Interaction of cultured human keratinocytes with liposomes encapsulating silver sulphadiazine: proof of the uptake of intact vesicles.

Abstract

There is evidence to suggest that human keratinocytes grown in vitro are capable of engulfing and subsequently disintegrating intact liposomes. However, as the liposomes used in this context did not carry an electron-dense marker, the possibility that the lamellar structures seen within the keratinocytes were composed of material produced within the cell could not be excluded. We therefore decided to investigate liposome-keratinocyte interaction using an electron-dense marker. Human keratinocytes obtained from juvenile foreskins were cultured in a serum-free medium, and subconfluent cultures were exposed to liposomally encapsulated and free silver sulphadiazine 1% (SSD), and a corresponding vehicle, for 5 min to 24 h. After fixation ultra-thin sections were analysed electron microscopically at magnifications of up to x85,000. Many keratinocytes treated with liposomal and free SSD showed marked damage to the plasma membranes and the cell organelles. The phagocytosis of intact liposomes was demonstrated by the appearance of silver-labelled unilamellar vesicles within the cytoplasm of undamaged keratinocytes. The labelled liposomes were found enclosed in cellular unit membranes, i.e. in lysosomes. In addition, perinuclear disintegration and release of the entrapped marker were observed. Silver particles, as present in liposomally encapsulated SSD, were found to be adequate markers for electron microscopy. Our results confirm the phagocytosis of intact liposomes by keratinocytes in vitro. In addition, the cytotoxic effects of liposomal (intended for the treatment of burns) and free SSD on human keratinocytes were studied in detail. Many keratinocytes treated for 10 min or more were severely affected.