Abstract：
Doxorubicin (DOX) is a chemotherapeutic with considerable efficacy, but its application is limited due to cardiotoxicity. Nanoparticles can improve DOX efficacy and prevent its adverse effects. Herein, DOX-loaded extracellular vesicles (DOX-EVs) were prepared using different loading methods including incubation, electroporation, and sonication in different hydration buffers to permeabilize nanostructures or desalinize DOX for improved entrapment. Different protein:drug (µg:µg) ratios of 1:10, 1:5, and 1:2, and incubation parameters were also investigated. The optimal formulation was characterized by western blotting, electron microscopy, Zetasizer, infrared spectroscopy, and release study. The cellular uptake and efficacy were investigated in MCF-7 spheroids via MTS assay, spheroid formation assay (SFA), confocal microscopy, and flow cytometry. The percentage of entrapment efficiency (EE) of formulations was improved from 1.0 ± 0.1 to 22.0 ± 1.4 using a protein:drug ratio of 1:2 and sonication in Tween 80 (0.1%w/v) containing buffer. Characterization studies verified the vesicles\' identity, spherical morphology, and controlled drug release properties. Cellular studies revealed the accumulation and cytotoxicity of DOX-EVs in the spheroids, and SFA and confocal microscopy confirmed the efficacy and cellular localization. Flow cytometry results revealed a comparable and amplified efficacy for DOX-EV formulations with different cell origins. Overall, the EV formulation of DOX can be applied as a promising alternative with potential advantages.
摘要：
多柔比星（DOX）是一种具有相当功效的化疗药物，但由于心脏毒性，其应用受到限制。纳米颗粒可以提高DOX的疗效并防止其不良反应。在这里，使用不同的加载方法制备DOX负载的细胞外囊泡（DOX-EV），包括孵育，电穿孔,并在不同的水合缓冲液中进行超声处理以使纳米结构透化或使DOX脱盐以改善截留。还研究了1：10、1：5和1：2的不同蛋白质：药物（µg：µg）比例以及孵育参数。最佳配方通过蛋白质印迹法表征，电子显微镜，泽塔西泽,红外光谱,和释放研究。通过MTS测定在MCF-7球体中研究细胞摄取和功效，球体形成测定(SFA)，共聚焦显微镜，和流式细胞术。使用1:2的蛋白质:药物比率和在含有吐温80(0.1%w/v)的缓冲液中超声处理,制剂的包封效率(EE)百分比从1.0±0.1提高至22.0±1.4。表征研究证实了囊泡的身份，球形形态，和受控的药物释放特性。细胞研究揭示了DOX-EV在球体中的积累和细胞毒性，SFA和共聚焦显微镜证实了疗效和细胞定位。流式细胞术结果揭示了具有不同细胞来源的DOX-EV制剂的相当和放大的功效。总的来说,DOX的EV配方可以用作具有潜在优势的有希望的替代品。
