Dynamic fluorescence investigations of the effect of osmotic shocks on the microenvironments of charged and uncharged dipalmitoyl-D,L-α-phosphatidylcholine liposomes

Abstract

Neutral methylanthracene (MA), anionic trisodium 8-hydroxy-1,3,6-pyrenetrisulfonate, (pyranine), and cationic 3,6-diamino-10-methylacridinium chloride (acriflavine), have been used as fluorescence probes to investigate effects of osmotic shrinkage on neutral, cationic and anionic dipalmitoyl-D,L-α-phosphatidylcholine liposomes. The determined fluorescence polarizations in the liposomes and in solvents of known viscosities afforded the estimation of the microviscosities of the environments of these probes. The viscosity reported by pyranine for anionic and that by acriflavine for cationic single compartment liposomes, ∼1.0 cP, indicate the aqueous environments of these probes. Increased viscosities following osmotic shrinkages have been rationalized in terms of changing the nature of the liposome entrapped water. Following the release of free water, some bound water is also released as the result of osmotic shrinkage. The determined shrinkage rates support this postulate. The viscosity of the environment of pyranine in cationic, 9.6 ± 0.3 cP, and that of acriflavine in anionic single compartment liposomes, 74 ± 5 cP, indicate electrostatic attractions of the probes to the charged liposome surface. Osmotic shrinkage results in lowering the viscosity of the environments of the probes presumably because the more concentrated sodium chloride replaces them from their sites. The high viscosities reported by MA, ∼ 1000 cP, suggest the intercalation of this probe in the phospholipid bilayers. Osmotic shrinkage does not alter the environment of MA. However, in the presence of cholesterol, the viscosities reported by MA are greater than in its absence. These data contradict previous NMR, ESR and X-ray results as well as those obtained in the present work from osmotic shrinkage rates. The need for care in interpreting data obtained by the use of fluorescence probes is emphasized.