Poly(ethylene glycol) (PEG) decorated lipid bilayers are widely used in biomembrane and pharmaceutical research. The success of PEG-lipid stabilized liposomes in drug delivery is one of the key factors for the interest in these polymer/lipid systems. From a more fundamental point of view, it is essential to understand the effect of the surface grafted polymers on the physical-chemical properties of the lipid bilayer. Herein we have used cryo-transmission electron microscopy and dynamic light scattering to characterize the aggregate structure and phase behavior of mixtures of PEG-lipids and distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The PEG-lipids contain PEG of molecular weight 2000 or 5000. We show that the transition from a dispersed lamellar phase (liposomes) to a micellar phase consisting of small spherical micelles occurs via the formation of small discoidal micelles. The onset of disk formation already takes place at low PEG-lipid concentrations (<5 mol %) and the size of the disks decreases as more PEG-lipid is added to the lipid mixture. We show that the results from cryo-transmission electron microscopy correlate well with those obtained from dynamic light scattering and that the disks are well described by an ideal disk model. Increasing the temperature, from 25 degrees C to above the gel-to-liquid crystalline phase transition temperature for the respective lipid mixtures, has a relatively small effect on the aggregate structure.

Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present.

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The effect of Triton X-100 on small unilamellar lecithin vesicles has been studied by means of quasi-elastic light scattering and cryogenic transmission electron microscopy. Low concentrations of Triton have little effect on the vesicle size, and very high concentrations give rise to the formation of mixed micelles. Addition of intermediate concentrations of surfactant results in the formation of very large vesicles with radii of up to 1000 A. The two methods give aggregate sizes in good agreement. Time-resolved cryo-TEM experiments showed that the time for the formation of the large vesicles is about 2 min. The use of a controlled environment vitrification system (CEVS) for the cryo-TEM sample preparation made it possible to visualize the aggregates formed without introducing artifacts due to changes in temperature or saturation.

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