The Effect of Mixed Lipid Concentrations and Sucrose on the Size of the Lipid Nanoparticles Containing mRNAs

The development of messenger RNA (mRNA) vaccines has been transformed using lipid nanoparticles (LNPs) as delivery systems. This study evaluates the impact of lipid mixture molar concentration and the addition of sucrose as a cryoprotectant on the size and stability of LNPs encapsulating mRNA. Using a microfluidic mixing technique, we formulated LNPs with varying lipid concentrations and analyzed their size, size distribution, and encapsulation efficiency through dynamic light scattering and a RiboGreen assay. Results indicated that higher lipid concentrations not only improved encapsulation efficiency but also maintained LNPs within the ideal size range of 80-120 nm, crucial for optimal biodistribution and immune cell uptake. Furthermore, the addition of sucrose significantly stabilized the LNPs against size changes during freeze-thaw cycles, particularly at lower temperatures. This study underscores the importance of optimizing lipid and sucrose concentrations to enhance the stability and functionality of LNPs, providing insights that could enhance the efficacy and storage stability of mRNA vaccines. HIGHLIGHTS The variation in molar concentrations of mixed lipids affects the particle size of LNP-mRNA. Buffer exchange using a membrane filter with centrifugation technique can reduce the size and uniformity of LNP particle distribution. The addition of sucrose affects the particle size of LNP-mRNA. The addition of 10 % sucrose as a cryoprotectant can maintain the particle size of LNP-mRNA at 4 and –20 °C. GRAPHICAL ABSTRACT