Dual-Channel NIR-II/Red Probe for Simultaneously Visualizing Mitochondrial Viscosity and NAD(P)H Dynamics Reveals Cuproptosis Mechanisms

Dual-Channel NIR-II/Red Probe for Simultaneously Visualizing Mitochondrial Viscosity and NAD(P)H Dynamics Reveals Cuproptosis Mechanisms | 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 Article Dual-Channel NIR-II/Red Probe for Simultaneously Visualizing Mitochondrial Viscosity and NAD(P)H Dynamics Reveals Cuproptosis Mechanisms baodui wang, Zhiwen Zhao, Ying Dang, Shihao Sun, Xinyue Li, Xue Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7386027/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cuproptosis, a copper-dependent mitochondrial cell death pathway, holds therapeutic potential for cancers reliant on mitochondrial respiration. While NAD(P)H, a core electron donor in mitochondrial metabolism, regulates this process, the dynamics of NAD(P)H levels and key microenvironments such as viscosity during cuproptosis remain unresolved, impeding mechanistic insight. Here, we developed Cy-N, a unimolecular fluorescent probe enabling simultaneous visualization of mitochondrial viscosity and NAD(P)H levels via distinct red (633 nm) and NIR-II (944 nm) emission channels. Utilizing Cy-N in vitro and in vivo within a hepatocellular carcinoma model, we uncovered that cuproptosis progression triggers a significant increase in mitochondrial viscosity coupled with NAD(P)H depletion. Mechanistically, lipoylated DLAT oligomerization drives viscosity elevation, while TCA cycle disruption, specifically downregulation of isocitrate dehydrogenase 3 (IDH3), impairs NAD(P)H synthesis. Our study establishes Cy-N as a powerful dual-parameter imaging tool for monitoring cuproptosis progression and delineates the roles of DLAT oligomerization and IDH3 suppression, advancing both mechanistic understanding and therapeutic targeting of copper-dependent cell death. Physical sciences/Chemistry/Analytical chemistry/Bioanalytical chemistry Biological sciences/Chemical biology/Chemical tools Cuproptosis Mitochondrial viscosity NAD(P)H Dual-channel fluorescence imaging NIR-II fluorescence probe Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterials.docx Supporting Information Cite Share Download PDF Status: Posted 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. 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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-7386027","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":548327299,"identity":"428b5456-d3c6-4bdd-93a8-e96f80b76567","order_by":0,"name":"baodui 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While NAD(P)H, a core electron donor in mitochondrial metabolism, regulates this process, the dynamics of NAD(P)H levels and key microenvironments such as viscosity during cuproptosis remain unresolved, impeding mechanistic insight. Here, we developed Cy-N, a unimolecular fluorescent probe enabling simultaneous visualization of mitochondrial viscosity and NAD(P)H levels via distinct red (633 nm) and NIR-II (944 nm) emission channels. Utilizing Cy-N in vitro and in vivo within a hepatocellular carcinoma model, we uncovered that cuproptosis progression triggers a significant increase in mitochondrial viscosity coupled with NAD(P)H depletion. Mechanistically, lipoylated DLAT oligomerization drives viscosity elevation, while TCA cycle disruption, specifically downregulation of isocitrate dehydrogenase 3 (IDH3), impairs NAD(P)H synthesis. Our study establishes Cy-N as a powerful dual-parameter imaging tool for monitoring cuproptosis progression and delineates the roles of DLAT oligomerization and IDH3 suppression, advancing both mechanistic understanding and therapeutic targeting of copper-dependent cell death.","manuscriptTitle":"Dual-Channel NIR-II/Red Probe for Simultaneously Visualizing Mitochondrial Viscosity and NAD(P)H Dynamics Reveals Cuproptosis Mechanisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 15:50:31","doi":"10.21203/rs.3.rs-7386027/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":"5c2feb6a-9494-45b0-9e41-5b820dab5358","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58346817,"name":"Physical sciences/Chemistry/Analytical chemistry/Bioanalytical chemistry"},{"id":58346818,"name":"Biological sciences/Chemical biology/Chemical tools"}],"tags":[],"updatedAt":"2026-04-22T23:56:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 15:50:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7386027","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7386027","identity":"rs-7386027","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}