{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T12:49:21Z","timestamp":1792500561132,"version":"4.5.0"},"reference-count":41,"publisher":"American Chemical Society (ACS)","issue":"41","license":[{"start":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T00:00:00Z","timestamp":1791158400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T00:00:00Z","timestamp":1791158400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T00:00:00Z","timestamp":1791158400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["RS-2024-00393195"],"award-info":[{"award-number":["RS-2024-00393195"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Institutes of Health (NIH)","award":["T32 CA251062"],"award-info":[{"award-number":["T32 CA251062"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Extracellular vesicles (EVs) are emerging nanoscale platforms for targeted drug delivery, yet rational design of their surface composition is limited by a lack of systematic, high-throughput approaches for identifying productive protein-display constructs. Here, we engineer the human tetraspanin CD9 as a modular scaffold for displaying nanobodies (VHHs) on recombinant EV surfaces, using a high-throughput single-EV imaging platform to screen candidate constructs at the individual vesicle level. Guided by conservation analysis and structural prediction, we mapped seven insertion sites across CD9\u2019s extracellular loops and termini and identified positions that support functional transgene display. Nonconserved regions within the short extracellular loop (SEL) yielded the highest counts of functional recombinant EVs \u2013 an empirical result that runs counter to the a priori expectation that the larger long extracellular loop (LEL) would be more permissive. We demonstrate the platform\u2019s modularity by interchangeably incorporating fluorescent proteins and VHHs against GFP, HER2, EGFR, and PD-L1 at optimized SEL positions, where the displayed VHHs bound their cognate antigens with high specificity (\u223c70\u201390% co-localization and minimal off-target binding). Co-transfection of host cells with dual constructs yielded EVs co-displaying two independent functional moieties on \u223c80% of vesicles. This work establishes single-EV imaging as a fast and quantitative screening tool for identifying productive surface-display constructs and provides a generalizable workflow for producing multifunctional, transgene-displaying EVs with potential downstream bioanalytical, diagnostic, and therapeutic applications.<\/jats:p>","DOI":"10.1021\/acsnano.6c12478","type":"journal-article","created":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T21:01:34Z","timestamp":1791234094000},"page":"27920-27930","source":"Crossref","is-referenced-by-count":0,"title":["Systematic Engineering\nof Human CD9 for Extracellular\nVesicle Surface Display of Nanobodies Using Single-Vesicle Imaging"],"prefix":"10.1021","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-7804-647X","authenticated-orcid":true,"given":"Arek V.","family":"Melkonian","sequence":"first","affiliation":[{"name":"Brigham and Women\u2019s Hospital , , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Wyss Institute for Biologically Inspired Engineering , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Harvard Medical School , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8266-318X","authenticated-orcid":true,"given":"Chungmin","family":"Han","sequence":"additional","affiliation":[{"name":"Brigham and Women\u2019s Hospital , , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Wyss Institute for Biologically Inspired Engineering , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Harvard Medical School , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5524-7348","authenticated-orcid":true,"given":"David R.","family":"Walt","sequence":"additional","affiliation":[{"name":"Brigham and Women\u2019s Hospital , , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Wyss Institute for Biologically Inspired Engineering , , ,","place":["Boston, Massachusetts, United States, 02115"]},{"name":"Harvard Medical School , , ,","place":["Boston, Massachusetts, United States, 02115"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,5]]},"reference":[{"issue":"5","key":"2026102008162782400_cit1","doi-asserted-by":"publisher","first-page":"379","DOI":"10.1038\/s41573-022-00410-w","article-title":"Therapeutically Harnessing Extracellular Vesicles","volume":"21","author":"Cheng","year":"2022","journal-title":"Nat. Rev. Drug Discovery"},{"key":"2026102008162782400_cit2","doi-asserted-by":"publisher","first-page":"148","DOI":"10.1016\/j.addr.2016.02.006","article-title":"Extracellular\nVesicles for Drug Delivery","volume":"106","author":"Vader","year":"2016","journal-title":"Adv. Drug Delivery\nRev."