In vivo Fluorescent Molecular Imaging Using Nanobodies Labeled with Next-Generation FNIR-Tag Dyes

In vivo Fluorescent Molecular Imaging Using Nanobodies Labeled with Next-Generation FNIR-Tag Dyes | 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 Article In vivo Fluorescent Molecular Imaging Using Nanobodies Labeled with Next-Generation FNIR-Tag Dyes Dora M. Chigoho, Marcus C.M. Stroet, Jelena Saliën, Sofie Pollenus, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8898776/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Purpose Fluorescently labeled Nanobodies (Nbs) provide rapid, specific, and high-contrast molecular imaging capabilities, making them well suited for applications such as fluorescence-guided surgery. As dye properties can sbstantially influence tracer performance, this study evaluated three next-generation FNIR-Tag dyes, each conjugated to an anti-EGFR Nb. Procedures The anti-EGFR Nb 7D12 was conjugated to FNIR-Tag-1.0, FNIR-Tag-766 or FNIR-Tag-804, and characterized in vitro. In vivo imaging and biodistribution studies were performed to assess pharmacokinetics, tumor uptake, tumor-to-background ratios, contrast-to-noise ratios, and renal clearance. Results All three Nb-based tracers exhibited similar overall pharmacokinetic behavior, characterized by fast tumor accumulation, rapid clearance from blood and non-target tissues, and predominant renal elimination. 7D12–FNIR-Tag-1.0 demonstrated slightly higher tumor uptake, whereas 7D12–FNIR-Tag-766 produced marginally improved tumor-to-background and contrast-to-noise ratios. In contrast, 7D12–FNIR-Tag-804 yielded significantly lower tumor signal intensity, although its imaging contrast remained comparable due to proportionally reduced background fluorescence. Despite differences in dye net charge, no substantial variation in kidney retention was observed over 24 h. Microscopic analysis revealed distinct renal handling: 7D12–FNIR-Tag-766 showed partial endosomal internalization within proximal tubule cells, while 7D12–FNIR-Tag-1.0 remained primarily luminal. Conclusions FNIR-Tag–labeled Nbs enable effective tumor visualization as early as 1 h post-injection. Although overall pharmacokinetics were comparable across dyes, differences in tissue uptake, intrinsic brightness, and compatibility with imaging system optics can influence detection sensitivity and should inform dye selection for clinical imaging applications. Single-Domain Antibodies Fluorescence Molecular Imaging Near-Infrared Fluorescent Dye FNIR-Tag Pharmacokinetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations Table 1 is available in the Supplementary Files section. Supplementary Files Table1.docx Table 1: Chemical, spectral and affinity characteristics of the fluorescent Nb-conjugates COID.Chigoho.pdf COID.Li.pdf COIMSchnermann.pdf COIM.Stroet.pdf COIS.Hernot.pdf COIS.Pollenus.pdf Supplementaryinformation.pdf renamedd7c04.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 27 Mar, 2026 Reviewers agreed at journal 26 Feb, 2026 Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 18 Feb, 2026 First submitted to journal 17 Feb, 2026 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-8898776","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597218800,"identity":"875b03d1-bfb9-4aa9-a361-aed76b66ff7d","order_by":0,"name":"Dora M. Chigoho","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Dora","middleName":"M.","lastName":"Chigoho","suffix":""},{"id":597218801,"identity":"a4bee142-9147-4f19-b0aa-17fe6a578d75","order_by":1,"name":"Marcus C.M. Stroet","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Marcus","middleName":"C.M.","lastName":"Stroet","suffix":""},{"id":597218802,"identity":"cac87512-011c-4feb-bccf-baffdffcac9d","order_by":2,"name":"Jelena Saliën","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Jelena","middleName":"","lastName":"Saliën","suffix":""},{"id":597218803,"identity":"e8e8f644-42de-4056-93fd-016f68e46649","order_by":3,"name":"Sofie Pollenus","email":"","orcid":"","institution":"Vrije Universiteit Brussel","correspondingAuthor":false,"prefix":"","firstName":"Sofie","middleName":"","lastName":"Pollenus","suffix":""},{"id":597218804,"identity":"7cd20eb8-1189-4a9a-827c-d8ca749d80aa","order_by":4,"name":"Dong-Hao Li","email":"","orcid":"","institution":"National Cancer Institute Center for Cancer Research","correspondingAuthor":false,"prefix":"","firstName":"Dong-Hao","middleName":"","lastName":"Li","suffix":""},{"id":597218805,"identity":"0e67d2e6-7d2c-4b14-b512-5e00d042fabf","order_by":5,"name":"Martin Schnermann","email":"","orcid":"","institution":"National Cancer Institute Center for Cancer Research","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Schnermann","suffix":""},{"id":597218806,"identity":"3adca6f3-741e-4ecd-8d91-e15b43ac5c8e","order_by":6,"name":"Sophie Hernot","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYHACNoYEBhACgQoGBgMGHlK0HDhDrBYGmJaDbURokW/vPfbgQQVDHr/02YOPP847nLidgffgA3xaDM6cSzdIOMNQLNmXl2xwcNvhxJ0NfMkGeLVI5JhJJLYxJG44w2MmcXBbWuKGA0AGXofNAGn5x5C4H6xlDliL+Q+8nrkB0tIAtIUHpKXBBmwLXh1Av6RJJByTSJxxhsfY4MwxG+OdzXzJ+B0GDDHJHzU2if09PIYPKmokZLez9x78gNcaSCwgG8uMXz1MyygYBaNgFIwCPAAAEb5KG3DzWHQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8739-6137","institution":"Vrije