Ln(III)-Catalyzed Cleavage of Phosphate-Functionalized Synthetic Lipids: Real Time Monitoring of Vesicle Decapsulation

Abstract

Double-chain zwitterionic phosphate lipids (1a,b) have been synthesized. Upon their hydrolysis, two single-chain surfactants, soluble in water, are produced. The presence in the cationic counterpart of a p-nitrophenol subunit allows one to monitor spectrophotometrically the cleavage process. By sonication, lipids 1 form stable vesicles which are in the fluid state in the temperature interval explored (5−55 °C) and are too leaky to trap the fluorescent dye 4(5)-carboxyfluoresceine (CF). In the case of 1a, addition of at least 10% cholesterol increases the packing of the lipids making the vesicles less permeable to CF although at up to 40% additive the membrane remains in its fluid state. Holovesicular compounds 1 are hydrolytically very stable at pH 7, but the addition of different Ln(III) ions (Eu, Tm, Tb) efficiently catalyzes the hydrolytic cleavage (t1/2 ca. 45 min at 25 °C with 1 mM Eu(III)), which follows a regular monoexponential kinetic process. In the case of the vesicular preparation of 1a containing cholesterol, the kinetic profile is more complicated and can be divided into three regions: a first monoexponential part accounting for the cleavage of ca. 30% of the lipids residing in the external leaflet of the membrane; a second part in which all lipids start to be exposed to the metal ion; a third part, again monoexponential, in which all the remaining lipids are cleaved. Experiments with trapped CF show that in correspondence with the time in which the metal ions start to interact with all lipids, the rate of dye release increases although the aggregate maintains its vesicular structure. Analysis of the data strongly suggests that in the hydrolytic process the exposure of all lipids to the action of the metal ions is the result of both permeation and increased flip-flop of the phosphate-functionalized lipids. This would indicate that the hydrolytic process affects lipid mobility more than the permeability of the bilayer.