},{"key":"2026102008162782400_cit3","doi-asserted-by":"publisher","first-page":"1330400","DOI":"10.3389\/fmolb.2023.1330400","article-title":"Biogenesis and Delivery\nof Extracellular Vesicles: Harnessing the Power of EVs for Diagnostics\nand Therapeutics","volume":"10","author":"Yu","year":"2024","journal-title":"Front. Mol. Biosci."},{"issue":"1","key":"2026102008162782400_cit4","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1038\/s41565-024-01774-3","article-title":"Harnessing\nExtracellular Vesicle Heterogeneity for Diagnostic and Therapeutic\nApplications","volume":"20","author":"Carney","year":"2025","journal-title":"Nat. Nanotechnol."},{"issue":"1","key":"2026102008162782400_cit5","doi-asserted-by":"publisher","DOI":"10.1080\/20013078.2017.1324730","article-title":"Comprehensive Toxicity and Immunogenicity\nStudies Reveal\nMinimal Effects in Mice Following Sustained Dosing of Extracellular\nVesicles Derived from HEK293T Cells","volume":"6","author":"Zhu","year":"2017","journal-title":"J. Extracell.\nVesicles"},{"key":"2026102008162782400_cit6","doi-asserted-by":"publisher","first-page":"2403199","DOI":"10.1002\/adma.202403199","article-title":"Immunogenicity of Extracellular\nVesicles","volume":"36","author":"Xia","year":"2024","journal-title":"Adv. Mater."},{"key":"2026102008162782400_cit7","doi-asserted-by":"publisher","first-page":"851","DOI":"10.3390\/cells9040851","article-title":"Targeting\nand Crossing the Blood-Brain Barrier with Extracellular Vesicles","volume":"9","author":"Saint-Pol","year":"2020","journal-title":"Cells"},{"key":"2026102008162782400_cit8","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0232442","article-title":"Plasma-Derived Exosome-like Vesicles\nAre Enriched in\nLyso-Phospholipids and Pass the Blood-Brain Barrier","volume":"15","author":"Jakubec","year":"2020","journal-title":"PLoS One"},{"issue":"9","key":"2026102008162782400_cit9","doi-asserted-by":"publisher","first-page":"939","DOI":"10.1016\/j.jcyt.2023.04.007","article-title":"Registered\nClinical Trials Investigating Treatment with Cell-Derived Extracellular\nVesicles: A Scoping Review","volume":"25","author":"Duong","year":"2023","journal-title":"Cytotherapy"},{"key":"2026102008162782400_cit10","doi-asserted-by":"publisher","first-page":"255","DOI":"10.1038\/s41392-025-02312-w","article-title":"Extracellular\nVesicle-Based Drug Overview: Research Landscape, Quality\nControl and Nonclinical Evaluation Strategies","volume":"10","author":"Xu","year":"2025","journal-title":"Signal Transduct. Target. Ther."},{"key":"2026102008162782400_cit11","doi-asserted-by":"publisher","first-page":"18","DOI":"10.1016\/j.jconrel.2015.03.033","article-title":"Exosomes\nas Drug Delivery Vehicles for Parkinson\u2019s\nDisease Therapy","volume":"207","author":"Haney","year":"2015","journal-title":"J. Controlled Release"},{"issue":"4","key":"2026102008162782400_cit12","doi-asserted-by":"publisher","first-page":"341","DOI":"10.1038\/nbt.1807","article-title":"Delivery of SiRNA\nto the Mouse Brain by Systemic Injection\nof Targeted Exosomes","volume":"29","author":"Alvarez-Erviti","year":"2011","journal-title":"Nat. Biotechnol."},{"issue":"10","key":"2026102008162782400_cit13","doi-asserted-by":"publisher","first-page":"1777","DOI":"10.1021\/bc500291r","article-title":"Surface\nFunctionalization of Exosomes Using Click Chemistry","volume":"25","author":"Smyth","year":"2014","journal-title":"Bioconjugate Chem."},{"issue":"4","key":"2026102008162782400_cit14","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12057","article-title":"Covalent Conjugation of Extracellular Vesicles with\nPeptides and Nanobodies for Targeted Therapeutic Delivery","volume":"10","author":"Pham","year":"2021","journal-title":"J. Extracell. Vesicles"},{"key":"2026102008162782400_cit15","doi-asserted-by":"publisher","DOI":"10.3402\/jev.v5.31053","article-title":"Display of GPI-Anchored Anti-EGFR\nNanobodies on Extracellular Vesicles Promotes Tumour Cell Targeting","volume":"5","author":"Kooijmans","year":"2016","journal-title":"J. Extracell. Vesicles"},{"issue":"5","key":"2026102008162782400_cit16","doi-asserted-by":"publisher","first-page":"1729","DOI":"10.1016\/j.ymthe.2021.01.020","article-title":"A Versatile Platform for Generating Engineered Extracellular\nVesicles with Defined Therapeutic Properties","volume":"29","author":"Dooley","year":"2021","journal-title":"Mol. Ther."