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Freeze-Anneal-Thaw Cycling of Unilamellar Liposomes: Effect on Encapsulation Efficiency

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ABSTRACT

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.

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Abbreviations

Chol:

Cholesterol

Cryo-SEM:

Cryogenic scanning electron microscopy

DPPC:

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

DSC:

Differential scanning calorimetry

DSPC:

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-thaw

PDI:

Polydispersity index

PFE:

Pre-formed empty

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ACKNOWLEDGMENTS AND DISCLOSURES

We express our gratitude to Dr. Marie Cantino and Stephen Daniels from the Biosciences Electron Microscope Laboratory of the Physiology and Neurobiology Department at the University of Connecticut for their work on the cryo-SEM images. The views expressed are those of authors and do not necessarily represent the official position of the Agency (FDA). The authors declare no competing financial interest.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd U3092, Storrs, Connecticut, 06269, USA

    Antonio P. Costa & Diane J. Burgess

  2. FDA/CDER/DPQR, 10903 New Hampshire Ave, WO64 RM1076, Silver Spring, Maryland, 20993, USA

    Xiaoming Xu

Authors
  1. Antonio P. Costa
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  2. Xiaoming Xu
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  3. Diane J. Burgess
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Correspondence to Diane J. Burgess.

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Costa, A.P., Xu, X. & Burgess, D.J. Freeze-Anneal-Thaw Cycling of Unilamellar Liposomes: Effect on Encapsulation Efficiency. Pharm Res 31, 97–103 (2014). https://doi.org/10.1007/s11095-013-1135-z

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  • DOI: https://doi.org/10.1007/s11095-013-1135-z

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