POLY(ETHYLENE GLYCOL)-LIPID CONJUGATES PROMOTE BILAYER FORMATION IN MIXTURES OF NON-BILAYER-FORMING LIPIDS

The influence of poly(ethylene glycol)-lipid conjugates on phospholipi d polymorphism has been examined using P-31-NMR and freeze-fracture el ectron microscopy. An equimolar mixture of dioleoylphosphatidylethanol amine (DOPE) and cholesterol adopts the hexagonal (H-II) phase when hy drated under physiological conditions but can be stabilized in a bilay er conformation when a variety of PEG-lipid conjugates are included in the lipid mixture, These PEG conjugates produced an increase in the b ilayer to hexagonal (H-II) phase transition temperature and a broadeni ng of the temperature: range over which both phases coexisted. Further , the fraction of phospholipid adopting the bilayer phase increased wi th increasing mole fraction of PEG-lipid such that at 20 mole % DOPE-P EG(2000) no H-II phase phospholipid was observed up to at least 60 deg rees C. Increasing the size of the PEG moiety from 2000 to 5000 Da (wh ile maintaining the PEG-lipid molar ratio constant) increased the prop ortion of lipid in the bilayer phase. In contrast, varying the acyl ch ains of the PE anchor had no effect on polymorphic behavior. PEG-lipid conjugates in which ceramide provides the hydrophobic anchor also pro moted bilayer formation in DOPE:cholesterol mixtures but at somewhat h igher molar ratios compared to the corresponding PEG-PE species. The s lightly greater effectiveness of the PE conjugates may result from the fact that these derivatives also possess a net negative charge. Phosp horus NMR spectroscopy indicated that a proportion of the phospholipid in DOPE:cholesterol:PEG-PE mixtures experienced isotropic motional av eraging with this proportion being sensitive to both temperature and P EG molecular weight. Surprisingly, little if any isotropic signal was observed when PEG-ceramide was used in place of PEG-PE. Consistent wit h 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 e ffects of PEG-lipid conjugates on DOPE:cholesterol mixtures are 2-fold , First, the complementary ''inverted cone'' shape of the conjugate he lps to accommodate the ''cone-shaped'' lipids, DOPE and cholesterol, i n the bilayer phase. Second, the steric hindrance caused by the PEG gr oup inhibits close apposition of bilayers, which is a prerequisite for the bilayer to H-II phase transition.