{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T23:56:27Z","timestamp":1789516587415,"version":"build-2803163510"},"reference-count":40,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T00:00:00Z","timestamp":1759968000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Intramural funding from Hualien Tzu Chi Hospital","award":["TCRD 113-065"],"award-info":[{"award-number":["TCRD 113-065"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Extracellular vesicles (EVs) from mesenchymal stem cells hold therapeutic promise for inflammatory and degenerative diseases; however, limited delivery and targeting capabilities hinder their clinical use. In this study, we sought to enhance the anti-inflammatory and chondroprotective effects of EVs through CAP-LAMP2b (chondrocyte affinity peptide fused to an EV membrane protein) engineering and ADAMTS4 gene editing hybrid vesicle formation. Human umbilical cord MSCs (hUCMSCs) were characterized via morphology, immunophenotyping, and trilineage differentiation. EVs from control and CAP-LAMP2b-transfected hUCMSCs were fused with liposomes carrying CRISPR-Cas9 ADAMTS4 gRNA. DiI-labeled EV uptake was assessed via fluorescence imaging. CAP-LAMP2b was expressed in hUCMSCs and their EVs. EVs exhibited the expected size (~120 nm), morphology, and exosomal markers (CD9, CD63, CD81, HSP70). CAP-modified hybrid EVs significantly enhanced chondrocyte uptake compared to control EVs and liposomes. IL-1\u03b2 increased ADAMTS4 expression, whereas CAP-LAMP2b-ADAMTS4 EVs, particularly clone SG3, reversed these effects by reducing ADAMTS4 and restoring aggrecan. Western blotting confirmed suppressed ADAMTS4 and elevated aggrecan protein. CAP-LAMP2b-ADAMTS4 EVs, therefore, showed superior uptake and therapeutic efficacy in inflamed chondrocytes, attenuating inflammatory gene expression and preserving matrix integrity. These results support engineered EVs as a promising cell-free approach for cartilage repair and osteoarthritis treatment.<\/jats:p>","DOI":"10.3390\/ijms26199812","type":"journal-article","created":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:50:16Z","timestamp":1760107816000},"page":"9812","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["CAP-LAMP2b\u2013Modified Stem Cells\u2019 Extracellular Vesicles Hybrid with CRISPR-Cas9 Targeting ADAMTS4 to Reverse IL-1\u03b2\u2013Induced Aggrecan Loss in Chondrocytes"],"prefix":"10.3390","volume":"26","author":[{"given":"Kun-Chi","family":"Wu","sequence":"first","affiliation":[{"name":"Department of Orthopedics, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Tzu Chi University, Hualien 970, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Hsun","family":"Chang","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Tzu Chi University, Hualien 970, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raymond Yuh-Shyan","family":"Chiang","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynecology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Tzu Chi University, Hualien 970, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5105-068X","authenticated-orcid":false,"given":"Dah-Ching","family":"Ding","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynecology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Tzu Chi University, Hualien 970, Taiwan"},{"name":"Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,9]]},"reference":[{"key":"ref_1","unstructured":"GBD 2021 Osteoarthritis Collaborators (2023). Global, Regional, and National Burden of Osteoarthritis, 1990\u20132020 and Projections to 2050: A Systematic Analysis for the Global Burden of Disease Study 2021. Lancet Rheumatol., 5, e508\u2013e522."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"He, Y., Li, Z., Alexander, P.G., Ocasio-Nieves, B.D., Yocum, L., Lin, H., and Tuan, R.S. (2020). Pathogenesis of Osteoarthritis: Risk Factors, Regulatory Pathways in Chondrocytes, and Experimental Models. Biology, 9.","DOI":"10.3390\/biology9080194"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Chang, W.-Y., Choi, S., Yoo, S.J., Lee, J., and Lim, C. (2023). Factors Associated with Osteoarthritis and Their Influence on Health-Related Quality of Life in Older Adults with Osteoarthritis: A Study Based on the 2020 Korea National Health and Nutrition Examination Survey. Int. J. Environ. Res. Public Health, 20.","DOI":"10.3390\/ijerph20126073"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Mort, J.S., Geng, Y., Fisher, W.D., and Roughley, P.J. (2016). Aggrecan Heterogeneity in Articular Cartilage from Patients with Osteoarthritis. BMC Musculoskelet. Disord., 17.","DOI":"10.1186\/s12891-016-0944-8"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"13505","DOI":"10.1021\/acs.jmedchem.2c01177","article-title":"Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition","volume":"65","author":"Cuffaro","year":"2022","journal-title":"J. Med. