Bone-targeted extracellular vesicles from mesenchymal stem cells for osteoporosis therapy

Bone-targeted extracellular vesicles from mesenchymal stem cells for osteoporosis therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Bone-targeted extracellular vesicles from mesenchymal stem cells for osteoporosis therapy Yayu Wang, Jie Yao, Lizhao Cai, Tong Liu, Xiaogang Wang, Ye Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-18279/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Oct, 2020 Read the published version in International Journal of Nanomedicine → Version 1 posted You are reading this latest preprint version Abstract Background Osteoporosis (OP) is one of the most common chronic diseases, but the drugs used to treat OP have strong side effects. Recently, bone regeneration in stem cell derivatives represented by extracellular vesicles (EVs) has provided a new strategy for the prevention and treatment of OP. EVs derived from mouse mesenchymal stem cells (mMSCs) have a positive effect on bone regeneration, yet their clinical application has been hampered by the lack of bone-targeting. Alendronate (Ale) has a specifically affinity for bone tissue through a high affinity with hydroxyapatite. Herein, we used copper-free "click chemistry” to combine EVs with Ale together for OP targeted therapy. Bone targeting was facilitated via Ale binding to hydroxyapatite, which is highly expressed on the bone surface. Methods In vitro , bone targeting of Ale-EVs was confirmed by flow cytometry. Also, Ex vivo fluorescent imaging data revealed strong fluorescent signals in bone tissues in mice treated with Ale-EVs-DiD compared to bone tissues of mice treated with EVs-DiD. Importantly, the modified EVs were well tolerated and showed no evidence of nonspecific side effects or immune response. Besides, our results showed that Ale-EVs could promote the proliferation and differentiation of mouse mesenchymal stem cells in vitro . And it had the antiosteoporotic effects in ovariectomy (OVX)-induced osteoporosis rat model. Conclusions A novel bone-targeting nanoparticle delivery system was developed for osteoporosis therapy. We used the Ale-N3 to modify mMSCs derived EVs by copper-free "click chemistry” to generate a Ale-EVs system. The Ale-EVs had a high affinity for bone and have great potential for clinical applications in osteoporosis therapy with low systemic toxicity. Nanoscience Osteoporosis bone regeneration stem cell derivatives extracellular vesicles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Supplementary Files Supplementarydata.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Oct, 2020 Read the published version in International Journal of Nanomedicine → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-18279","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":449924,"identity":"de957920-67e7-4f15-911e-d19652e526b9","order_by":1,"name":"Yayu Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDADNvbm4z8+GNjYEa+Fj+dYguSMgrRk4rXISeQYSPN8OMTYQEilwfGzh1/z1BxObJNISzC2MTjAzMB++OgGvFrO5KVZ8xwDauF5fCA5x+AOHwNPWtoNfFrMDuSYGfOwAbWwpyUczjF4xswgwWOGX8v5N0At/4BaGHIMmy0MDjM2ENRyI8f4MW8bUAtHjjEzAzFa7G+8MWOc25du3MZzLI2xxyAtmY2QXyT7c4w/vPlmLTu/vfkYw48/Nnb87IeP4dUCBGxSPAzNSFwCykGA+eMPhjoi1I2CUTAKRsGIBQDtFU8GqJINFAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2359-537X","institution":"Jinan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yayu","middleName":"","lastName":"Wang","suffix":""},{"id":449925,"identity":"6e3f3062-6c63-4299-ba3d-6b27d871781e","order_by":2,"name":"Jie Yao","email":"","orcid":"","institution":"Jinan University First Affiliated 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Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiangning","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2020-03-20 10:25:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-18279/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-18279/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.2147/IJN.S263756","type":"published","date":"2020-10-01T19:41:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":809193,"identity":"cfd0a5d7-ba23-489d-aca6-607b5ff5fafc","added_by":"auto","created_at":"2020-03-31 18:06:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":730961,"visible":true,"origin":"","legend":"S ynthesis and characterization of Ale EVs .\nA.\nSchematic illustration for synthesis of Ale EVs.\nB.\nFluorescence microscopy analysis of the N3 Cy5.5 conjugation with EVs. EVs were conjugation with N3 Cy5.5 via “Click Chemistry”. Then, the Cy5.5-EVs were captured by Dynabeads® with CD63 antibody. The resulting fluorescence signal was very strong (Scale bars: 10 0 μm μm).