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Herein, we present a facile, versatile anti-PEG single-chain variable-fragment antibody (PEG-scFv)-based method enabling quantitative measurement of both forms in various biofluids (e.g., interstitial fluid, cytoplasm). By this method, we map the in vivo process of PEGylated liposomal doxorubicin (sLip\/Dox) at unprecedented resolution. In the bloodstream, doxorubicin remains largely encapsulated in liposomes (&amp;gt;99%). In liver as the main organ for drug elimination, less drug was distributed in the interstitium (&amp;gt;80% encapsulated) but more in liver cells (mainly in Kupffer cells) released in a time-dependent manner, accompanying doxorubicin transferred to hepatocytes most in free form by 12 h postinjection. After extravasation into tumors, there was a limited access of sLip\/Dox to tumor cells, confining most of the drug in the interstitium mainly being encapsulated (more than 75%), and the internalized fraction underwent a gradual release process in both tumor-associated macrophages and tumor cells. These findings revealed that for sLip\/Dox, which primarily underwent drug release intracellularly, cellular internalization rates could be the key factor in determining its in vivo performance. Given widespread PEGylation on developing nanomedicines and the cost-effectiveness of scFv production, PEG-scFv offers a broadly applicable tool for dissecting in vivo processes of nanomedicines to establish dose\u2013effect relationships like small-molecule drugs, further to guide rational nanotherapeutic design.<\/jats:p>","DOI":"10.1021\/acsnano.6c09408","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T05:13:14Z","timestamp":1785129194000},"page":"22215-22232","source":"Crossref","is-referenced-by-count":0,"title":["Anti-PEG\nSingle-Chain Variable-Fragment Antibody-Assisted\n                    <i>In Vivo<\/i>\n                    Process Decoding of PEGylated Nanomedicines"],"prefix":"10.1021","volume":"20","author":[{"given":"Feng","family":"Pan","sequence":"first","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Fudan University and Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education , , ,","place":["Shanghai, P. R. China, 201203"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingyuan","family":"Xu","sequence":"additional","affiliation":[{"name":"Zhongshan Hospital, Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Key Laboratory of Lung Inflammation and Injury , ,","place":["Shanghai, P. R. China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaisong","family":"Tian","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gan","family":"Luo","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Institute of Infectious Disease and Biosecurity , ,","place":["Shanghai, P. R. China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yawen","family":"Qi","sequence":"additional","affiliation":[{"name":"Zhongshan Hospital, Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Key Laboratory of Lung Inflammation and Injury , ,","place":["Shanghai, P. R. China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yinyu","family":"Ma","sequence":"additional","affiliation":[{"name":"Fudan University and Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education , , ,","place":["Shanghai, P. R. China, 201203"]},{"name":"Quzhou Fudan Institute , , ,","place":["Quzhou, Zhejiang Province, P. R. China, 324000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianlei","family":"Ying","sequence":"additional","affiliation":[{"name":"Zhongshan Hospital, Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Key Laboratory of Lung Inflammation and Injury , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Institute of Infectious Disease and Biosecurity , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Engineering Research Center for Synthetic Immunology , ,","place":["Shanghai, P. R. China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-8131-9704","authenticated-orcid":true,"given":"Jun","family":"Qian","sequence":"additional","affiliation":[{"name":"Fudan University and Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education , , ,","place":["Shanghai, P. R. China, 201203"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenlin","family":"Yang","sequence":"additional","affiliation":[{"name":"Zhongshan Hospital, Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Key