Using ionizable guanidine to develop cholesterol-free three-component lipid nanoparticles for spleen-targeted mRNA delivery

Using ionizable guanidine to develop cholesterol-free three-component lipid nanoparticles for spleen-targeted mRNA delivery | 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 Using ionizable guanidine to develop cholesterol-free three-component lipid nanoparticles for spleen-targeted mRNA delivery Mao Li, He Zhang, Dejing Liu, Zhuoying Liang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4805293/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Lipid nanoparticle (LNP) represents a key breakthrough in the realm of RNA therapeutics, particularly for the development of mRNA therapeutics and vaccines. Most LNPs developed to date rely on tertiary-amine based ionizable lipids and are mainly limited to liver delivery. Current strategies to alter the targeting performances of LNPs require four or more components, with cholesterol being a crucial element in the formulation. In this study, we introduce a novel approach utilizing an ionizable guanidine analogue, guanidino-carbonyl-pyrrole (GCP), to construct ionizable lipids. These GCP-based lipids can form multilayered, onion-like LNPs. These guanidine-based LNPs are highly sensitive to pH changes in the environment and demonstrate superior mRNA delivery capabilities both in vitro and in vivo. Unlike the commonly used amine-based lipids, GCP-lipids enable the formation of three-component, cholesterol-free LNPs that efficiently deliver functional mRNA to the spleen. Furthermore, we show that the new LNP system could transport mRNA to splenic immune cells with high efficacy, thus providing an exciting opportunity for future applications. Our findings underscore the advantages of ionizable guanidine-based lipids and could pave the way for the development of LNPs capable of targeting tissues beyond the liver. Physical sciences/Chemistry/Chemical biology/Drug delivery Physical sciences/Chemistry/Chemical biology/Nucleic acids Full Text Additional Declarations There is NO Competing Interest. Supplementary Files GLNPsupporting0725.pdf Cite Share Download PDF Status: Under Review 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. 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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-4805293","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":336797955,"identity":"543af6a3-4201-403b-b708-ba604a0857b9","order_by":0,"name":"Mao Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACPmYQWcHAYMBOrBY2sJYzQC3MRGsBEYxtJGlh50788HFenb05MwPjhx8MdnlEOIx3s+TMbYeZLZsZmCV7GJKLidGyQZp32wE2g8MMDNIMDAcSG4ix5TfvnDoeoBbm38Rq2SbN28AsAdTCRrQt2yxnHDtsYNnM2GbZY5BMWAs//9nNNz7UAEOMvfnwjR8VdoS1IAFGoGIDEtSPglEwCkbBKMANABxmLzHDMGMnAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9598-9776","institution":"Shenzhen Bay Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Mao","middleName":"","lastName":"Li","suffix":""},{"id":336797956,"identity":"28dbdb13-72d8-493a-949f-62c92825c881","order_by":1,"name":"He Zhang","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Zhang","suffix":""},{"id":336797957,"identity":"8d8d7cb9-da55-404a-aa9a-acafcf09c583","order_by":2,"name":"Dejing Liu","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Dejing","middleName":"","lastName":"Liu","suffix":""},{"id":336797958,"identity":"c97d927d-c8a9-40f4-895c-5bfecc9933f1","order_by":3,"name":"Zhuoying Liang","email":"","orcid":"","institution":"Shenzhen Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Zhuoying","middleName":"","lastName":"Liang","suffix":""}],"badges":[],"createdAt":"2024-07-26 04:35:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4805293/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4805293/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63424798,"identity":"5ef17aa8-e2f1-4b07-bd91-5c74e54112e8","added_by":"auto","created_at":"2024-08-28 03:32:40","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1077246,"visible":true,"origin":"","legend":"","description":"","filename":"GLNPNcommmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805293/v1_covered_581888be-1e74-4fb2-962f-2a3e21611258.pdf"},{"id":63424377,"identity":"dd59b0ed-6e27-406b-b21f-7e3d123a746d","added_by":"auto","created_at":"2024-08-28 03:24:39","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2766427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"GLNPsupporting0725.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805293/v1/02095dd93595af45ffd5a73f.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Using ionizable guanidine to develop cholesterol-free three-component lipid nanoparticles for spleen-targeted mRNA delivery","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4805293/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4805293/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Lipid nanoparticle (LNP) represents a key breakthrough in the realm of RNA therapeutics, particularly for the development of mRNA therapeutics and vaccines. 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