Dried rehydrated vesicles

DRV Dried rehydrated vesicles or Dried Reconstituted vesicles... 

The abbreviation DRV stands for Dehydration-Rehydration Vesicles as initially named by the inventors of this liposome preparation technique (1). However, one will find several other explanations in the relevant literature as Dried Rehydrated Vesicles and Dried Reconstituted Vesicles, which are actually the same type of liposomes. 

Entrapment of plasmid DNA and/or protein into liposomes entails the preparation of a lipid film from which multilamellar vesicles and, eventually, small unilamellar vesicles (SUVs) are produced. SUVs are then mixed with the plasmid DNA and/or protein destined for entrapment and dehydrated. The dry cake is subsequently broken up and rehydrated to generate multilamellar dehydration-rehydration vesicles (DRV) containing the plasmid DNA and/or protein. On centrifugation, liposome-entrapped vaccines are separated from nonentrapped materials. When required, the DRV are reduced in size by microfluidization in the presence or absence of nonentrapped materials or by employing an alternative method (7) of DRV production, which utilizes sucrose (see below). 

Quantitative entrapment of vaccines into small (up to about 200 nm diameter) liposomes in the absence of microfluidization (which can damage DNA and other labile materials when extensive) can be carried out by a novel one-step method (7) as follows SUVs (e.g., cationic) prepared as in section Preparation of Small Unilamellar Vesicles are mixed with sucrose to give a range of sucrose-to-lipid weight/weight ratio of 1.0 to 5.0 and the appropriate amount of plasmid DNA (e.g., 10-500 pg) and/or protein (e.g., up to 1 mg). The mixture is then rapidly frozen and subjected to dehydration by freeze-drying, followed by rehydration as in section Preparation of Vaccine-Containing Dehydration-Rehydration Vesicles. ... 

The content of vaccine within the small liposomes is estimated as in the section Estimation of Vaccine Entrapment in Dehydration-Rehydration Vesicles Liposomes for both microfluidized and sucrose liposomes and expressed as percentage of DNA and/or protein in the mixture subjected to freeze drying as in the section Preparation of Vaccine-Containing Small Liposomes by the Sucrose Method in the case of sucrose small liposomes or in the original DRV preparation (obtained in the section Estimation of Vaccine Entrapment in DRV Liposomes ) for microfluidized liposomes. Vesicle size measurements are carried out by PCS as described elsewhere (6,8,17). Liposomes can also be subjected to microelectrophoresis in a Zetasizer to determine their zeta potential. This is often required to determine the net surface charge of DNA-containing cationic liposomes. 

In order to prepare liposomes, the lipid preparation is dried at low temperature under an inert gas atmosphere (protect the lipid from oxidation). The lipid film is swollen with water or buffered aqueous solution and several freeze-thaw cycles are carried out to get optimal rehydration of the lipid. The rehydrated lipid preparation is filtered using membrane filters with defined pore size. After repeated filtration steps (extrusion) an unilamellar liposome preparation with a defined size distribution is obtained. Large unilamellar vesicles (LUV) are produced in this way. LUV s are about 100 nm in size the thickness of the lipid bilayer is about 4 nm. Even smaller liposomes can be derived from sonication (sonication probe or ultra-sonication bath). Separation of the prepared liposomes... 

The high entrapment capability of DRV s is due to the fact that preformed empty small unilamellar vesicles are disrupted during a freeze-drying cycle in the presence of the solute destined for entrapment. Subsequently, during controlled rehydration, which is carried out in the presence of a concentrated solution of the solute (to be encapsulated), the vesicles fuse into large oligolamellar... 

Fluoromax-3 spectrofluorometer (Edison, NJ). The total calcein content of the vesicles was determined after lysis of the membranes with Triton X-100 (1% (v/v)). Calcein retention of vesicles was estimated from the fluorescence before and after the addition of Triton X-100. Prior to calcein retention analysis, the freeze-dried samples were rehydrated at room temperature. 

Keeping the residual water content of the lyophilized liposomes at minimum may also increase the shelf life of freeze-dried formulation and prevent increases of vesicle size on rehydration [45]. A dehydration-rehydration protocol has been introduced by Kirby and Gregoriadis [46] to produce a mixture of OLVs and MLVs, with entrapment efficiencies between 40% and 50% being reported. Ambisome is an antifungal freeze-dried liposome formulation that is currently available in the maiket for treatment of systemic fungal infections [47]. 

Figure 9.4 Lipid rehydrated in 90mM salt solution, (a) Typical partially rehydrated myelinlike structures that are obtained when 90 mM salt solution is added to a dried lipid film. The film does rehydrate, but only to the extent of forming the partially hydrated refractory lipid structures, (b) The only vesicle-like structures that could be seen were very multilamellar, aggregated, and clearly not suitable for micropipet manipulation experiments.

Figure 9,5 Zeta potential versus concentration of the charged lipid DOPG in DOPC vesicles. Eight samples, each containing 10mg of lipid (of different DOPC DOPG ratios) were dried down, rehydrated with 1 ml of 0.1 mM NaCl and then extruded through 200 nm filters in a Lipex Biomembranes extruder 11 times. The samples were then diluted with 1 ml of 0.1 mM NaCl. 1.3 ml of each sample was placed in a different cuvette. The zeta potential of the extruded vesicles was measured 10 times for each sample using a Brookhaven Zeta PlusPCS machine.

FIGURE 6.15 A schematic demonstrating the electroformation method for giant unilamellar vesicle (GUV) preparation. Dry lipid films are rehydrated in the presence of an alternating electric field. ITO, indium tin oxide. 

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