Liposomes leaflet

Razinkov, V. I., HernandezJimenez, E. I., Mikhalyov, 1.1., Cohen, F. S. and Molotkovsky, J. G. (1997). New fluorescent lysolipids Preparation and selective labeling of inner liposome leaflet. Biochim. Biophys. Acta-Biomembranes 1329, 149-158. 

J. Sunamoto 110> reported a coating of the outer liposome leaflet with partially palmitoyl-derivatized polysaccharides according to route 2 in Fig. 53. These arti-... 

Lipids also show asymmetrical distributions between the inner and outer leaflets of the bilayer. In the erythrocyte plasma membrane, most of the phosphatidylethanolamine and phosphatidylserine are in the inner leaflet, whereas the phosphatidylcholine and sphingomyelin are located mainly in the outer leaflet. A similar asymmetry is seen even in artificial liposomes prepared from mixtures of phospholipids. In liposomes containing a mixture of phosphatidylethanolamine and phosphatidylcholine, phosphatidylethanolamine localizes preferentially in the inner leaflet, and phosphatidylcholine in the outer. For the most part, the asymmetrical distributions of lipids probably reflect packing forces determined by the different curvatures of the inner and outer surfaces of the bilayer. By contrast, the disposition of membrane proteins reflects the mechanism of protein synthesis and insertion into the membrane. We return to this topic in chapter 29. 

Another group of naturally occurring lipids with applications in liposome technology is comprised of the sphingophospholipids (mainly sphingomyelin) which are derivatives of ceramides [17]. Sphingomyelin (SM) is found in the outer leaflet of plasma membranes [17] and has many similarities with PC since they both have the same zwitterionic polar group and two hydrophobic acyl chains. 

Is lipid-assisted folding a widespread phenomenon and possibly applicable to soluble proteins The erythrocyte membrane contains about 20-mole % of PE that is almost exclusively localized in the inner leaflet and is in contact with highly concentrated heme-containing proteins. The refolding of the denatured soluble and heme-containing enzyme horseradish peroxidase (HRP) was followed in the presence and absence of liposomes made up of different phospholipids (Debnath et al., 2003). Remarkably, dimyristoyl-PE (a bilayer-forming... 

Direct experiments to examine the transbilayer movement of phospholipids (R.D. Kornberg, 1971) made use of spin-labeled analogs of PC in which the choline moiety was replaced with the tempocholine probe,, A -dimethyl-A -(l -oxyI-2, 2, 6, 6 -tetramethyl-4 -piperidyl)-ethanolamine (Fig. 2). These workers found that only the electron spin resonance signal generated by molecules in the outer leaflet of unilamellar liposomes could be rapidly quenched by ascorbate. The electron spin resonance signal from lipid molecules initially residing at the inner leaflet of liposomes was accessible to ascorbate with a r,/2 of >6.5 h, indicating slow transbilayer lipid movement (Fig. 3). 

Additional evidence for slow transbilayer phospholipid movement in liposomes came from experiments using [ H]PC-labeled liposomes and PC transfer protein. In the presence of excess unlabeled acceptor membranes, only the PC in the outer leaflet of the liposome membrane was rapidly transferred (J.E. Rothman, 1975). The [ H]PC initially present in the inner leaflet of the membrane moved to the outer leaflet with a r,/2 of... 

Fig. 3. Summary of key experiments examining transbilayer lipid movement in liposomes. Reaction of dithio-bisnitrobenzoic acid (DTNBA) with R-SH gives R-S-SNBA. Each of the experiments was specifically designed to initially sample only the outer leaflet of the bilayer and then at subsequent periods detect the movement of lipid from the inner to the outer leaflet of the bilayer. lAA, isethionylacetimidate TNBS, trinitrobenzenesulfonate DG, diacylglycerol DTNBA, dithiobisnitrobenzoic acid (Ellman s reagent).

Vesicles (from the Latin vesicula, small bubble) belong to the most extensively studied amphiphilic aggregates. Not only because the bilayer membrane is the building block of cell membranes, but also because vesicles or liposomes offer the unique possibility for solutes to bind to the outer- and inner leaflets of the bilayer whereas the hydrophobic interior of the bilayer can be used to entrap hydrophobic solutes and also membrane proteins. 

In some experiments, the liposomes are supplemented with lipopep-tides that imitate the cytosolic tail of the transmembrane protein p23. Lipopeptide synthesis has been described elsewhere (Bremser et al, 1999 Nickel and Wieland, 2001). The lipopeptide (1 mM in DMSO) is incorporated by a 100-fold dilution in the extruded liposomes suspension (1 mM), giving a surface concentration of 2 mol % (considering the outer leaflet of the liposomes). 

These results demonstrate that an increase in the ionic strength of the buffer outside the liposome will decrease the molecular density in the DOPC monolayer. The expansion in the monolayer should indicate that the outer leaflet expands more than the inner leaflet in the liposomes. Hence, it is expected that the difference of area between leaflets contributes to the outward protmsion of the membrane in liposome. 

By considering these ti-A results, the surface area of inner leaflet in the liposome would exceed the area of inner leaflet when the external pH of the solution is smaller than the internal pH. As a result, the inner leaflet becomes pressed into the inside of liposome by the outer leaflet, creating an inward protrusion into the liposome. Notably, this deformation is observed in the absence of osmotic pressure differences across the liposome membrane wall. 

Fig 3(b) shows the n-A curve of a DOPC monolayer over a buffer solution with pH3.5 and ionic strength of 0.6. The ti-A curve for this experiment slowly increases with a decrease in surface area, as was the result in case 2. However, in this experiment, the curve begins to rise to a surface area of 1.2nm /molecules, which is larger than that of the reference curve. This new profile can be interpreted as a summation of the ion strength effect suggested in case 1 and the pH effect in case 2. Therefore, the combination of a rise in ionic strength and a decrease in pH expanded the DOPC mono-layers and also reduced their compressibility modulus to 7 mN/m [28], which is about half the value of the reference monolayers. If these monolayer properties for this third case are used to interpret the properties of each leaflet of the liposomes, it can be deduced that the outer leaflet area exceeds area of inner leaflet because the area of outer monolayer at case 3 have larger occupied area than that of inner monolayer at the reference. This excess in outer monolayer on liposome may drive the protrusion into outside of liposome. Furthermore,... 

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