Liposomes separation

Sephadex G-50 (medium) (Phase Separations, Pharmacia, Sweden). The powder is dispersed in PBS buffer for swelling and the dispersion is subsequently degassed under vacuum. Gel chromatography columns are packed and used for DRV liposome separation from nonencapsulated molecules (as described in detail in the following section). 

A study by Bames and co-workers of the equilibrium spreading behavior of dimyristol phosphatidylcholine (DMPC) reconciles the differences between spreading of bulk solids and dispersions of liposomes [41]. This study shows the formation of multibilayers below the monolayer at the air-water interface. An incipient phase separation, undetectable by microscopy, in DMPC-cholesterol... 

In water, a particle of lecithin exhibits myelin growth, ie, cylindrical sheets that are formed by bdayers and are separated by water which may break up into liposomes (vesicles with a single bilayer of Hpid enclosing an aqueous space). PhosphoHpids more generally form multilamellar vesicles (MLV) (5). These usually are converted to unilamellar vesicles (ULV) upon treatment, eg, sonication. Like other antipolar, surface-active agents, the phosphoHpids are... 

Phospholipids e.g. form spontaneously multilamellar concentric bilayer vesicles73 > if they are suspended e.g. by a mixer in an excess of aqueous solution. In the multilamellar vesicles lipid bilayers are separated by layers of the aqueous medium 74-78) which are involved in stabilizing the liposomes. By sonification they are dispersed to unilamellar liposomes with an outer diameter of 250-300 A and an internal one of 150-200 A. Therefore the aqueous phase within the liposome is separated by a bimolecular lipid layer with a thickness of 50 A. Liposomes are used as models for biological membranes and as drug carriers. 

FIGURE H Proposed mechanism for the separation of "free" DXR from DXR in negative liposomes with a cation exchange resin. (From Crommelin and Storm, 1987.)... 

Size exclusion chromatography has been used to analyse the size distribution of liposomes. For example, SUV can be separated from MLV, which elute in the void volume, by using a Sepharose 4B gel. 

ATPase also catalyzed a passive Rb -Rb exchange, the rate of which was comparable to the rate of active Rb efflux. This suggested that the K-transporting step of H,K-ATPase is not severely limited by a K -occluded enzyme form, as was observed for Na,K-ATPase. Skrabanja et al. [164] also described the reconstitution of choleate solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. With the use of a pH electrode to measure the rate of H transport they observed not only an active transport, which is dependent on intravesicular K, but also a passive H exchange. This passive transport process, which exhibited a maximal rate of 5% of the active transport process, could be inhibited by vanadate and the specific inhibitor omeprazole, giving evidence that it is a function of gastric H,K-ATPase. The same authors demonstrated, by separation of non-incorporated H,K-ATPase from reconstituted H,K-ATPase on a sucrose gradient, that H,K-ATPase transports two protons and two ions per hydrolyzed ATP [112]. 

In the discussion above it has been shown that the lipid can been polymerized through UV irradiation of its aqueous suspension. The polymerization of the system improves the stability of the synthetic liposomes. Since there is an acetal linkage introduced between the polymer chain and the amphiphilic structure, this linkage can be slowly hydrolyzed in aqueous systems to separate the polymer chain from the lipid. 

Liposomes have been, and continue to be, of considerable interest in drug-delivery systems. A schematic diagram of their production is shown in Fig. 10. Liposomes are normally composed of phospholipids that spontaneously form multilamellar, concentric, bilayer vesicles, with layers of aqueous media separating the lipid layers. These systems, commonly referred to as multilamellar vesicles (MLVs), have diameters in the range of 15 pm. Sonication of MLVs... 

The determination of partition coefficients using liposomes as a lipid phase require that the sample be equilibrated with a suspension of liposomes, followed by a separation procedure, before the sample is quantitated in the fraction free of the lipid component. 

The pH-metric method, which also requires no phase separation, has been used to determine dmg-liposome partitioning [149,162,385-387], The method is the same as that described in Section 4.14, except that FAT-LUV-ET liposomes are used in place of octanol. SUV liposomes have also been used [385,386]. To allow for pH gradients to dissipate (Section 5.6) in the course of the titration, at least 5-10 min equilibration times are required between successive pH readings. 

Liposomes are artificial structures composed of phospholipid bilayers exhibiting amphiphilic properties (Chapter 22). In complex liposome morphologies, concentric spheres or sheets of lipid bilayers are usually separated by aqueous regions that are sequestered or compartmentalized... 

The modified liposomes may be separated from excess protein by gel filtration using Sephadex G-75 or by centrifugal floatation in a polymer gradient (Derksen and Scherphof, 1985). 

The next level of sophistication involved studies of systems where separate donors and acceptors interact across an interface. Chlorophyll-quinone photochemical studies have been conducted using liposomes (23-25) and acetate films (26). Calvin and his coworkers (27) have conducted a variety of experiments... 

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