An Advanced Lyophilization Toward Intact Lipid Nanovesicles: Liquid-mediated Freezing With Cryoprotectant to Retain the Integrity of Lipid Nanovesicles

Abstract A novel lyophilization method using liquid-mediated freezing (LMF) with cryoprotectant (CPA) was designed to achieve intact lipid nanovesicles after lyophilization. During the freezing step, CPA prevented water supercooling, and the freezing rate was controlled by LMF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933°C/min) was the optimal medium for the LMF system. Lyophilized lipid nanovesicle using both LMF and CPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 135.5 nm, polydispersity index = 0.074), similar to intact vesicles (112.3 nm and 0.184, respectively), after rehydration. Only lyophilized lipid nanovesicle using both LMF and CPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs such as a mRNA vaccine, while retaining its original activity.