Membrane vesicle lipid uptake

The results in Figure 3, obtained with membrane vesicles, show that imposition of a membrane potential difference (inside negative) greatly enhanced the peak uptake of a solute taken up by a Na+-dependent route. The potential difference could be increased by using lipid soluble anions or valinomycin in vesicles with Kf KJ (Colombini and Johnstone, 1974 Murer and Hopfer, 1974 Sigrist-Nelson et al., 1975 Lever, 1977 Hammerman and Sacktor, 1978 Hopfer, 1978 Wright et al 1983 Kimmich etal., 1991). 

F Bellemare, J Noel, C Malo. Characteristics of exogenous lipid uptake by renal and intestinal brush border membrane vesicles. Biochem Cell Biol 73 171—179,1995. 

In particular, the use of intestinal brush border membranes has offered much potential for the detailed investigation of absorptive phenomena. It is likely that the absorption of lipid nutrients including fat-soluble vitamins for example, which may intercalate transiently into the bilayer and affect the physical properties of the membrane, will concurrently influence the entry of other substances. Alternatively, interactions between nutrients in the gut might be expected to mutually influence absorption of these substances. Also, there may be competition between nutrients for the same transport system. Membrane vesicles are very much suited for studying this type of problem and their use has helped resolve, for example, the mechanism whereby lecithins inhibit cholesterol uptake (Merrill et al, 1987). Again, the use of isolated brush order membranes has been conducive to a better understanding of the mechanisms of interaction between lipid micelles or lipid vesicles and the intestinal luminal membrane. 

Other systems like electroporation have no lipids that might help in membrane sealing or fusion for direct transfer of the nucleic acid across membranes they have to generate transient pores, a process where efficiency is usually directly correlated with membrane destruction and cytotoxicity. Alternatively, like for the majority of polymer-based polyplexes, cellular uptake proceeds by clathrin- or caveolin-dependent and related endocytic pathways [152-156]. The polyplexes end up inside endosomes, and the membrane disruption happens in intracellular vesicles. It is noteworthy that several observed uptake processes may not be functional in delivery of bioactive material. Subsequent intracellular obstacles may render a specific pathway into a dead end [151, 154, 156]. With time, endosomal vesicles become slightly acidic (pH 5-6) and finally fuse with and mature into lysosomes. Therefore, polyplexes have to escape into the cytosol to avoid the nucleic acid-degrading lysosomal environment, and to deliver the therapeutic nucleic acid to the active site. Either the carrier polymer or a conjugated endosomolytic domain has to mediate this process [157], which involves local lipid membrane perturbation. Such a lipid membrane interaction could be a toxic event if occurring at the cell surface or mitochondrial membrane. Thus, polymers that show an endosome-specific membrane activity are favorable. 

A highly stable and shielded polyplex should circulate in the blood stream without undesired interactions until it reaches the target cell. At that location, specific interactions with the cell surface should trigger intracellular uptake. While lipid membrane interaction is undesired at the cell surface, it should happen subsequently within the endosomal vesicle and mediate polyplex delivery into the cytosol. During or after intracellular transport to the site of action, the polyplex stability should be weakened to an extent that the nucleic acid is accessible to exert its function. 

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