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However, the lack of direct comparisons between functionalization strategies has hindered the rational design of EV delivery systems. To address this gap, we developed a nano\u2010flow cytometry\u2010based methodology to quantitatively evaluate ligand conjugation and its relationship to targeting efficiency across three representative post\u2011production EV engineering strategies: lipid modification, protein modification, and membrane insertion. All three strategies achieved high conjugation efficiencies (&gt;90%) with ligand densities ranging from several to tens of ligands per 100 nm\n                    <jats:sup>2<\/jats:sup>\n                    under optimized conditions. Beyond ligand density, functionalization strategies resulted in varying degrees of ligand clustering and reduced accessibility of endogenous cell\u2010binding proteins on EVs, such as MFGE8, leading to differences in targeting performance. For milk\u2010derived EVs, lipid modification achieved the highest ligand density, the most uniform conjugation, and minimal disruption to surface protein accessibility, yielding superior cancer\u2010targeting efficiency. These findings highlight the importance of precise quantification of ligand conjugation performance and provide a robust methodology for optimizing surface functionalization to advance EV\u2010based drug delivery.\n                  <\/jats:p>","DOI":"10.1002\/jev2.70174","type":"journal-article","created":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T12:13:19Z","timestamp":1760443999000},"source":"Crossref","is-referenced-by-count":13,"title":["Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein, and Membrane Modifications"],"prefix":"10.1002","volume":"14","author":[{"given":"Xueping","family":"Pan","sequence":"first","affiliation":[{"name":"College of Ocean Food and Biological Engineering Jimei University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siqin","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Ocean Food and Biological Engineering Jimei University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Xia","sequence":"additional","affiliation":[{"name":"Xiamen Cell Therapy Research Center, The First Affiliated Hospital of Xiamen University, School of Medicine Xiamen University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengdi","family":"Sun","sequence":"additional","affiliation":[{"name":"College of Ocean Food and Biological Engineering Jimei University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xueqi","family":"Su","sequence":"additional","affiliation":[{"name":"College of Ocean Food and Biological Engineering Jimei University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingying","family":"Ke","sequence":"additional","affiliation":[{"name":"College of Ocean Food and Biological Engineering Jimei University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianyu","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Ocean Food and Biological Engineering Jimei University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liyun","family":"Su","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology, MOE Key Laboratory of Spectrochemical Analysis &amp; Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering Xiamen University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiabian","family":"Lian","sequence":"additional","affiliation":[{"name":"Xiamen Cell Therapy Research Center, The First Affiliated Hospital of Xiamen University, School of Medicine Xiamen University  Xiamen Fujian China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuqi","family":"Wu","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Key Laboratory of Space Bioscience &amp; Biotechnology Northwestern Polytechnical University  Xi'an China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7482-6863","authenticated-orcid":false,"given":"Xiaomei","family":"Yan","sequence":"additional","affiliation":[{"name":"Department of Chemical Biology, MOE Key Laboratory of Spectrochemical Analysis &amp; 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