},{"key":"2026102008162782400_cit17","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-40453-0","article-title":"Identification\nof Scaffold Proteins\nfor Improved Endogenous Engineering of Extracellular Vesicles","volume":"14","author":"Zheng","year":"2023","journal-title":"Nat. Commun."},{"issue":"2","key":"2026102008162782400_cit18","doi-asserted-by":"publisher","first-page":"158","DOI":"10.1016\/j.bcmd.2005.07.003","article-title":"Exosome\nDisplay Technology: Applications to the Development of New Diagnostics\nand Therapeutics","volume":"35","author":"Delcayre","year":"2005","journal-title":"Blood Cells Mol. Dis."},{"issue":"4","key":"2026102008162782400_cit19","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1038\/nrm.2017.125","article-title":"Shedding Light on the\nCell Biology of Extracellular Vesicles","volume":"19","author":"Van Niel","year":"2018","journal-title":"Nat.\nRev. Mol. Cell Biol."},{"key":"2026102008162782400_cit20","doi-asserted-by":"publisher","first-page":"442","DOI":"10.3389\/fimmu.2014.00442","article-title":"Tetraspanins in Extracellular Vesicle\nFormation and Function","volume":"5","author":"Andreu","year":"2014","journal-title":"Front. Immunol."},{"issue":"8","key":"2026102008162782400_cit21","doi-asserted-by":"publisher","first-page":"E968","DOI":"10.1073\/pnas.1521230113","article-title":"Proteomic Comparison Defines Novel\nMarkers to Characterize\nHeterogeneous Populations of Extracellular Vesicle Subtypes","volume":"113","author":"Kowal","year":"2016","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"issue":"1","key":"2026102008162782400_cit22","doi-asserted-by":"publisher","DOI":"10.1080\/10717544.2022.2162161","article-title":"The Cell Type\nDependent Sorting of\nCD9- and CD81 to Extracellular Vesicles Can Be Exploited to Convey\nTumor Sensitive Cargo to Target Cells","volume":"30","author":"Zuppone","year":"2023","journal-title":"Drug\nDelivery"},{"issue":"1","key":"2026102008162782400_cit23","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15459-7","article-title":"Structural Insights into Tetraspanin CD9 Function","volume":"11","author":"Umeda","year":"2020","journal-title":"Nat. Commun."},{"issue":"4","key":"2026102008162782400_cit24","doi-asserted-by":"publisher","first-page":"1041","DOI":"10.1016\/j.cell.2016.09.056","article-title":"Crystal Structure of a Full-Length\nHuman Tetraspanin Reveals a Cholesterol-Binding Pocket","volume":"167","author":"Zimmerman","year":"2016","journal-title":"Cell"},{"issue":"7","key":"2026102008162782400_cit25","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12248","article-title":"Extracellular\nVesicles Engineered to Bind Albumin Demonstrate Extended Circulation\nTime and Lymph Node Accumulation in Mouse Models","volume":"11","author":"Liang","year":"2022","journal-title":"J. Extracell. Vesicles"},{"issue":"22","key":"2026102008162782400_cit26","doi-asserted-by":"publisher","first-page":"12014","DOI":"10.1039\/D0NR00362J","article-title":"Sequential\nDeletion of CD63 Identifies\nTopologically Distinct Scaffolds for Surface Engineering of Exosomes\nin Living Human Cells","volume":"12","author":"Curley","year":"2020","journal-title":"Nanoscale"},{"issue":"11","key":"2026102008162782400_cit27","doi-asserted-by":"publisher","first-page":"1453","DOI":"10.1038\/s41551-024-01214-6","article-title":"Antibody-Displaying Extracellular\nVesicles for Targeted Cancer Therapy","volume":"8","author":"Wiklander","year":"2024","journal-title":"Nat. Biomed.\nEng."},{"key":"2026102008162782400_cit28","doi-asserted-by":"publisher","first-page":"446","DOI":"10.1038\/363446a0","article-title":"Naturally Occurring\nAntibodies Devoid of Light Chains","volume":"363","author":"Hamers-Casterman","year":"1993","journal-title":"Nature"},{"key":"2026102008162782400_cit29","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","article-title":"Nanobodies:\nNatural Single-Domain Antibodies","volume":"82","author":"Muyldermans","year":"2013","journal-title":"Annu. Rev.