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.

Liposomes or Extracellular Vesicles: A Comprehensive Comparison of Both Lipid Bilayer Vesicles for Pulmonary Drug Delivery

“…In contrast, in order to achieve lyophilized intact lipid nanovesicles (i.e., LPs and EVs), an advanced lyophilization technology has been developed that combines liquid-mediated freezing (LMF), e.g., isopropanol was optimal, and lyoprotectant, e.g., trehalose for internal- and sucrose for an external-aqueous phase in order to retain the lipid nanovesicles integrity. During the freezing step, the lyoprotectant prevents supercooling of the water, while LMF controls the freezing rate, which is a critical factor for membrane disruption [ 199 ] ( Figure 10 ). However, further investigation is needed to apply these techniques on a large scale to manufacture and store EV-based therapeutics.…”