Reverse phase evaporation

Reverse Phase Evaporation Szoka and Papahadjopoulos (1978) developed the so-called reverse phase evaporation method. Vesicles prepared with this technique (REV) show higher encapsulation efficiencies of hydrophilic compounds than unextruded MLV. 

Szoka, F., and Papahadjopoulos, D. (1978). Procedure for preparation of liposomes with large aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Acad. Sci. USA. 75, 4194-4198. 

REVs Reverse-phase evaporation vesicles LUVs prepared by reverse-phase evaporation method, high encapsulation efficiency 11,12... 

Reverse-phase evaporation in a nitrogen atmosphere was used to prepare lipids. A lipid film previously formed was redissolved in diethyl ether and an aqueous phase containing the dyebath components added to the phospholipid solution. The resulting two-phase system was sonicated at 70 W and 5 °C for 3 minutes to obtain an emulsion. The solvent was removed at 20 °C by rotary evaporation under vacuum, the material forming a viscous gel and then an aqueous solution. The vesicle suspension was extruded through a polycarbonate membrane to obtain a uniform size distribution (400 nm). 

Nagata, T., Okabe, K., Takebe, I. and Matsui, C. (1981). Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (liposomes). Mol. Genet. Genomics 184, 161-5. 

Cortesi, R., Esposito, E., Gambarin, S., Telloli, P., Menegatti, E., and Nastruzzi, C., Preparation of liposomes by reverse-phase evaporation using alternative organic solvents, Journal of Microencapsulation, 1999, 16, 251-256. 

Szoka, F., Olson, F., Heath, T., Vail, W., Mayhew, E., and Paphadjopoulos, D. (1980) Preparation of unilamellar liposomes of intermediate size (0.1-0.2 pm) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes. Biocbim. Biophys. Acta 601, 559-571. 

Figure 10.10 Transmission electron micrograph of ferritin entrapped in POPC liposomes (palmitoyloleoylphosphatidylcholine). Cryo-TEM micrographs of (a) ferritin-containing POPC liposomes prepared using the reverse-phase evaporation method, followed by a sizing down by extrusion through polycarbonate membranes with 100 nm pore diameters ([POPC] = 6.1 mM) and (b) the vesicle suspension obtained after addition of oleate to pre-formed POPC liposomes ([POPC] = 3 mM, [oleic acid - - oleate] = 3 mM). (Adapted from Berclaz et al, 2001a, b.)...

Sada, E., Katoh, S., Terashima, M., Shiraga, H., and Miura, Y. (1990). Stability and reaction characteristics of reverse-phase evaporation vesicles (revs) as enzyme containers. Biotechnol. Bioeng., 36, 665-71. 

In this case, unilamellar vesicles with a large capture volume were prepared by the reverse phase evaporation technique and alginate was used to microencapsulate the liposome s. The alginate spheres were double coated, first with poly-L-lysine and then with polyvinyl amine (Wheatley and Langer in press). 

Imura, T., Otake, K., Hashimoto, S., Gotoh, T., Yuasa, M., Yokoyama, S., Sakai, H., Rathman, J. F., and Abe, M. (2003). Preparation and physicochemical properties of various soybean lecithin liposomes using supercritical reverse phase evaporation meOnWsfcids. Surf. B-Biointerfaces, 27, 133-140. 

One of the major drawbacks of liposomes is related to their preparation methods [3,4]. Liposomes for topical delivery are prepared by the same classic methods widely described in the literature for preparation of these vesicles. The majority of the liposome preparation methods are complicated multistep processes. These methods include hydration of a dry lipid film, emulsification, reverse phase evaporation, freeze thaw processes, and solvent injection. Liposome preparation is followed by homogenization and separation of unentrapped drug by centrifugation, gel filtration, or dialysis. These techniques suffer from one or more drawbacks such as the use of solvents (sometimes pharmaceutically unacceptable), an additional sizing process to control the size distribution of final products (sonication, extrusion), multiple-step entrapment procedure for preparing drug-containing liposomes, and the need for special equipment. 

