{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:02:39Z","timestamp":1787029359223,"version":"build-2736575974"},"reference-count":34,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,3,29]],"date-time":"2025-03-29T00:00:00Z","timestamp":1743206400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background: Lipid nanoparticles (LNPs) have proven effective in delivering RNA-based modalities. Rapid approval of the COVID-19 vaccines highlights the promise of LNPs as a delivery platform for nucleic acid-based therapies and vaccines. Nevertheless, improved LNP designs are needed to advance next-generation vaccines and other gene therapies toward greater clinical success. Lipid components and LNP formulation excipients play a central role in biodistribution, immunogenicity, and stability. Therefore, it is important to understand, identify, and assess the appropriate lipid components for developing a safe and effective formulation. Herein, this study focused on developing a novel Polysorbate-80 (PS-80)-based LNP. We hypothesized that substituting conventional linear PEG-lipids with PS-80, a widely used, biocompatible injectable surfactant featuring a branched PEG-like structure, may change the LNPs biodistribution pattern and enhance long-term stability. By leveraging PS-80\u2019s unique structural properties, this study aimed to develop an mRNA-LNP platform with improved extrahepatic delivery and robust freeze\/thaw tolerance. Methods: We employed a stepwise optimization to establish both the lipid composition and formulation buffer to yield a stable, high-performing PS-80-based SARS-CoV-2 mRNA-LNP (SC2-PS80 LNP). We compared phosphate- versus tris-based buffers for long-term stability, examined multiple lipid ratios, and evaluated the impact of incorporating PS-80 (a branched PEG-lipid) on in vivo biodistribution. Various analytical assays were performed to assess particle size, encapsulation efficiency, mRNA purity, and in vitro potency of the developed formulation and a humanized mouse model was used to measure its immunogenicity over six months of storage at \u221280 \u00b0C. Results: Replacing the standard 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol-2000 (PEG-DMG) lipid with PS-80 increased spleen-specific expression of the mRNA-LNPs after intramuscular injection. Formulating in a tris-sucrose-salt (TSS) buffer preserved the LNP\u2019s physicochemical properties and in vitro potency over six months at \u221280 \u00b0C, whereas a conventional PBS-sucrose (PSS) buffer was less protective under frozen conditions. Notably, TSS-based SC2-PS80 LNPs elicited potent humoral immunity in mice, including high anti-spike IgG titers and robust pseudovirus neutralization, comparable to freshly prepared formulations. Conclusions: A PS-80-based mRNA-LNP platform formulated in TSS buffer confers improved extrahepatic delivery, long-term frozen stability, and strong immunogenicity against SARS-CoV-2 following six months. These findings offer a promising pathway for the design of next-generation mRNA vaccines and therapeutics with enhanced stability and clinical potential.<\/jats:p>","DOI":"10.3390\/pharmaceutics17040441","type":"journal-article","created":{"date-parts":[[2025,3,31]],"date-time":"2025-03-31T05:21:04Z","timestamp":1743398464000},"page":"441","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A Polysorbate-Based Lipid Nanoparticle Vaccine Formulation Induces In Vivo Immune Response Against SARS-CoV-2"],"prefix":"10.3390","volume":"17","author":[{"given":"Aishwarya","family":"Saraswat","sequence":"first","affiliation":[{"name":"Formulation Development Group, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0028-5870","authenticated-orcid":false,"given":"Alireza","family":"Nomani","sequence":"additional","affiliation":[{"name":"Formulation Development Group, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lin-Kin","family":"Yong","sequence":"additional","affiliation":[{"name":"Vaccine Technology, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jimmy Chun-Tien","family":"Kuo","sequence":"additional","affiliation":[{"name":"Regeneron Genetic Medicines, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heather","family":"Brown","sequence":"additional","affiliation":[{"name":"Regeneron Genetic Medicines, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muralikrishna","family":"Narayanareddygari","sequence":"additional","affiliation":[{"name":"Vaccine Technology, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avery","family":"Peace","sequence":"additional","affiliation":[{"name":"Infectious Diseases, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rizan","family":"Fazily","sequence":"additional","affiliation":[{"name":"Formulation Development Group, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy","family":"Blake","sequence":"additional","affiliation":[{"name":"Regeneron Genetic Medicines, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher D.","family":"Petro","sequence":"additional","affiliation":[{"name":"Infectious Diseases, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bindhu","family":"Rayaprolu","sequence":"additional","affiliation":[{"name":"Formulation Development Group, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johanna","family":"Hansen","sequence":"additional","affiliation":[{"name":"Vaccine Technology, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amardeep Singh","family":"Bhalla","sequence":"additional","affiliation":[{"name":"Formulation Development Group, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohammed","family":"Shameem","sequence":"additional","affiliation":[{"name":"Formulation Development Group, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Liu, Y., Huang, Y., He, G., Guo, C., Dong, J., and Wu, L. 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