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Pharm Res (2014) 31:97103 DOI 10.1007/s11095-013-1135-z
RESEARCH PAPER
Freeze-Anneal-Thaw Cycling of Unilamellar Liposomes: Effect on Encapsulation Efficiency
Antonio P. Costa & Xiaoming Xu & Diane J. Burgess
Received: 24 April 2013 /Accepted: 24 June 2013 /Published online: 24 July 2013 # Springer Science+Business Media New York 2013
ABSTRACTPurpose 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 196C, annealing at 1.4C for ~30 min, thawing at 65C) resulted in the maximum predicted encapsulation efficiency without causing any significant change in particle size or zeta potential. Annealing at 22C 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.
KEY WORDS annealing . cryo-concentration . freeze-drying . freeze-thaw cycling . Tenofovir
ABBREVIATIONSChol CholesterolCryo-SEM Cryogenic scanning electron microscopy DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DSC Differential scanning calorimetryDSPC 1,2-distearoyl-sn-glycero-3-phosphocholine DSPG 1,2-dioctadecanoyl-sn-glycero-3-phospho-
(1-rac-glycerol)
EE% Encapsulation efficiency percentage FANNT Freeze-anneal-thaw
FT Freeze-thawPDI Polydispersity index PFE Pre-formed empty
INTRODUCTION
Freeze-thaw (FT) cycling is a technique often used in the preparation of liposomes to increase encapsulation efficiency (1,2). A common procedure is freezing the liposomes with liquid nitrogen (196C) and thawing at a temperature above the phase transition temperature of the lipids (35). In the preparation of liposomes, freeze-thaw cycling is implemented to reduce the lamellarity of liposomes (6), form a less polydispersed system and/or disrupt the liposomal bilayer (7) to allow drug molecules to diffuse into the liposome, promoting encapsulation (5,8,9). The number of freeze-thaw cycles needed to...