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Updated: May 9, 2026
Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins
Published on: August 31, 2019
Freeze-anneal-thaw cycling of unilamellar liposomes: effect on encapsulation efficiency
Antonio P Costa1, Xiaoming Xu, Diane J Burgess
1Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd U3092, Storrs, Connecticut, 06269, USA.
Purpose:
Freeze-thaw cycling is an important processing step in the preparation of liposomes that leads to the encapsulation of drug molecules. There is considerable variability in the number of freeze-thaw cycles reported in the literature. This work is designed to aid in liposomal formulation design by gaining an insight into the drug encapsulation process and an understanding of liposome stabilization during various thawing conditions.
Methods:
The effects of different thawing temperatures, as well as "annealing" at subzero temperatures on a liposome formulation, are reported here.
Results:
Two freeze-anneal-thaw (FANNT) cycles (freezing to -196°C, annealing at -1.4°C for ~30 min, thawing at 65°C) resulted in the maximum predicted encapsulation efficiency without causing any significant change in particle size or zeta potential. Annealing at -22°C was shown to be destabilizing due to limited hydration of the liposomes in the frozen state.
Conclusions:
It was shown that two important processes are occurring during the FANNT cycling that affect liposome encapsulation efficiency. The first is drug diffusion in the frozen state and the second is fusion/destabilization of the liposomes. This is the first report on the annealing of liposomes and understanding the mechanism of drug encapsulation using the freeze-thaw cycling method.
