Morphological characterization of liposome suspensions by stereological analysis of freeze‐fracture replicas from spray‐frozen samples

Abstract

A method for size distribution analysis of liposome suspensions is described. Spray-freezing was used in order to avoid cryoprotectants and fixatives. Light scattering showed that, depending on their composition, liposomes can be modified by the spraying procedure. Dipalmitoylphosphatidylcholine liposome suspensions display large modifications of light scattering upon spraying. The commonly used unilamellar phosphatidylserine/phosphatidylcholine (1:4) liposomes, with or without cholesterol, are, however, only slightly affected by the spraying procedure. For liposome suspensions stable upon spraying, size distribution analysis can be performed on freeze-fracture replicas by standard procedures and average size, area and volume of particles are obtained. The number of particles per unit volume cannot be determined by stereological procedures, since the fracture face does not correspond to a plane section through the sample. By combining stereological data with phospholipid mass and specific molecular area of phospholipids, abundance of particles, fractional volume and total membrane area can nevertheless be evaluated. Data on small unilamellar liposomes prepared by sonication are presented as an illustration of the method. In suspensions obtained by this procedure, vesicles may be considered at thermodynamic equilibrium in the aqueous phase. The experimental size distribution compared favourably with that obtained from self-assembly theory for liposomes, considering natural lecithin heterogeneity. For comparison purposes, data on large unilamellar liposomes, obtained by reverse-phase evaporation, are also presented.