Phospholipids liposome preparation

Sodium chlorite and chlorate did not induce lipid peroxidation at concentrations up to 400 pmol/l in an in vitro system of multilayer phospholipid liposomes prepared from egg phosphatidylcholine (Panasenko et al., 1997). 

Peroxidation of lipids is another factor which must be considered in the safety evaluation of liposome administration. Smith and coworkers (1983) demonstrated that lipid peroxides can play an important role in liver toxicity. Allen et al. (1984) showed that liposomes protected by an antioxidant caused less MPS impairment than liposomes subjected to mild oxidizing conditions. From the study of Kunimoto et al. (1981) it can be concluded that the level of peroxidation in freshly prepared liposome preparations and those on storage strongly depends both on the phospholipid fatty acid composition and on the head group of the phospholipid. Addition of appropriate antioxidants to liposomes composed of lipids which are liable to peroxidation and designed for use in human studies is therefore necessary. 

Liposomes are formed due to the amphiphilic character of lipids which assemble into bilayers by the force of hydrophobic interaction. Similar assemblies of lipids form microspheres when neutral lipids, such as triglycerides, are dispersed with phospholipids. Liposomes are conventionally classified into three groups by their morphology, i.e., multilamellar vesicle (MLV), small unilamellar vesicle (SUV), and large unilamellar vesicle (LUV). This classification of liposomes is useful when liposomes are used as models for biomembranes. However, when liposomes are used as capsules for drugs, size and homogeneity of the liposomes are more important than the number of lamellars in a liposome. Therefore, "sized" liposomes are preferred. These are prepared by extrusion through a polycarbonate... 

Osterherg, T. Svensson, M. Lundahl, P, Chromatographic retention of drug molecules on immohilized liposomes prepared from egg phospholipids and from chemically pure phospholipids, Eur. J. Pharm. Sci. 12, 427 139 (2001). 

Liposome Formation. The pioneering investigations of Bang-ham (5) have shown that thin films of natural phospholipids form bilayer assemblies if they are lyophilized in excess water by simple handshaking above the phase transition temperature. While this procedure results in the formation of large, multibilayered spherical structures, by ultrasonication of such lipid dispersions small unilamellar liposomes are formed (16), which are schematically shown in Figure 10. Additional metTiods for liposome preparation are described in a number of reviews (17,44,45,46). 

Several cell lines were used to inveshgate the role of PS oxidahon in apoptosis. Preferenhal oxidahon of PS was observed in human leukemia HL-60 cells (Fabisiak et al, 1998, 2000 Kawai et al, 2000), and normal human keratinocytes (Shvedova et al, 2001). Similarly, in pheochromocytoma PC 12 cells exposed to a radical-generating anhneoplashc dmg, neocarzinostatin, extemalizahon of PS was potentiated by its selechve oxidation in whole cells (Schor et al, 1999). In contrast, this selechve PS oxidahon did not occur in liposomes prepared from mixtures of PnA-labeled phospholipids extracted from the ceUs and exposed to oxidants imder the same conditions (Fabisiak et al, 1998 Kagan et al, 2000 Shvedova et al,... 

Phospholipid concentration was determined using our modification of Bartlett s procedure (49,53). Cholesterol concentration and purity were determined by HPLC or enzymatically by cholesterol oxidase (49,53). Purity of phospholipids as raw materials, and the extent of their hydrolysis during various steps of liposome preparation and liposome storage, were assessed by TLC and enzymatic determination of the increase in level of nonesterified fatty acids (10,38,49-51,53). 

Glycolipid incorporated liposomes have found extensive use as sensors for the detection of Escherichia coli bacteria. Liposomes prepared using a diacetylene and a glucosyl lipid underwent a chromatic transition upon the addition of E. coli (Ma et al. 1998). The chromatic transition is sensitive to the diyne and glycolipid stmc-ture (Ma et al. 2000). An optimized vesicle assembly, consisting of a maltotriosyl lipid, phospholipid, and diyne, detected E. coli at a concentration of 2x10 cells/mL... 

By inserting hydrophobic antiobesity compounds into the liposomal bilayers, marine phospholipids would boost the effect of the inserted hydrophobic antiobesity compounds. When marine phospholipids are served as liposomal drinks, they would be more effective than adding into solid foods or feeds. These facts were borne out by Okada et al. (2011). They carried out the following experiment. Brown seaweed (Undaria pinnatifida) lipid containing fucoxanthin (UL) encapsulated into scallop midgut gland phospholipids (PL) liposomes were prepared to see the promotional effect of marine phospholipid liposome on antiobesity. Animal model used in their study was 3-week-old male diabetic-obese... 

As an example of an asymmetric membrane integrated protein, the ATP synthetase complex (ATPase from Rhodospirillum Rubrum) was incorporated in liposomes of the polymerizable sulfolipid (22)24). The protein consists of a hydrophobic membrane integrated part (F0) and a water soluble moiety (Ft) carrying the catalytic site of the enzyme. The isolated ATP synthetase complex is almost completely inactive. Activity is substantially increased in the presence of a variety of amphiphiles, such as natural phospholipids and detergents. The presence of a bilayer structure is not a necessary condition for enhanced activity. Using soybean lecithin or diacetylenic sulfolipid (22) the maximal enzymatic activity is obtained at 500 lipid molecules/enzyme molecule. With soybean lecithin, the ATPase activity is increased 8-fold compared to a 5-fold increase in the presence of (22). There is a remarkable difference in ATPase activity depending on the liposome preparation technique (Fig. 41). If ATPase is incorporated in-... 

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. 

Despite the large variety of potential fatty acid components in natural-occurring phosphodiglycerides, only three major fatty acid derivatives of synthetic phospholipids are commonly used in liposome preparation (1) myristic acid (w-tetradecanoic acid containing 14 carbons), (2) palmitic acid (w-hexadecanoic acid containing 16 carbons), and (3) stearic acid (w-octadccanoic acid containing 18 carbons) (Fig. 334). 

Another significant component of many liposome preparations is cholesterol. In natural cell membranes, cholesterol makes up about 10—50% of the total lipid on a molar basis. For liposome preparation, it is typical to include a molar ratio of about 50% cholesterol in the total lipid recipe. The addition of cholesterol to phospholipid bilayers alters the properties of the resultant membrane in important ways. As it dissolves in the membrane, cholesterol orients itself with its polar hydroxyl group pointed toward the aqueous outer environment, approximately even, in a three-dimensional sense, with the glyceryl backbone of the bilayer s phosphodiglyceride components (Fig. 337). Structurally, cholesterol is a rigid component in membrane construction, not having the same freedom of movement that the fatty acid tails of... 

The vaccine was incorporated into phospholipid-based liposomes prior to administration the mice that were immunized with the liposome preparations elicited IgM and IgG antibodies against the Tn antigen. 

In a related study [62], in vivo skin hydration was monitored by FUR after occlusive application of either NAT 106 liposomes prepared in D20 or pure D20. Liposomes were superior, compared with pure D20, in driving D20 into the skin, and the phospholipid components could be detected in deeper SC layers. The presence of phospholipids deep in the SC confirmed the results observed with freeze fracture, in which strong interactions between phospholipid layers and SC were observed. 

As phospholipid bilayers form spontaneously when water is added, the important challenge in liposome preparation is not the assembly of simple bilayers (which happens automatically), but in causing the bilayers to form stable vesicles of the desired size, structure and physicochemical properties, with a high drag encapsulation efficiency. There are many different approaches to the preparation of liposomes however, they all have in common that they are based on the hydration of lipids ... 

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