\nC.\nTransmission electron microscopy of A le EVs . The morphology of A le EVs was intact and the size\nwas approximately 30 2 00 nm (Arrows indicate A le EVS , Scale bars: 2 00 nm).\nD.\nSize distribution of Ale EVs measured by nanoparticle tracking analysis. The peak diameter was at\n136 nm for Ale EVs.","description":"","filename":"1.PNG","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/1.PNG"},{"id":809195,"identity":"af084012-35b8-4e13-8140-3616eb867647","added_by":"auto","created_at":"2020-03-31 18:06:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230447,"visible":true,"origin":"","legend":"B one targeting of Ale EVs in vitro and in vivo\nA. Binding\nof Ale EVs Di D with HA beads detected by flow cytometry Ale EVs or EVs were loaded with\nDi D and then incubated with the HA beads at room temperature for 30 minutes. The result showed that\nthe fluorescent signal was relatively stronger in HA beads incubated with Ale EVs Di D\nB.\nEx vivo fluorescence imaging of major organs from mice at 6 h after intravenous injection with 15 0 μg\nof A le EVs Di D EVs Di D or PBS. In A le EVs Di D group s , bone tissue s had strong fluorescence signals\nIn EVs Di D group s , bone tissue s had weak er fluorescence signal relatively\nC.\nQuantification of average fluorescence signal intensity of the bone in figure B by MI SE software Data\nare presented as the mean ± s.e.m. (n=3)","description":"","filename":"2.PNG","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/2.PNG"},{"id":809196,"identity":"ca8b83f0-e7c5-4d9b-b0bf-0da698ca8934","added_by":"auto","created_at":"2020-03-31 18:06:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":743281,"visible":true,"origin":"","legend":"Evaluation of the toxicity profile of Ale EVs\nA. H\u0026E staining of various organs Scale bar = 100 μm\nB. Serum\nmarkers of organ damage. Each bar represents means with SD of three replicates. NS, not\nsignificant, CK MB: creatine kinase MB isoenzyme BUN: blood urea nitrogen.\nC. Serum\nassociated inflammatory cytokines (TNF α and INF α). No significant difference note d between\nthe 2 treatment groups.","description":"","filename":"3.PNG","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/3.PNG"},{"id":809197,"identity":"71065ed3-fccd-48d8-9abe-03b9a01a4a47","added_by":"auto","created_at":"2020-03-31 18:06:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":286302,"visible":true,"origin":"","legend":"Antiosteoporosis efficacy of Ale EVs in vitro\nMMSC s were incubated with PBS, Ale , EVs and A le EVs ( 3 0 0 μg /ml ). After 48 hours, Ale EVs and EVs\npromoted cells growth ( A). After 14 Days, c ell s ALP activity were assessed using ALP staining A le EVs\nand EVs promoted c ell s ALP activity remarkably, but almost had no effect in other groups (B After 7 Days,\nthe expression levels of RUNX 2 and COL1 were examined by ( C ) Q PCR and ( D ) Western Ale EVs\nand EVs promoted the c ell s expression levels of RUNX 2 and COL1 remarkably\nStatistical analyses were performed using the Student's\nt test. *, P \u003c 0.05; **, P\u003c0.01","description":"","filename":"4.PNG","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/4.PNG"},{"id":809198,"identity":"0844cb31-07d8-438a-aee0-d0da17f30974","added_by":"auto","created_at":"2020-03-31 18:06:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":439606,"visible":true,"origin":"","legend":"Antiosteoporosis efficacy of Ale EVs in vivo\nA. representative images showing three dimensional trabecular architecture by micro CT reconstruction\nin distal femora (bars 1 mm);\nB. micro CT measurements of BMD in distal femora .\nC. micro CT measurements of BV/TV in distal femora\nStatistical analyses\nwere performed using the Student's t test. *, P\u003c 0.05; **, P\u003c 0.01.","description":"","filename":"5.PNG","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/5.PNG"},{"id":1075543,"identity":"94645373-ccec-43bb-a731-d1c04516f536","added_by":"auto","created_at":"2020-05-12 12:01:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":908929,"visible":true,"origin":"","legend":"","description":"","filename":"journalofnanobiotechnologymanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/journalofnanobiotechnologymanuscript.pdf"},{"id":809229,"identity":"a4cf6a32-f078-464c-9e69-82112a79eea8","added_by":"auto","created_at":"2020-03-31 18:06:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":911382,"visible":true,"origin":"","legend":"","description":"","filename":"journalofnanobiotechnologymanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/Manuscript.pdf"},{"id":809194,"identity":"fdc29327-14b0-4525-99d3-332754edb462","added_by":"auto","created_at":"2020-03-31 18:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":908929,"visible":true,"origin":"","legend":"","description":"","filename":"journalofnanobiotechnologymanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/journal of nanobiotechnology-manuscript.pdf"},{"id":809191,"identity":"3e46ec1a-4e7d-47dd-96d6-3bd5c61a045a","added_by":"auto","created_at":"2020-03-31 18:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":893192,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/manuscript.pdf"},{"id":13496246,"identity":"423cc0d7-6269-40bd-936a-a8e8b61b89b7","added_by":"auto","created_at":"2021-09-16 22:48:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1111736,"visible":true,"origin":"","legend":"","description":"","filename":"journalofnanobiotechnologymanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1_covered.pdf"},{"id":809192,"identity":"702ef823-0bbd-4402-aaf7-9c32cf953406","added_by":"auto","created_at":"2020-03-31 18:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":119164,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-18279/v1/Supplementary data.pdf"}],"financialInterests":"","formattedTitle":"Bone-targeted extracellular vesicles from mesenchymal stem cells for osteoporosis therapy","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThis preprint is available for \u003ca href='/article/rs-18279/latest.pdf' target='_blank'\u003edownload as a PDF\u003c/a\u003e.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoporosis, bone regeneration, stem cell derivatives, extracellular vesicles","lastPublishedDoi":"10.21203/rs.3.rs-18279/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-18279/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground Osteoporosis (OP) is one of the most common chronic diseases, but the drugs used to treat OP have strong side effects. Recently, bone regeneration in stem cell derivatives represented by extracellular vesicles (EVs) has provided a new strategy for the prevention and treatment of OP. EVs derived from mouse mesenchymal stem cells (mMSCs) have a positive effect on bone regeneration, yet their clinical application has been hampered by the lack of bone-targeting. Alendronate (Ale) has a specifically affinity for bone tissue through a high affinity with hydroxyapatite. Herein, we used copper-free \"click chemistry” to combine EVs with Ale together for OP targeted therapy. Bone targeting was facilitated via Ale binding to hydroxyapatite, which is highly expressed on the bone surface. \u003c/p\u003e\u003cp\u003eMethods \u003cem\u003eIn vitro\u003c/em\u003e, bone targeting of Ale-EVs was confirmed by flow cytometry. Also, Ex vivo fluorescent imaging data revealed strong fluorescent signals in bone tissues in mice treated with Ale-EVs-DiD compared to bone tissues of mice treated with EVs-DiD. Importantly, the modified EVs were well tolerated and showed no evidence of nonspecific side effects or immune response. Besides, our results showed that Ale-EVs could promote the proliferation and differentiation of mouse mesenchymal stem cells \u003cem\u003ein vitro\u003c/em\u003e. And it had the antiosteoporotic effects in ovariectomy (OVX)-induced osteoporosis rat model. \u003c/p\u003e\u003cp\u003eConclusions A novel bone-targeting nanoparticle delivery system was developed for osteoporosis therapy. We used the Ale-N3 to modify mMSCs derived EVs by copper-free \"click chemistry” to generate a Ale-EVs system. The Ale-EVs had a high affinity for bone and have great potential for clinical applications in osteoporosis therapy with low systemic toxicity.\u003c/p\u003e","manuscriptTitle":"Bone-targeted extracellular vesicles from mesenchymal stem cells for osteoporosis therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-03-31 18:05:53","doi":"10.21203/rs.3.rs-18279/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"21ddfa59-522f-4cec-bd31-d7c4e3f65fca","owner":[],"postedDate":"March 31st, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":76886,"name":"Nanoscience"}],"tags":[],"updatedAt":"2021-07-22T19:41:31+00:00","versionOfRecord":{"articleIdentity":"rs-18279","link":"https://doi.org/10.2147/IJN.S263756","journal":{"identity":"international-journal-of-nanomedicine","isVorOnly":true,"title":"International Journal of Nanomedicine"},"publishedOn":"2020-10-01 19:41:31","publishedOnDateReadable":"October 1st, 2020"},"versionCreatedAt":"2020-03-31 18:05:53","video":"","vorDoi":"10.2147/IJN.S263756","vorDoiUrl":"https://doi.org/10.2147/IJN.S263756","workflowStages":[]},"version":"v1","identity":"rs-18279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-18279","identity":"rs-18279","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}