\nBiochem."},{"key":"2026102008162782400_cit30","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-026-72179-0","article-title":"High-Throughput\nSingle Extracellular Vesicle Imaging Platform for Direct Extracellular\nVesicle Profiling of Human Plasma","volume":"17","author":"Han","year":"2026","journal-title":"Nat. Commun."},{"key":"2026102008162782400_cit31","doi-asserted-by":"publisher","first-page":"1584","DOI":"10.1016\/j.yexcr.2008.09.020","article-title":"Trafficking\nand Function of the Tetraspanin CD63","volume":"315","author":"Pols","year":"2009","journal-title":"Exp. Cell\nRes."},{"issue":"10","key":"2026102008162782400_cit32","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12130","article-title":"Quantification of Protein Cargo Loading into Engineered\nExtracellular Vesicles at Single-Vesicle and Single-Molecule Resolution","volume":"10","author":"Silva","year":"2021","journal-title":"J. Extracell. Vesicles"},{"issue":"2","key":"2026102008162782400_cit33","doi-asserted-by":"publisher","DOI":"10.1002\/jev2.12404","article-title":"Minimal Information\nfor Studies of Extracellular Vesicles (MISEV2023): From Basic to Advanced\nApproaches","volume":"13","author":"Welsh","year":"2024","journal-title":"J. Extracell. Vesicles"},{"issue":"9","key":"2026102008162782400_cit34","doi-asserted-by":"publisher","first-page":"1073","DOI":"10.1093\/bioinformatics\/btm076","article-title":"COBALT: Constraint-Based Alignment Tool for Multiple\nProtein Sequences","volume":"23","author":"Papadopoulos","year":"2007","journal-title":"Bioinformatics"},{"issue":"7873","key":"2026102008162782400_cit35","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly Accurate Protein\nStructure Prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"issue":"6","key":"2026102008162782400_cit36","doi-asserted-by":"publisher","first-page":"509","DOI":"10.1016\/j.tcb.2023.09.003","article-title":"Tetraspanins: Structure, Dynamics,\nand Principles of Partner-Protein Recognition","volume":"34","author":"Susa","year":"2024","journal-title":"Trends Cell Biol."},{"issue":"12","key":"2026102008162782400_cit37","doi-asserted-by":"publisher","first-page":"2389","DOI":"10.1002\/pro.519","article-title":"Structural and Thermodynamic\nAnalysis of the GFP:GFP-Nanobody Complex","volume":"19","author":"Kubala","year":"2010","journal-title":"Protein\nSci."},{"issue":"7","key":"2026102008162782400_cit38","doi-asserted-by":"publisher","first-page":"1214","DOI":"10.1016\/j.str.2013.05.008","article-title":"Structural Evaluation of EGFR Inhibition Mechanisms\nfor Nanobodies\/VHH Domains","volume":"21","author":"Schmitz","year":"2013","journal-title":"Structure"},{"key":"2026102008162782400_cit39","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-46599-9","article-title":"SpyMask Enables Combinatorial Assembly\nof Bispecific Binders","volume":"15","author":"Driscoll","year":"2024","journal-title":"Nat. Commun."},{"key":"2026102008162782400_cit40","unstructured":"Zhang, Y.; Wu, S.; Yang, S.; Chou, C.-C.\n          Single-Domain Antibodies and\nVariants\nThereof Against PD-L1. U.S. Patent US20210221891A1, 2021."},{"key":"2026102008162782400_cit41","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.86394","article-title":"Improved\nIsolation of Extracellular Vesicles by Removal\nof Both Free Proteins and Lipoproteins","volume":"12","author":"Ter-Ovanesyan","year":"2023","journal-title":"eLife"}],"container-title":["ACS Nano"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsnano.6c12478","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancac3\/article-pdf\/20\/41\/27920\/68750408\/acsnano.6c12478.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancac3\/article-pdf\/20\/41\/27920\/68750408\/acsnano.6c12478.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T12:30:16Z","timestamp":1792499416000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancac3\/article\/20\/41\/27920\/5444832\/Systematic-Engineering-of-Human-CD9-for"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,5]]},"references-count":41,"journal-issue":{"issue":"41","published-online":{"date-parts":[[2026,10,5]]},"published-print":{"date-parts":[[2026,10,20]]}},"URL":"https:\/\/doi.org\/10.1021\/acsnano.6c12478","relation":{},"ISSN":["1936-0851","1936-086X"],"issn-type":[{"value":"1936-0851","type":"print"},{"value":"1936-086X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,5]]}}}