels in HepG2 cells. CONCLUSIONS: This study suggests that CAF-FA-CS-NPs (D4) in combination with MTX may be a promising candidate for cancer immunotherapy, by inhibiting A2aR signaling and leading to improved immune activation and anti-tumor activity of MTX.

背景：甲氨蝶呤（MTX）是一种常见的化疗药物，可抑制 DNA 合成并诱导细胞凋亡。用 MTX 治疗可增加 CD73 的表达，从而导致细胞外腺苷水平升高。癌细胞代谢也会使肿瘤微环境中的腺苷水平升高。这促进了癌细胞的存活，并通过腺苷 2a 受体促进肿瘤免疫逃逸。A2A 受体拮抗剂是一类新兴的药物，通过增强免疫疗法，无论是作为单一疗法还是与其他治疗药物联合使用，来治疗癌症。咖啡因是一种腺苷受体拮抗剂。本文中，我们展示了一种新型的、经过精心制备和表征的纳米配方 CAF-FA-CS-NPs（D4）的能力，当与 MTX 联合使用时，通过增强免疫系统和消除免疫抑制来提高其抗肿瘤疗效，从而阻断 A2aR。

方法：制备并表征 CAF-FA-CS-NPs（D4）的粒径、载药量和释放曲线。分子对接用于验证咖啡因和叶酸与 A2A 受体的结合亲和力。评估纳米配方对人肝癌细胞（HepG2）、乳腺癌细胞（MCF-7）和 MDA-MB-231 以及正常人类细胞（WI-38）的影响。研究了 MTX 和 D4 的不同组合比例，以确定进一步遗传研究的最佳组合。

结果：分子对接结果验证了咖啡因和叶酸与 A2A 受体具有结合亲和力。使用离子凝胶化法成功制备了 CS-NPs，通过静电相互作用将咖啡因和叶酸载入并连接到纳米颗粒上。D4 中 CAF 的载药量为 77.9±4.37%，包封效率为 98.5±0.37。通过比例变化优化了粒径。所得纳米颗粒经过充分表征。结果表明，（D4）具有抗氧化活性和细胞毒性，可针对不同的癌细胞。D4 与 MTX 联合（IC50 D4+0.5 IC50 MTX）可下调 HepG2 细胞中 Bcl-2、FOXP3、CD39 和 CD73 基因的表达水平，并上调 Bax 和 A2AR 基因的表达水平。

结论：本研究表明，CAF-FA-CS-NPs（D4）与 MTX 联合使用可能是癌症免疫治疗的一种有前途的候选药物，通过抑制 A2aR 信号通路，提高 MTX 的免疫激活和抗肿瘤活性。