Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, TX, United States.
Department of Biomedical Engineering, Cockrell College of Engineering, The University of Texas at Austin, Austin, TX, United States.
Int J Pharm. 2024 Sep 30;663:124576. doi: 10.1016/j.ijpharm.2024.124576. Epub 2024 Aug 10.
Extracellular vesicles (EVs) have emerged as a promising drug delivery system. Connectosomes are a specialized type of EVs that contain connexins in their membranes. Connexin is a surface transmembrane protein that forms connexin hemichannels. When a connexin hemichannel on a connectosome docks with another connexin hemichannel of a target cell, they form a gap junction that allows direct intracellular delivery of therapeutic cargos from within the connectosome to the cytoplasm of the recipient cell. In the present study, we tested the feasibility of converting connectosomes into dry powders by (thin-film) freeze-drying to enable their potential storage in temperatures higher than the recommended -80 °C, while maintaining their activity. Connectosomes were isolated from a genetically engineered HeLa cell line that overexpressing connexin-43 subunit protein tagged with red fluorescence protein. To facilitate the testing of the function of the connectosomes, they were loaded with calcein green dye. Calcein green-loaded connectosomes were thin-film freeze-dried with trehalose alone or trehalose and a polyvinylpyrrolidone polymer as lyoprotectant(s) to produce amorphous powders with high glass transition temperatures (>100 °C). Thin-film freeze-drying did not significantly change the morphology and structure of the connectosomes, nor their particle size distribution. Based on data from confocal microscopy, flow cytometry, and fluorescence spectrometry, the connexin hemichannels in the connectosomes reconstituted from the thin-film freeze-dried powder remained functional, allowing the passage of calcein green through the hemichannels and the release of the calcein green from the connectosomes when the channels were opened by chelating calcium in the reconstituted medium. The function of connectosomes was assessed after one month storage at different temperatures. The connexin hemichannels in connectosomes in liquid lost their function when stored at -19.5 ± 2.2 °C or 6.0 ± 0.5 °C for a month, while those in dry powder form remained functional under the same storage conditions. Finally, using doxorubicin-loaded connectosomes, we showed that the connectosomes reconstituted from thin-film freeze-dried powder remained pharmacologically active. These findings demonstrate that (thin-film) freeze-drying represents a viable method to prepare stable and functional powders of EVs that contain connexins in their membranes.
细胞外囊泡 (EVs) 已成为一种有前途的药物递送系统。连接体是一种特殊类型的 EVs，其膜中含有连接蛋白。连接蛋白是一种表面跨膜蛋白，形成连接蛋白半通道。当连接体上的连接蛋白半通道与靶细胞上的另一个连接蛋白半通道对接时，它们形成一个间隙连接，允许治疗货物从连接体内部直接递送到受体细胞的细胞质中。在本研究中，我们通过（薄膜）冷冻干燥将连接体转化为干粉的可行性进行了测试，以使它们能够在高于推荐的-80°C 的温度下储存，同时保持其活性。连接体是从过表达红色荧光蛋白标记的连接蛋白-43 亚基蛋白的基因工程 HeLa 细胞系中分离出来的。为了方便连接体功能的测试，它们被加载了 calcein 绿色染料。用海藻糖单独或海藻糖和聚乙烯吡咯烷酮聚合物作为冷冻保护剂（s）对 calcein 绿色负载的连接体进行薄膜冷冻干燥，以产生具有高玻璃化转变温度（>100°C）的无定形粉末。薄膜冷冻干燥不会显著改变连接体的形态和结构，也不会改变它们的粒径分布。基于共聚焦显微镜、流式细胞术和荧光光谱的数据，从薄膜冷冻干燥粉末中重建的连接体中的连接蛋白半通道仍然具有功能，允许 calcein 绿色通过半通道，并在重建介质中螯合钙打开通道时从连接体中释放 calcein 绿色。在不同温度下储存一个月后评估了连接体的功能。当储存在-19.5±2.2°C 或 6.0±0.5°C 一个月时，液体中的连接体中的连接蛋白半通道失去了功能，而干粉形式的连接体中的半通道在相同的储存条件下仍然具有功能。最后，使用阿霉素负载的连接体，我们表明从薄膜冷冻干燥粉末中重建的连接体仍然具有药理活性。这些发现表明，（薄膜）冷冻干燥是一种可行的方法，可以制备含有膜连接蛋白的 EV 的稳定且功能性粉末。
