The spontaneous, dynamic formation of hydrophobic active sites in lipid bilayer membranes is studied and characterized. It is shown that the rates of formation and consumption of these active sites control at least two important properties of liposomes: their affinity for hydrophobic surfaces and the rate by which they spontaneously release encapsulated molecules. The adhesion and spreading of liposomes onto hydrophobic polystyrene nanoparticles and the spontaneous leakage of an encapsulated fluorescent dye were monitored for different liposome compositions employing Cryo-TEM, DLS, and fluorescence measurements. It was observed that an apparently homogeneous, monodisperse liposome suspension behaves as if composed by two different populations: a fast leaking population that presents affinity for the hydrophobic substrate employed, and a slow leaking population that does not attach immediately to it. The results reported here suggest that the proportion of liposomes in each population changes over time until a dynamic equilibrium is reached. It is shown that this phenomenon can lead to irreproducibility in, for example, spontaneous leakage experiments, as extruded liposomes leak much faster just after preparation than 24 h afterward. Our findings account for discrepancies in several experimental results reported in the literature. To our knowledge, this is the first systematic study addressing the issue of an existing intrinsic heterogeneity of liposome suspensions.
© 2011 American Chemical Society