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Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named \u201cSonication and Extrusion\u2010assisted Active Loading\u201d (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin\u2010loaded milk\u2010derived EVs (Dox\u2010mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10\u2010fold enhancement of drug encapsulation efficiency compared with passive loading. Single\u2010particle analysis by nano\u2010flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox\u2010mEVs which would otherwise be overlooked by bulk\u2010based approaches. Correlation analysis between doxorubicin auto\u2010fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid\u2010enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid\u2010probe\u2010 and immuno\u2010mediated magnetic isolation techniques to selectively remove the contaminants of non\u2010lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL\u2010prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as\u2010developed cargo\u2010loading approach and nano\u2010flow cytometry\u2010based characterization method will provide an instructive insight in the development of EV\u2010based delivery systems.<\/jats:p>","DOI":"10.1002\/jev2.12163","type":"journal-article","created":{"date-parts":[[2021,10,31]],"date-time":"2021-10-31T23:31:08Z","timestamp":1635723068000},"source":"Crossref","is-referenced-by-count":160,"title":["Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour"],"prefix":"10.1002","volume":"10","author":[{"given":"Chaoxiang","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Biological Engineering, College of Food and Biological Engineering Jimei University  Xiamen Fujian People's Republic of China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengdi","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Biological Engineering, College of Food and Biological 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