Universiteit Brussel","correspondingAuthor":true,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Hernot","suffix":""}],"badges":[],"createdAt":"2026-02-17 07:55:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8898776/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8898776/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103864530,"identity":"e2cea627-1b51-4ddc-94e4-fa8b059c2f84","added_by":"auto","created_at":"2026-03-03 21:26:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1167396,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular structure of FNIR-Tag-1.0 (A), FNIR-Tag-766 (B) and FNIR-Tag-804 (C) with their previously reported photophysical properties*[19-21]. λ\u003csub\u003eEx/Em\u003c/sub\u003e = Maximal emission and excitation wavelength, ɸ = Quantum yield; 𝛆 = Molar extinction coefficient at the maximal absorption wavelength; CF\u003csub\u003e280nm \u003c/sub\u003e= Correction factor applied for absorbance at 280 nm when calculating protein concentration.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1/ce03814297e11c59d33ac10b.png"},{"id":103864538,"identity":"02dfe0e9-e0f5-430b-bd95-b35f6e4fac83","added_by":"auto","created_at":"2026-03-03 21:26:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2542968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eQuality control of the fluorescent Nb conjugates by SEC, with absorbance at 280 nm for the Nb shown in blue and at 766/804 nm for the dye shown in red. (B) Excitation (solid lines) and emission spectra (dashed lines) of 7D12-FNIR-Tag-1.0 (orange), 7D12-FNIR-Tag-766 (green), and 7D12-FNIR-Tag-804 (red). The 780 nm laser excitation wavelength and emission filters transmitting wavelengths \u0026gt;820 nm employed by the FluoBeam800 imaging system are indicated in blue. (C) \u003cstrong\u003eF\u003c/strong\u003eluorescence image of freshly prepared 10 µM solutions of labeled 7D12-Nb constructs, acquired using the FluoBeam800 (exposure time 12 ms) and corresponding quantification of the fluorescence intensity. (D) Binding kinetics of labeled 7D12-Nb conjugates to human EGFR/His as determined by surface plasmon resonance.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1/53d21d3602922e3594841ee8.png"},{"id":104401156,"identity":"72e999c8-69cf-47bf-8043-257d4b71909d","added_by":"auto","created_at":"2026-03-11 12:12:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3780979,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative \u003cem\u003ein vivo\u003c/em\u003e fluorescence images (exposure time 75 ms) of FaDu tumor-bearing mice (n=5), intravenously injected 1 h before with 2 nmol of 7D12-FNIR-Tag1.0, 7D12-FNIR-Tag-766 or 7D12-FNIR-Tag-804. Kidneys (green arrows), tumor (yellow arrow) and contralateral muscle (orange arrow) are indicated, as well as regions-of-interest used for quantification (gray circles). Quantification of mean fluorescent intensity at the level of the tumor and contralateral muscle (B) and the kidneys (C), as well as calculated tumor-to-muscle (D) and contrast-to-noise ratios (E). Statistical significance was defined as p \u0026lt; 0.05 (ns: not significant; p \u0026lt; 0.05; *p \u0026lt; 0.01; **p \u0026lt; 0.005; ***p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1/bba185254f26dc124559f0f7.png"},{"id":104401553,"identity":"2bd20871-1acf-429a-a29e-ed19cd9cfba6","added_by":"auto","created_at":"2026-03-11 12:12:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4407886,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative \u003cem\u003eex vivo\u003c/em\u003efluorescence images (exposure time 100 ms) of major organs and tissues collected from FaDu tumor-bearing mice (n=5) intravenously injected with 2 nmol of 7D12-FNIR-Tag1.0, 7D12-FNIR-Tag-766 or 7D12-FNIR-Tag-804 1 h before. (B) Quantification of mean fluorescent intensity of the different organs. Statistical comparisons were performed both between the three tracers and between tumor and individual organs for each tracer. (D) Calculated tumor-to-organ and (E) contrast-to-noise ratios, where statistical comparisons were made between the three tracers. Statistical significance was defined as p \u0026lt; 0.05 (ns: not significant; p \u0026lt; 0.05; *p \u0026lt; 0.01; **p \u0026lt; 0.005; ***p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1/1c3c836807791c6f29664c0b.png"},{"id":103864542,"identity":"a5316ddf-7a8d-4076-bd7a-9665b2937f94","added_by":"auto","created_at":"2026-03-03 21:26:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16395937,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Mesoscopic imaging of sagittal kidney sections from mice injected 1 h before with 7D12-Nb-FNIR-Tag-1.0, 7D12-Nb-FNIR-Tag-766 or 7D12-Nb-FNIR-Tag-804. (B) Confocal imaging of kidney sections with 7D12-Nb-FNIR-Tag-1.0 and 7D12-Nb-FNIR-Tag-766. (C\u003cstrong\u003e)\u003c/strong\u003e Representative fluorescence images (exposure time 100 ms) of mice at 1, 6 and 24 h post-intravenous injection of 7D12-FNR-Tag-1.0, 7D12-FNR-Tag-766 or 7D12-Nb-FNR-Tag-804 (n=3 per group). (D\u003cstrong\u003e)\u003c/strong\u003eQuantification of in vivo renal fluorescence signal over time. (E\u003cstrong\u003e)\u003c/strong\u003e Quantification of ex vivo renal fluorescence signal 24 h post injection.