Laboratory of Lung Inflammation and Injury , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Shanghai Engineering Research Center for Synthetic Immunology , ,","place":["Shanghai, P. R. China, 200032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5215-2829","authenticated-orcid":true,"given":"Changyou","family":"Zhan","sequence":"additional","affiliation":[{"name":"Fudan University , , ,","place":["Shanghai, P. R. China, 200032"]},{"name":"Fudan University and Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education , , ,","place":["Shanghai, P. R. China, 201203"]},{"name":"Shanghai Engineering Research Center for Synthetic Immunology , ,","place":["Shanghai, P. R. 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Med."},{"issue":"4","key":"2026081111144166000_cit17","doi-asserted-by":"publisher","first-page":"566","DOI":"10.3390\/pharmaceutics13040566","article-title":"Encapsulation\nof \u03b5-Viniferin into Multi-Lamellar Liposomes: Development of\na Rapid, Easy and Cost-Efficient Separation Method to Determine the\nEncapsulation Efficiency","volume":"13","author":"Beaumont","year":"2021","journal-title":"Pharmaceutics"},{"key":"2026081111144166000_cit18","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.jconrel.2019.09.016","article-title":"Predicting Drug Release Kinetics\nfrom Nanocarriers\nInside Dialysis Bags","volume":"315","author":"Yu","year":"2019","journal-title":"J. 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Pharm."},{"key":"2026081111144166000_cit21","doi-asserted-by":"publisher","DOI":"10.1016\/j.talanta.2019.120358","article-title":"Uptake and Release\nProfiles of PEGylated Liposomal Doxorubicin Nanoparticles: A Comprehensive\nPicture Based on Separate Determination of Encapsulated and Total\nDrug Concentrations in Tissues of Tumor-Bearing Mice","volume":"208","author":"Wang","year":"2020","journal-title":"Talanta"},{"key":"2026081111144166000_cit22","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2021.462688","article-title":"Fast and Versatile Analysis of Liposome\nEncapsulation Efficiency\nby NanoParticle Exclusion Chromatography","volume":"1662","author":"Bian","year":"2022","journal-title":"J.\nChromatogr. 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Pharm.\nSci."},{"issue":"1","key":"2026081111144166000_cit35","doi-asserted-by":"publisher","DOI":"10.1002\/btm2.10600","article-title":"PEGylated\nTherapeutics in the Clinic","volume":"9","author":"Gao","year":"2023","journal-title":"Bioeng. Transl.\nMed."},{"issue":"1","key":"2026081111144166000_cit36","doi-asserted-by":"publisher","DOI":"10.1080\/10717544.2025.2494775","article-title":"PEGylation\nTechnology: Addressing\nConcerns, Moving Forward","volume":"32","author":"Simberg","year":"2025","journal-title":"Drug Delivery"},{"issue":"8","key":"2026081111144166000_cit37","doi-asserted-by":"publisher","first-page":"12567","DOI":"10.1021\/acsnano.1c02103","article-title":"Nanotechnology\nfor Boosting Cancer Immunotherapy and Remodeling Tumor Microenvironment:\nThe Horizons in Cancer Treatment","volume":"15","author":"Gao","year":"2021","journal-title":"ACS Nano"},{"issue":"Pt 4","key":"2026081111144166000_cit38","doi-asserted-by":"publisher","first-page":"293","DOI":"10.1107\/S0907444911007773","article-title":"Data Processing and\nAnalysis with the AutoPROC Toolbox","volume":"67","author":"Vonrhein","year":"2011","journal-title":"Acta Crystallogr.,\nSect. D:Biol. Crystallogr."},{"issue":"Pt 2","key":"2026081111144166000_cit39","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1107\/S0907444909047337","article-title":"XDS","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta Crystallogr., Sect.\nD:Biol. Crystallogr."},{"issue":"Pt\n7","key":"2026081111144166000_cit40","doi-asserted-by":"publisher","first-page":"1204","DOI":"10.1107\/S0907444913000061","article-title":"How Good Are My Data and What Is\nthe Resolution?","volume":"69","author":"Evans","year":"2013","journal-title":"Acta Crystallogr., Sect. D:Biol.\nCrystallogr."},{"issue":"Pt 10","key":"2026081111144166000_cit41","doi-asserted-by":"publisher","first-page":"861","DOI":"10.1107\/S2059798319011471","article-title":"Macromolecular Structure Determination Using X-Rays,\nNeutrons and Electrons: Recent Developments in Phenix","volume":"75","author":"Liebschner","year":"2019","journal-title":"Acta Crystallogr., Sect. D:Biol. 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