Exclusion of the strongly hygroscopic
polymer, poly(
ethylene glycol) (PEG), from the surface of
phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the
liposome surface and the bulk
polymer solution, thus causing a partial
dehydration of the
phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large
unilamellar liposomes (LUVs). This leads to, in addition to the
dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of
liposomes. Under these conditions phase separation of the fluorescent
lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-
phosphocholine (
PPDPC) embedded in various
phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent
lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the
dehydration of the
lipid. Importantly, phase separation of
PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (
DMPC),
1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (
SOPC), and
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent
lipid below Tm was otherwise not achieved. In the different
lipid matrices the segregation of
PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
DMPC,
SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of
PPDPC cannot be accounted for the 1 degree C increase in Tm for
DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of
PPDPC in the different
lipid matrices) decreased in the sequence
1,2-dihexadecyl-sn-glycero-3-phosphocholine (
DHPC) > DPPC >
DMPC >
SOPC > POPC, whereas no evidence for phase separation in
1,2-dioleoyl-sn-glycero-3-phosphocholine (
DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced
dehydration of liposomal membranes provides the driving force for the segregation of the
pyrene lipid. In brief, phase separation of
PPDPC from the matrix
lipid could be attributed to the diminishing effective size of the
phosphatidylcholine polar headgroup resulting from its partial
dehydration by PEG. This in turn would allow for enhanced van der Waals interactions between the acyl chains of the matrix
lipid, which then caused the exclusion of
PPDPC due to the perturbing bulky
pyrene moiety. Phase separation in
DMPC/
PPDPC liposomes was abolished by the inclusion of 25 mol %
cholesterol and to a lesser extent by
epicholesterol.