Liposome Preparation Techniques In most cases, liposomes are named by the preparation method used for their formation, Such as sonicated, dehydrated-rehy-drated vesicle (DRV), reverse-phase evaporation (REV), one step, and extruded. Several reviews have summarized available liposome preparation methods [91,124, 125], Liposome formation happens spontaneously when phospholipids are dispersed in water. However, the preparation of drug-encapsulating liposomes with high drug encapsulation and specific size and lamellarity is not always an easy task. The most important methods are highlighted below. 

Reverse-Phase Evaporation The REV method was developed by Szoka and Papa-hadjopoulos [132], Lipids are dissolved in organic solvent and the solvent is removed with evaporation. The thin film is resuspended in diethyl ether (1 mL solvent/mL liposomes) followed by the addition of one-third of water and sonication in a bath sonicator for 1 min. This water-in-oil (w/o)-emulsion is evaporated until a dry gel is formed, and finaly the gel is broken by agitation and water addition. Sometimes this step is quite difficult. The remnants of the organic solvent are removed by evacuation and the resulting dispersion is REV liposomes. 

In another study, the antibiotic cefoxitine was incorporated in DMPC/Chol (2 1) liposomes prepared using the reverse-phase evaporation technique in order to... 

Elorza, B., Elorza, M. A., Frutos, G., and Chantres, J. R. (1993), Characterization of 5-flourouracil loaded liposomes prepared by reverse phases evaporation or freezing thawing extrusion methods Study of release, Biochim. Biophys. Acta, 1153, 135-142. 

Based on size and lamellarity, liposomes can be categorized into four groups (175, 205) (as indicated in Figure 8.22) (i) multilanellar vesicles (MLVs) (ii) large unilamellar vesicles (LUVs) (iii) small unilamellar vesicles (SUVs) and (iv) intermediate-size unilamellar vesicles (lUVs), which are also called reverse-phase evaporation vesicles (REV). 

Canova-Davis, E., Redemann, C. T., Vollmer, Y. P., and Kung, V. T. (1986). Use of a reversed-phase evaporation vesicle formulation for a homogeneous liposome immunoassay. Clin. Chem. 32 1687-1691. 

Cortesi R, Esposito E, Gamharin S, Telloli P, Menegatti E, Nastruzzi C (1999) Preparation of liposomes hy reverse-phase evaporation using alternative organic solvents. J Microencapsul 16 251-256... 

The formation of liposomes [or better arsonoliposomes (ARSL)], composed solely of arsonolipids (Ars with R=lauric acid (C12) myristic acid (C14) palmitic acid (C16) and stearic acid (C18) (Fig. 1) have been used for ARSL construction), mixed or not with cholesterol (Choi) (plain ARSL), or composed of mixtures of Ars and phospholipids (as phosphatidylcholine [PC] or l,2-distearoyl- -glyceroyl-PC [DSPC]) and containing or not Choi (mixed ARSL), was not an easy task. Several liposome preparation techniques (thin-film hydration, sonication, reversed phase evaporation, etc.) were initially tested, but were not successful to form vesicles. Thereby a modification of the so called one step or bubble technique (8), in which the lipids (in powder form) are mixed at high temperature with the aqueous medium, for an extended period of time, was developed. This technique was successfiil for the preparation of arsonoliposomes (plain and mixed) (9). If followed by probe sonication, smaller vesicles (compared to those formed without any sonication [non-sonicated]) could be formed [sonicated ARSL] (9). Additionally, sonicated PEGylated ARSL (ARSL that contain polyethyleneglycol [PEG]-conjugated phospholipids in their lipid bilayers) were prepared by the same modified one-step technique followed by sonication (10). 

Emulsions of water and CO2 have been used to form liposomes in one step without any organic solvent (57). The liposomes were made by forming a W/C emulsion stabilized with/L-i -dipalmitoylphosphatidylcholine. The pressure was reduced to form the liposomes by a reversed phase evaporation method. Large unilamellar liposomes with diameters of 0.1 to 1.2 pm were formed and used to trap D-( + )-glucose. 

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