title: Enhanced permeability of freeze-dried liposomal bilayers upon rehydration.
abstract: Until now, studies on the protection of liposomes against freeze-drying damage have mainly focused on the bilayer integrity during the freezing or drying step of this process. Here, we investigated the bilayer permeability of freeze-dried, lyoprotected liposomes to a nonbilayer interacting compound after rehydration, by monitoring the leak-in kinetics of externally added carboxyfluorescein (CF). The results showed that freeze-drying and rehydration of DPPC:DPPG 10:1 liposomes with sucrose in- and outside the vesicles caused a temporary increase in the bilayer permeability for CF, which leveled off after approximately 20 h. The amount of CF/mol phospholipid which leaked into the vesicles increased with vesicle size (range 0.1-1 micro m) / lamellarity. Reduction of the number of bilayers in 1-1 micro m) vesicles enhanced the permeability to CF after freeze-drying and rehydration. The presence of CHOL decreased CF leak-in rates into 1 micro m MLVs consisting of DPPC:DPPG 10:1, but not into 0.1-micro m unilamellar vesicles. In the absence of sucrose similar leak-in profiles as a function of time were found after rehydration, suggesting that repacking processes of the bilayer were responsible for the enhanced permeability after freeze-drying and dehydration both with and without sucrose. The effect of size and lamellarity on the CF leak-in correlated with the retention of encapsulated CF after freeze-drying and rehydration, but no correlation was found with the effect of lipid composition. Both small (0.1 micro m) lyoprotected liposomes made of DPPC:DPPG 10:1 and DPPC:DPPG:CHOL 10:1:4 were highly permeable during the rehydration step itself. The results indicate that, despite the presence of the lyoprotectant, "repacking" of the bilayer components takes place both during and after rehydration. This eventually leads to regaining of its barrier function.
authors.affiliation: Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, 3508 TB, The Netherlands.
authors.forename: W
authors.initials: W
authors.lastname: Zhang
authors.forename: E C
authors.initials: EC
authors.lastname: van Winden
authors.forename: J A
authors.initials: JA
authors.lastname: Bouwstra
authors.forename: D J
authors.initials: DJ
authors.lastname: Crommelin
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine; D005452:Fluoresceins; D005612:Freeze Drying; D066298:In Vitro Techniques; D008051:Lipid Bilayers; D008081:Liposomes; D010316:Particle Size; D010539:Permeability; D010715:Phosphatidylglycerols; D012542:Scattering, Radiation; D013395:Sucrose; D013997:Time Factors; D014867:Water; D014965:X-Rays
chemical_list: D005452:Fluoresceins; D008051:Lipid Bilayers; D008081:Liposomes; D010715:Phosphatidylglycerols; D014867:Water; D015060:1,2-Dipalmitoylphosphatidylcholine; C024098:6-carboxyfluorescein; D013395:Sucrose; C030345:1,2-dipalmitoylphosphatidylglycerol