Effects of disaccharide and cationic lipid types on reverse transfection with lyophilized mRNA lipoplexes

Recently, the development of messenger RNA (mRNA) therapeutics has received increased attention, and mRNA/cationic liposome complexes (mRNA lipoplexes) have been studied as effective mRNA delivery carriers. However, owing to the vast diversity of the mRNA and carrier components employed, establishing a comprehensive analysis of their efficacy remains a critical issue. Reverse transfection using lyophilized mRNA lipoplexes has the potential to simplify and automate the efficacy evaluation processes. The present study investigated the effects of cationic lipids in cationic liposomes and disaccharides as cryoprotectants during reverse transfection with lyophilized mRNA lipoplexes. Overall, five types of dialkyl or trialkyl cationic lipids were used to prepare cationic liposomes. The mRNA lipoplexes were lyophilized in the presence of trehalose or sucrose in multi-well plates. An increase in the concentration of the disaccharide solution during the lyophilization of mRNA lipoplexes enhanced the transfection activity. Furthermore, mRNA lipoplexes lyophilized in 150 mM sucrose solution exhibited long-term stability for up to 1 month. The transfection activity of mRNA lipoplexes composed of dialkyl cationic lipids was largely unaffected by lyophilization, whereas a significant reduction in transfection activity was observed for mRNA lipoplexes composed of trialkyl cationic lipids. These findings suggest that although dependent on the lipid type used for the preparation of cationic liposomes, the reverse transfection method using lyophilized mRNA lipoplexes has the potential to be applied for screening the transfection efficiency of mRNA lipoplexes and the function of proteins translated from mRNA.