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1/d7652f85716d1742b40cbfb1.png"},{"id":104779243,"identity":"ca13e288-41a3-4a53-864d-db62302f6447","added_by":"auto","created_at":"2026-03-17 07:37:14","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2908660,"visible":true,"origin":"","legend":"","description":"","filename":"Chigoho2026NbslabeledwithFNIRTagDyes2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1_covered_731a1603-d013-4d0a-a8b0-322db69525f8.pdf"},{"id":103864529,"identity":"eb939358-8fe6-4190-bed1-42b09a2c82fb","added_by":"auto","created_at":"2026-03-03 21:26:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Chemical, spectral and affinity characteristics of the fluorescent Nb-conjugates\u003c/p\u003e","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1/4b596e74317755a0938fa77e.docx"},{"id":103864531,"identity":"f9d659db-8cb2-4529-9814-03993515e452","added_by":"auto","created_at":"2026-03-03 21:26:27","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1250469,"visible":true,"origin":"","legend":"","description":"","filename":"COID.Chigoho.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8898776/v1/a850716c58688376675680c4.pdf"},{"id":104401183,"identity":"bea63e62-b9f9-4952-a91a-03bad01c3a64","added_by":"auto","created_at":"2026-03-11 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Dyes","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Single-Domain Antibodies, Fluorescence Molecular Imaging, Near-Infrared Fluorescent Dye, FNIR-Tag, Pharmacokinetics","lastPublishedDoi":"10.21203/rs.3.rs-8898776/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8898776/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescently labeled Nanobodies (Nbs) provide rapid, specific, and high-contrast molecular imaging capabilities, making them well suited for applications such as fluorescence-guided surgery. As dye properties can sbstantially influence tracer performance, this study evaluated three next-generation FNIR-Tag dyes, each conjugated to an anti-EGFR Nb.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe anti-EGFR Nb 7D12 was conjugated \u003c/strong\u003eto FNIR-Tag-1.0, FNIR-Tag-766 or FNIR-Tag-804, and characterized in vitro. In vivo imaging and biodistribution studies were performed to assess pharmacokinetics, tumor uptake, tumor-to-background ratios, contrast-to-noise ratios, and renal clearance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll three Nb-based tracers exhibited similar overall pharmacokinetic behavior, characterized by fast tumor accumulation, rapid clearance from blood and non-target tissues, and predominant renal elimination. 7D12–FNIR-Tag-1.0 demonstrated slightly higher tumor uptake, whereas 7D12–FNIR-Tag-766 produced marginally improved tumor-to-background and contrast-to-noise ratios. In contrast, 7D12–FNIR-Tag-804 yielded significantly lower tumor signal intensity, although its imaging contrast remained comparable due to proportionally reduced background fluorescence. Despite differences in dye net charge, no substantial variation in kidney retention was observed over 24 h. Microscopic analysis revealed distinct renal handling: 7D12–FNIR-Tag-766 showed partial endosomal internalization within proximal tubule cells, while 7D12–FNIR-Tag-1.0 remained primarily luminal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFNIR-Tag–labeled Nbs enable effective tumor visualization as early as 1 h post-injection. Although overall pharmacokinetics were comparable across dyes, differences in tissue uptake, intrinsic brightness, and compatibility with imaging system optics can influence detection sensitivity and should inform dye selection for clinical imaging applications.\u003c/p\u003e","manuscriptTitle":"In vivo Fluorescent Molecular Imaging Using Nanobodies Labeled with Next-Generation FNIR-Tag Dyes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 21:26:22","doi":"10.21203/rs.3.rs-8898776/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2026-03-27T23:25:10+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-26T19:09:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-26T01:20:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-19T01:38:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Imaging and Biology","date":"2026-02-18T02:31:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a799a354-af55-494c-bcab-2a654e984c1b","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T21:16:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 21:26:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8898776","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8898776","identity":"rs-8898776","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}