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/j.cger.2021.11.015","article-title":"Osteoarthritis Pathophysiology: Therapeutic Target Discovery May Require a Multifaceted Approach","volume":"38","author":"Vincent","year":"2022","journal-title":"Clin. Geriatr. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e184","DOI":"10.52225\/narra.v3i2.184","article-title":"Application of CRISPR-Cas9 Genome Editing Technology in Various Fields: A Review","volume":"3","author":"Ansori","year":"2023","journal-title":"Narra J."},{"key":"ref_8","first-page":"353","article-title":"Mechanism and Applications of CRISPR\/Cas-9-Mediated Genome Editing","volume":"15","author":"Asmamaw","year":"2021","journal-title":"Biologics"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Zhang, M.-L., Li, H.-B., and Jin, Y. (2024). Application and Perspective of CRISPR\/Cas9 Genome Editing Technology in Human Diseases Modeling and Gene Therapy. Front. Genet., 15.","DOI":"10.3389\/fgene.2024.1364742"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Jia, S., Liang, R., Chen, J., Liao, S., Lin, J., and Li, W. (2024). Emerging Technology Has a Brilliant Future: The CRISPR-Cas System for Senescence, Inflammation, and Cartilage Repair in Osteoarthritis. Cell. Mol. Biol. Lett., 29.","DOI":"10.1186\/s11658-024-00581-x"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Wu, K.-C., Yang, H.-I., Chang, Y.-H., Chiang, R.Y.-S., and Ding, D.-C. (2025). Extracellular Vesicles Derived from Human Umbilical Mesenchymal Stem Cells Transfected with miR-7704 Improved Damaged Cartilage and Reduced Matrix Metallopeptidase 13. Cells, 14.","DOI":"10.3390\/cells14020082"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"339","DOI":"10.3727\/096368915X686841","article-title":"Human Umbilical Cord Mesenchymal Stem Cells: A New Era for Stem Cell Therapy","volume":"24","author":"Ding","year":"2015","journal-title":"Cell Transplant."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2837","DOI":"10.1080\/15548627.2023.2235196","article-title":"LAMP2A, LAMP2B and LAMP2C: Similar Structures, Divergent Roles","volume":"19","author":"Qiao","year":"2023","journal-title":"Autophagy"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"749","DOI":"10.2217\/nnm-2017-0335","article-title":"Chondrocyte Affinity Peptide Modified PAMAM Conjugate as a Nanoplatform for Targeting and Retention in Cartilage","volume":"13","author":"Hu","year":"2018","journal-title":"Nanomedicine"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e2100639","DOI":"10.1002\/adhm.202100639","article-title":"Liposomes and Extracellular Vesicles as Drug Delivery Systems: A Comparison of Composition, Pharmacokinetics, and Functionalization","volume":"11","author":"Gandek","year":"2022","journal-title":"Adv. Healthc. Mater."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1016\/j.jconrel.2014.07.049","article-title":"Extracellular Vesicles as Drug Delivery Systems: Lessons from the Liposome Field","volume":"195","author":"Fens","year":"2014","journal-title":"J. Control. Release"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"jcs260201","DOI":"10.1242\/jcs.260201","article-title":"Extracellular Vesicles and Nanoparticles at a Glance","volume":"137","author":"Jeppesen","year":"2024","journal-title":"J. Cell Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"12735","DOI":"10.1021\/acs.langmuir.0c02491","article-title":"Physicochemical Characterization of Liposomes That Mimic the Lipid Composition of Exosomes for Effective Intracellular Trafficking","volume":"36","author":"Yoshida","year":"2020","journal-title":"Langmuir"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Varga, Z., Feh\u00e9r, B., Kitka, D., Wacha, A., B\u00f3ta, A., Ber\u00e9nyi, S., Pipich, V., and Fraikin, J.-L. (2020). Size Measurement of Extracellular Vesicles and Synthetic Liposomes: The Impact of the Hydration Shell and the Protein Corona. Colloids Surf. B Biointerfaces, 192.","DOI":"10.1016\/j.colsurfb.2020.111053"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1016\/j.addr.2020.03.002","article-title":"An Emerging Focus on Lipids in Extracellular Vesicles","volume":"159","author":"Skotland","year":"2020","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1007\/978-1-0716-2209-4_24","article-title":"Generation of Hybrid Extracellular Vesicles by Fusion with Functionalized Liposomes","volume":"2473","author":"Piffoux","year":"2022","journal-title":"Methods Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Dom\u00ednguez-Arca, V., Costa, R.R., Carvalho, A.M., Taboada, P., Reis, R.L., Prieto, G., and Pashkuleva, I. (2021). Liposomes Embedded in Layer by Layer Constructs as Simplistic Extracellular Vesicles Transfer Model. Mater. Sci. Eng. C Mater. Biol. Appl., 121.","DOI":"10.1016\/j.msec.2020.111813"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"17968","DOI":"10.1021\/acs.analchem.3c04306","article-title":"Using Functionalized Liposomes to Harvest Extracellular Vesicles of Similar Characteristics in Dermal Interstitial Fluid","volume":"95","author":"Cheng","year":"2023","journal-title":"Anal. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Li, T., Peng, J., Li, Q., Shu, Y., Zhu, P., and Hao, L. (2022). The Mechanism and Role of ADAMTS Protein Family in Osteoarthritis. Biomolecules, 12.","DOI":"10.3390\/biom12070959"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Rose, K.W.J., Taye, N., Karoulias, S.Z., and Hubmacher, D. (2021). Regulation of ADAMTS Proteases. Front. Mol. Biosci., 8.","DOI":"10.3389\/fmolb.2021.701959"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/s41374-021-00685-4","article-title":"Synergistic Upregulation of ADAMTS4 (aggrecanase-1) by Cytokines and Its Suppression in Knee Osteoarthritic Synovial Fibroblasts","volume":"102","author":"Cilek","year":"2022","journal-title":"Lab. Investig."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Clarissa, E.M., Karmacharya, M., Choi, H., Kumar, S., and Cho, Y.-K. (2025). Nature Inspired Delivery Vehicles for CRISPR-Based Genome Editing. Small, e2409353.","DOI":"10.1002\/smll.202409353"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/j.tibtech.2017.11.006","article-title":"Engineering the Delivery System for CRISPR-Based Genome Editing","volume":"36","author":"Glass","year":"2018","journal-title":"Trends Biotechnol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"4028","DOI":"10.1038\/s41467-025-59377-y","article-title":"Engineering of Extracellular Vesicles for Efficient Intracellular Delivery of Multimodal Therapeutics Including Genome Editors","volume":"16","author":"Liang","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"102040","DOI":"10.1016\/j.omtn.2023.102040","article-title":"CRISPR-Cas9 Delivery Strategies with Engineered Extracellular Vesicles","volume":"34","author":"Lu","year":"2023","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1007\/s11154-023-09860-y","article-title":"Potential Therapeutic Strategies for Osteoarthritis via CRISPR\/Cas9 Mediated Gene Editing","volume":"25","author":"Vlashi","year":"2024","journal-title":"Rev. Endocr. Metab. Disord."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1136\/annrheumdis-2018-214724","article-title":"Exploration of CRISPR\/Cas9-Based Gene Editing as Therapy for Osteoarthritis","volume":"78","author":"Zhao","year":"2019","journal-title":"Ann. Rheum. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1007\/s13770-018-0172-4","article-title":"Genome Engineering for Osteoarthritis: From Designer Cells to Disease-Modifying Drugs","volume":"16","author":"Choi","year":"2019","journal-title":"Tissue Eng. Regen. Med."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.jgg.2023.07.007","article-title":"Current Therapies for Osteoarthritis and Prospects of CRISPR-Based Genome, Epigenome, and RNA Editing in Osteoarthritis Treatment","volume":"51","author":"Chen","year":"2024","journal-title":"J. Genet. Genom."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"e12396","DOI":"10.1002\/jev2.12396","article-title":"Size Matters: Functional Differences of Small Extracellular Vesicle Subpopulations in Cardiac Repair Responses","volume":"13","author":"Manjikian","year":"2024","journal-title":"J. Extracell. Vesicles"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.cytogfr.2019.12.007","article-title":"Large Extracellular Vesicles: Size Matters in Tumor Progression","volume":"51","author":"Ciardiello","year":"2020","journal-title":"Cytokine Growth Factor Rev."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Sidhom, K., Obi, P.O., and Saleem, A. (2020). A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?. Int. J. Mol. Sci., 21.","DOI":"10.20944\/preprints202007.0485.v2"},{"key":"ref_38","first-page":"28","article-title":"Wharton\u2019s Jelly Derived-Mesenchymal Stem Cells: Isolation and Characterization","volume":"56","author":"Ranjbaran","year":"2018","journal-title":"Acta Med. Iran."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1700611","DOI":"10.1002\/advs.201700611","article-title":"Exosome-Liposome Hybrid Nanoparticles Deliver CRISPR\/Cas9 System in MSCs","volume":"5","author":"Lin","year":"2018","journal-title":"Adv. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1016\/j.omtn.2019.07.011","article-title":"Multi-Pathway Protective Effects of MicroRNAs on Human Chondrocytes in an in Vitro Model of Osteoarthritis","volume":"17","author":"Brinchmann","year":"2019","journal-title":"Mol. Ther. Nucleic Acids"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9812\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:51:58Z","timestamp":1760107918000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/19\/9812"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,9]]},"references-count":40,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["ijms26199812"],"URL":"https:\/\/doi.org\/10.3390\/ijms26199812","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,9]]}}}