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Here, structure\u2010guided engineering of the canonical EV tetraspanins, CD63 and CD9, is applied. Permissive sites for the internal insertion of the twin\u2010Strep tag are identified within unstructured regions of their large extracellular loops, using flexible linkers. This design preserves scaffold functionality and enables efficient antibody\u2010free affinity isolation of intact EVs using Strep technology. Fusing proteins of interest (POIs) to the C\u2010terminus of these scaffolds, either directly or via the photocleavable protein PhoCl\n                    <jats:sub>2<\/jats:sub>\n                    , enables efficient luminal POI loading with optional optogenetic control of POI release. Proteomic analysis confirms high purity of isolated EVs with more than 96% of contaminants removed, allowing sensitive detection of possibly novel EV\u2010associated proteins. Additionally, this approach effectively removes hepatitis C virus, which has EV\u2010like biophysical properties, and adeno\u2010associated virus, demonstrating high specificity. Vesicular stomatitis virus glycoprotein coating of EVs facilitates endosomal escape, while PhoCl\n                    <jats:sub>2<\/jats:sub>\n                    cleavage enables POI release after EV uptake by target cells, including primary human hepatocytes and hepatocyte\u2010like cells. This strategy provides a broadly applicable platform for producing intact, pure engineered EVs for basic and translational studies.\n                  <\/jats:p>","DOI":"10.1002\/smll.75973","type":"journal-article","created":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T08:31:31Z","timestamp":1791189091000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid and Efficient Affinity Isolation of Engineered Extracellular Vesicles Encapsulating Functional Proteins via Internal Twin\u2010Strep Tetraspanin Tagging"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5092-7347","authenticated-orcid":false,"given":"Minh\u2010Tu","family":"Pham","sequence":"first","affiliation":[{"name":"Department of Infectious Diseases Molecular Virology Center for Integrative Infectious Diseases Research (CIID) Heidelberg University  Heidelberg Germany"},{"name":"Heidelberg Partner Site German Center for 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