Preparation and biophysical characterization of lipid nanoparticles encapsulating mRNA for vaccine delivery applications

Lipid nanoparticles (LNPs) are currently the leading delivery platform for mRNA-based vaccines and therapeutics due to their ability to protect and efficiently deliver nucleic acids into target cells. In this study, we designed and characterized LNPs encapsulating mRNA (LNP-mRNA), using a lipid composition based on Moderna’s Spikevax mRNA vaccine. LNPs were prepared by thinfilm hydration of lipids followed by extrusion to achieve uniform nanoscale particles. Incorporation of mRNAwas performed during the hydration step in an acetate buffer. The resulting LNPs were characterized by dynamic light scattering and electrophoretic light scattering to determine their hydrodynamic size and zeta potential. The average diameter of empty LNPs was approximately 114 nm, while mRNA encapsulation increased the size to 134 nm, with both formulations exhibiting a zeta potential of+32 mV, indicating good colloidal stability. The structural organization and membrane dynamics of LNPs were further evaluated by electron paramagnetic resonance spectroscopy using spin-labeled fatty acids, which confirmed preserved bilayer fluidity and stability after mRNA incorporation. Additionally, fluorescently labeled LNP-mRNA nanoparticles were prepared to enable future cellular uptake studies. These results demonstrate the successful formulation of stable mRNA-loaded LNPs with biophysical properties suitable for further therapeutic development and functional testing. Authors are thankful for the financial support by theMinistry of Science, Technological Development and Innovation, Contract numbers 451-03-137/2025- 03, 451-03-136/2025-03, and the Science Fund of the Republic of Serbia, PROMIS REDIRECT, Contract number 11132. *The authors marked with an asterisk equally contributed to thework