多功能铜基纳米酶的制备及体外抗肿瘤活性

Preparation and in vitro anti-tumor activity of multifunctional copper-based nanozymes

摘要

为克服肿瘤细胞中过氧化氢（H₂O₂）匮乏和谷胱甘肽 (glutathione, GSH) 高表达对化学动力学疗法（chemodynamic therapy, CDT）效果的限制，通过一步法制备负载双硫仑（disulfiram, DSF）与 3-氨基-1,2,4-三唑 (3-animo-1,2,4-triazole, 3-AT) 的沸石咪唑框架（zeolitic imidazolate framework-8, ZIF-8），并经生物矿化在其表面原位生长透明质酸（hyaluronic acid, HA）修饰的过氧化铜（cupric peroxide, CuO₂），构建多功能铜基纳米酶 ADZCH（3-AT/DSF@ZIF-8@CuO₂-HA）。该纳米酶可通过级联催化破坏肿瘤 H₂O₂ 稳态、消耗 GSH 并同步递送 DSF 与 Cu²⁺，协同增强 CDT 和敏化 DSF 化疗效果。理化性能检测结果显示，ADZCH 呈均匀核壳结构、分散性良好，其粒径和 Zeta 电位分别为 196.5 nm 和−19.5 mV；具有微孔结构，比表面积为 81.860 0 m²/g，可有效负载 DSF 和 3-AT，对应载药率分别为 5.91% 与 45.07%；ADZCH 在酸性环境中可持续缓慢释放药物，且在不同生理条件下保持良好的稳定性。体外性能检测结果显示，ADZCH 具有浓度和孵育时间呈依赖性地产生 H₂O₂、羟基自由基并消耗 GSH。细胞摄取实验表明，HA 修饰显著提高了 4T1 细胞对纳米颗粒的摄取能力。细胞毒试验表明，80 μg/mL ADZCH 对 4T1 细胞有明显杀伤作用，对 L929 细胞无明显毒性作用。DCFH-DA 探针检测表明，ADZCH 可显著诱导细胞内 ROS 生成，有利于增强 CDT 疗效。活/死染色实验显示，ADZCH 高效诱导细胞凋亡，死细胞比例达 94.74%，具备良好的抗肿瘤应用潜力。本研究为克服肿瘤微环境屏障、提升 CDT 联合化疗的抗肿瘤效果提供了新的研究思路与实验依据。

Abstract

To address the constrains imposed by insufficient hydrogen peroxide (H₂O₂) and high glutathione (GSH) expression in tumor cells on the efficacy of chemodynamic therapy (CDT), zeolitic imidazolate framework-8 (ZIF-8) loaded with disulfiram (DSF) and 3-amino-1,2,4-triazole (3-AT) was synthesized via a one-pot approach. Subsequently, hyaluronic acid (HA)-modified cupric peroxide (CuO₂) was in-situ grown on its surface through biomineralization to construct a multifunctional copper-based nanozyme ADZCH (3-AT/DSF@ ZIF-8@CuO₂-HA). This nanoplatform disrupts the intratumoral H₂O₂ homeostasis, depletes GSH, and synchronously delivers DSF and Cu²⁺ via cascade catalysis, thereby enhancing CDT and sensitizing tumors to DSF-based chemotherapy. The results of physicochemical characterization indicated that ADZCH presented a uniform core-shell structure with favorable dispersibility. Its particle size and Zeta potential were 196.5 nm and −19.5 mV, respectively. It possessed a microporous structure with a specific surface area of 81.860 0 m 2/g, and demonstrated efficient loading capacity for DSF and 3-AT, achieving drug loading efficiencies of 5.91% and 45.07%, respectively. Moreover, ADZCH can continuously and slowly release drugs in an acidic environment and maintain good stability under diverse physiological conditions.In vitro functional assays verified that ADZCH catalytically generated H₂O₂ and hydroxyl radicals while concurrently depleting GSH in a concentration- and incubation time-dependent manner. Cellular uptake experiments showed that HA modification significantly improved the uptake of nanoparticles by 4T1 cells. Cytotoxicity tests showed that 80 μg/mL ADZCH had a significant cytotoxic effect on 4T1 cells but no significant toxicity on L929 cells. DCFH-DA probe detection indicated that ADZCH could significantly induce intracellular reactive oxygen species (ROS) generation, thereby enhancing CDT efficacy. Live/dead staining experiments showed that ADZCH efficiently induced apoptosis, with the proportion of dead cells reaching 94.74%, demonstrating its promising potential for anti-tumor applications.This study provides new research ideas and experimental basis for overcoming the tumor microenvironment barrier and enhancing the anti-tumor effect of CDT combined with chemotherapy.

关键词

肿瘤治疗 / 金属有机框架 / 化疗 / 化学动力学治疗 / 敏化

Key words

tumor treatment / metal-organic framework / chemotherapy / chemodynamic therapy / sensitization

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