Reverse phase evaporation vesicles

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

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. 

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. 

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. 

Qi, X.-R. Maitani, Y. Shimoda, N. Sakaguchi, K. Nagai, T. Evaluation of liposomal erythropoietin prepared with reverse-phase evaporation vesicle method by subcutaneous administration in rats. Chem.Pharm.Bull, 1995, 43, 295-299... 

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. 

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). 

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.)...

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). 

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. 

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). 

Fig. 3 Effect of external pH on internal vesicle pH and MCP hydrolysis. In all experiments, a vesicle sample of 1 ml, was eluted from a Sephadex G-25 column (1 x 50 cm) with the appropriate solution. The column was previously saturated with sonicated vesicles of (DODA)B or lecithin to avoid vesicle disruption due to adsorption on the Sephadex. The vesicles eluted in the void volume were pooled and a 0.05 ml aliquot was added to 1.5 ml buffer. Borate buffer (5 mM) was used at pH 10.2 and NaOH for all other pHs. The samples were maintained at 30 °C and the hydrolysis of MCP was followed at 265 nm. A Effect of pH on the hydrolysis of free (o) and (DODA)B-entrapped MCP ( ). Chloroformic (DODA)B vesicles were prepared in 5 mM MCP chloride and 0.95 M erythritol. The vesicles were eluted from the column with erythritol 0.95 M and NaCl 5 mM. Erythritol 0.95 M was used in all buffers and the ionic strength was maintained at 5 mM with NaCl in order to avoid osmotic and ionic stress. Final concentrations of MCP and (DODA)B were 5 x 10 M and 7 x 10 M, respectively. B Effect of, pH on hydrolysis of free (o) and Lecithin entrapped MCP ( ). Lecithin vesicles were prepared by reverse phase evaporation (0.01 M, with 10% DCP) in 0.16 M KCl and 5 mM MCP, and the sample was eluted from Sephadex with KCl (0.165 M). All buffers contained KCl (0.16 M) and the kinetics were obtained with lecithin (5.6 X 10" M) and MCP (5 x 10" M). (a) Lecithin vesicles eluted from the column were incubated with 5 x 10" M Val for 15 min. After pH change the rate of MCP hydrolysis was the same as in aqueous phase. In a separate experiment, lecithin vesicles containing MCP only in the internal compartment were incubated with Val for 90 min and refiltered in Sephadex. No free MCP was found. An aliquot of the refiltered vesicles was added to NaOH and the rate constant was measured (a). C Kinetics of the effect of pH on the absorbance of intravesicular 2-HTAB. (DODA)B vesicles (5 mM) prepared with 5 mM 2-HTAB, 5 mM NaCl and 0.95 M erythritol were eluted from Sephadex G-25 with NaCl 5 mM and erythritol 0.95 M. Aliquots (0.05 ml) of (DODA)B vesicles were added to 1.5 ml of a solution containing erythritol (0.95 M), NaOH, pH 11.59 and the absorbance was measured at 340 nm. At the end of the reaction, HCl was added. A new addition of NaOH promotes a similar increase of absorbance [42]...

Over 40 years since it what found that phospholipids can form closed bilayered structures in aqueous systems, liposomes have made a long way to become a popular pharmaceutical carrier for numerous practical applications. Liposomes are phospholipid vesicles, produced by various methods from lipid dispersions in water. Liposome preparation, their physicochemical properties and possible biomedical application have already been discussed in several monographs. Many different methods exist to prepare liposomes of different sizes, structure and size distribution. The most frequently used methods include ultrasonication, reverse phase evaporation and detergent removal from mixed lipid-detergent micelles by dialysis or gel-filtration. To increase liposome stability towards the physiological environment, cholesterol is incorporated into the liposomal membrane (up to 50% mol). The size of liposomes depends on their composition and preparation method and can vary from... 

Vesicle preparation. LUV were prepared in 100 mM NaCl buffered with 5 mM Hepes (pH 7.4) by the reverse-phase evaporation procedure of Szoka and Papahadjopoulos (1) from soybean PC, phosphatidylglycerol (PG) and sterol(s) in different molar ratios. PG was present at 10 mol % in all the assays. Sterol-free vesicles were also prepared. Stigmasterol, sitosterol and (24R)-methylcholesterol were obtained from commercial sources. 24-methylpollinas-tanol was extracted from fenpropimorph-treated maize roots as reported in Ref.2. Lipid phosphorus as well as sterol content were aetermined after LUV recovery. 

The size and smface properties of liposomes vary with types of lipids, their compositions, their modification, and methods of preparation. For example, multilamellar vesicles (MLVs) several hundred nanometers in size can be produced by a reverse phase evaporation and extrusion, but smaller unilamellar vesicles (SUVs), whose size is less than 100 mn, can be produced by a sonication process [15]. Further, the membrane state of a bilayer is of primary interest not only for surface treatment but also for recognition of a cell surface and delivery of active ingredients. We will briefly review the microfluidity of bilayers and the interaction of liposomes with a cell surface. 

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