Development of lyophilized mRNA-LNPs with high stability and transfection efficiency in specific cells and tissues.

Abstract

Lipid nanoparticles (LNPs) are critical for the delivery of drugs and nucleic acids. However, current mRNA-LNP formulations require stringent freezing for storage, which limits their global distribution. Our previous studies demonstrated that optimizing the lipid type or molar ratio of Comirnaty-type mRNA-LNPs could enhance their lyophilization stability, thus improving their long-term storage stability under mild conditions. This study aims to enhance the storage stability of Spikevax-type mRNA-LNPs by optimizing lipid compositions and utilizing lyophilization for storage at 4°C. Fifteen mRNA-LNP formulations were evaluated for their physicochemical properties and transfection efficiency (TE) in human embryonic kidney (HEK)-293T cells using the I-optimal design of mixture experiments. Mathematical models were developed to predict the relationships among encapsulation efficiency, transfection performance and lipid ratios. The optimized mRNA-LNP formulation (N4), with a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-to-cholesterol ratio of 0.36, exhibited superior stability and TE after lyophilization. N4 outperformed the original Spikevax formulation in several cell lines, including eye-derived ARPE-19 cells and lung-derived A549 cells. In vivo, N4 demonstrated high TE in the spleen of C57BL/6 mice both before and after lyophilization, with no signals observed in the kidneys, heart or eyes. These findings suggest that the optimized N4 formulation offers a robust, stable and efficient delivery system for gene therapy and vaccines, potentially overcoming the storage limitations of current Spikevax-type mRNA-LNPs and broadening their therapeutic applications.