A Polysorbate-Based Lipid Nanoparticle Vaccine Formulation Induces In Vivo Immune Response Against SARS-CoV-2

<b>Background:</b> 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's unique structural properties, this study aimed to develop an mRNA-LNP platform with improved extrahepatic delivery and robust freeze/thaw tolerance. <b>Methods</b>: 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 -80 °C. <b>Results:</b> 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's physicochemical properties and in vitro potency over six months at -80 °C, 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. <b>Conclusions:</b> 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.