Poly(ethylene glycol)-lipid conjugates promote bilayer formation in mixtures of non-bilayer-forming lipids

The influence of poly(ethylene glycol)-lipid conjugates on phospholipid polymorphism has been examined using P-31-NMR and freeze-fracture electron microscopy. An equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and cholesterol adopts the hexagonal (H-II) phase when hydrated under physiological conditions but can be stabilized in a bilayer conformation when a variety of PEG-lipid conjugates are included in the lipid mixture, These PEG conjugates produced an increase in the bilayer to hexagonal (H-II) phase transition temperature and a broadening of the temperature: range over which both phases coexisted. Further, the fraction of phospholipid adopting the bilayer phase increased with increasing mole fraction of PEG-lipid such that at 20 mole % DOPE-PEG(2000) no H-II phase phospholipid was observed up to at least 60 degrees C. Increasing the size of the PEG moiety from 2000 to 5000 Da (while maintaining the PEG-lipid molar ratio constant) increased the proportion of lipid in the bilayer phase. In contrast, varying the acyl chains of the PE anchor had no effect on polymorphic behavior. PEG-lipid conjugates in which ceramide provides the hydrophobic anchor also promoted bilayer formation in DOPE:cholesterol mixtures but at somewhat higher molar ratios compared to the corresponding PEG-PE species. The slightly greater effectiveness of the PE conjugates may result from the fact that these derivatives also possess a net negative charge. Phosphorus NMR spectroscopy indicated that a proportion of the phospholipid in DOPE:cholesterol:PEG-PE mixtures experienced isotropic motional averaging with this proportion being sensitive to both temperature and PEG molecular weight. Surprisingly, little if any isotropic signal was observed when PEG-ceramide was used in place of PEG-PE. Consistent with the P-31-NMR spectra, freeze-fracture electron microscopy showed the presence of small vesicles (diameter < 200 nm) and lipidic particles in DOPE:cholesterol mixtures containing PEG-PE, We conclude that the effects of PEG-lipid conjugates on DOPE:cholesterol mixtures are 2-fold, First, the complementary ''inverted cone'' shape of the conjugate helps to accommodate the ''cone-shaped'' lipids, DOPE and cholesterol, in the bilayer phase. Second, the steric hindrance caused by the PEG group inhibits close apposition of bilayers, which is a prerequisite for the bilayer to H-II phase transition.