细胞外囊泡（EVs）作为药物传递工具具有巨大潜力。虽然间充质/基质干细胞（MSC）条件培养基（CM）和牛奶潜在地是安全可扩展的EVs来源，但MSC EVs和牛奶EVs作为药物传递载体的适用性从未被比较，因此这是本研究的目标。在这里，EVs从MSC CM和牛奶中分离出来，并通过纳米粒子跟踪分析、透射电镜、总蛋白定量和免疫印迹进行表征。然后，将一种抗癌化疗药物多柔比星（Dox）通过三种方法之一（通过被动负载或通过电穿孔或声波破碎进行活性负载）装载到EVs中。Dox负载的EVs通过荧光分光光度计、高效液相色谱（HPLC）和成像流式细胞仪（IFCM）进行分析。我们的研究表明，EVs成功地从牛奶和MSC CM中分离出来，牛奶EVs/mL原料的产量显著高于MSC EVs/mL原料（p<0.001）。在每个比较中使用相同数量的EVs时，电穿孔比被动负载更有效地进行Dox负载（p <0.01）。实际上，对于250 µg提供的Dox，经由电穿孔为90.1±12µg装载到MSC EVs中，为68.0±10µg装载到牛奶EVs中，由HPLC分析。有趣的是，与被动负载和电穿孔方法相比，在声波处理后CD9 + EVs/mL（p <0.001）和CD63 + EVs/mL（p <0.001）都存在较少的EVs，由IFCM确定。这种观察结果表明，声波特别可能对EVs产生有害影响。总之，EVs可以成功地从MSC CM和牛奶中分离出来，其中牛奶是特别丰富的来源。在测试的三种方法中，电穿孔似乎是实现最大药物负载并不损害EV表面蛋白质的优越方法。

Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs' CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.