Dehydration-rehydration liposomes

Gregoriadis G, da Silva H, Florence AT. A procedure for the efficient entrapment of drugs in dehydration-rehydration liposomes (DRV). Int J Pharm... 

A method resulting in improved encapsulation of aqueous phase by MLV is the so-called dehydration-rehydration procedure (Kirby and Gregoriadis, 1984 Shew and Deamer, 1985). The lipid (usually preformed liposomes) is dried (by either lyophilization or evaporation) in the presence of the aqueous solute to be entrapped, thus forming a mixed film with solute trapped between layers. Subsequent gradual rehydration with a minimum of aqueous phase leads to the formation of MLV with a high entrapment of the aqueous solutes added. 

Foradada and Estelrich [3.63] studied the encapsulation of thioguanine (TG) in three types of liposomes produced by extrusion, ethanol injection and dehydration-rehydration vesicles. The entrapment has been examined at three different concentrations (1, 0.1 and 0.01 mM) and at three different pH values (4.7,7.4 and 9.2). The dehydration-rehydration vesicles were found to be the optimum method to encapsulate TG, independent of the pH value. At pH 4.7, 12 mmol/mol of lipid were entrapped, while with the other methods a maximum of 3 mmol/mol of lipid has been achieved. The authors related this behavior to the formation of hydrogen bridges between the TG and the liposomes. 

ENTRAPMENT OF PLASMID DNA AND PROTEIN VACCINES INTO LIPOSOMES BY THE DEHYDRATION-REHYDRATION PROCEDURE... 

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). 

Table 1 Incorporation of Plasmid DNA and Protein into Liposomes by the Dehydration-Rehydration Method... 

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. 

Figure 1 Anti-HA IgG titres ( SD) ( Y-axis) in mice immunized with a single subcutaneous injection of (Alum-adsorbed) DRV liposomes composed of PC, DOPE, and DOTAP (molar ratios 4 2 1) and containing pl.l7/SichHA DNA and killed influenza virus ( ), killed influenza virus only (A), or DNA only ( ). For other details see the text. Abbreviations HA, hemagglutinin DRV, dehydration-rehydration vesicles PC, phosphatidylcholine DOPE, dioleoyl phosphatidylcholine DOTAP, 1,2-dioleyloxy-3-(trimethylamonium propane).

Kirby C, Gregoriadis G. Dehydration-rehydration vesicles (DRV) a new method for high yield drug entrapment in liposomes. Biotechnology 1984 2 979-984. 

Seltzer SE, Gregoriadis G, Dick R (1988) Evaluation of the dehydration-rehydration method for production of contrast-carrying liposomes. Invest Radiol 23 131... 

Brandi, M. Gregoriadis, G. Entrapment of haemoglobin into liposomes by the dehydration-rehydration method vesicle characterization and in vivo behavior. Biochim. Biophys. Acta 1994,1196, 65-75. 

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. 

Senior , Gregoriadis G(1989) Dehydration-rehydration vesicle methodology facilitates a novel approach to antibody binding to liposomes. Biochim Biophys Acta 1003 58-62... 

DNA, coding for the GFP, was introduced in liposomes composed by a phospholipid mixture (a), together with the whole T T machinery (T7 RNA polymerase and E. coli cell extracts). GFP was synthesized inside large MLVs prepared by the dehydration/ rehydration method. 

E. coll Klenow fragment was entrapped in liposomes composed by the lipid mixture (c), together with template DNA and dNTPS. DNA was then produced within vesicles prepared by the dehydration-rehydration method. 

T7 RNA polymerase has been entrapped inside DMPC liposomes, together with DNA template, and fed by externally added NTPs. In order to increase the entrance of NTPs, liposomes were kept at 23 °C (at the TJ. RNA was therefore synthesized within DMPC vesicles prepared by the dehydration-rehydration and extrusion method. 

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]. 

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