Dehydration/rehydration

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. 

A critical property of minimum protocells in the prebiotic environment would be their ability to sequester other molecules, including macromolecules. [142] In 1982, Deamer and Barchfeld [143] subjected phospholipid vesides to dehydration-rehydration cycles in the presence of either monomeric 6-carboxyfluorescein molecules or polymeric salmon sperm DNA molecules as extraneous solutes. The experiment modeled a prebiotic tidal pool containing dilute dispersions of phospholipids in the presence of external solutes, with the dehydration-rehydration cydes representing episodic dry and wet eras. They found that the vesides formed after rehydration... 

The continuous availability of trillions of independent microreactors greatly multiplied the initial mixture of extraterrestrial organics and hydrothermal vent-produced chemicals into a rich variety of adsorbed and transformed materials, including lipids, amphiphiles, chiral metal complexes, amino add polymers, and nudeo-tide bases. Production and chiral amplification of polypeptides and other polymeric molecules would be induced by exposure of absorbed amino adds and organics to dehydration/rehydration cydes promoted by heat-flows beneath a sea-level hydro-thermal field or by sporadic subaerial exposure of near-shore vents and surfaces. In this environment the e.e. of chiral amino adds could have provided the ligands required for any metal centers capable of catalyzing enantiomeric dominance. The auto-amplification of a small e.e. of i-amino adds, whether extraterrestrially delivered or fluctuationally induced, thus becomes conceptually reasonable. 

Defat and dehydrate/rehydrate Take slides through a series of graded ethanol washes and xylene, and back through the ethanols to defat, dehydrate, and rehydrate the tissue sections. 

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

The dehydration-rehydration procedure is characterized by its mildness and is thus compatible with most labile materials. The amounts of lipids and vaccine materials described below are typical for the preparations made for animal work described here but could be scaled up or down depending on the number of animals and the amount of antigens used. 

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... 

Entrapment value was obtained by the dehydration-rehydration method carried out in the presence of sucrose (see text). z-Average vesicle size... 

Abbreviations. HA, hemagglutinin DRV, dehydration-rehydration vesicles PC, phosphatidylcholine DOPE, dioleoyl phosphatidylcholine DOTAP, l,2-dioleyloxy-3-(trimethylamonium propane) BisHOP,, 2-bis (hexadecylcycloxy)-3-trimethylamino propane DC-CHOL, 3p(V,V,-dimethylami-noethane)-carbamyl cholesterol (DC-CHOL) DOTMA, V-[l-(2,3-dioleyloxy) propyl]-iV,V,V-triethylammonium PG, phosphatidyl glycerol PS, phosphatidylserine SA, stearylamine DODAP, l,2-dioleyloxy-3-(dimethylamonium propane). 

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 2 Anti-HBsAg IgG titres ( SD) (Y-axis) in mice immunized with a single subcutaneous injection of small DRV liposomes composed of PC, DOPE, DOTAP, and DOGMann (molar ratios 4 2 1 1) containing pRc/CMV HBS DNA and the encoded antigen HBsAg ( ) or with HBsAg in the form of Engerix ( ). For other details, see the text. Abbreviations DRV, dehydration-rehydration vesicles PC, phosphatidylcholine DOPE, dioleoyl phosphatidylcholine DOTAP, 1,2-dioleyloxy-3-(trimethylamonium propane).

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

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

Aconitase [citrate(isocitrate) hydro-lyase, EC 4.2.1.3] is the second enzyme of the citric acid cycle, which plays a central role in metabolism for all aerobic organisms. This enzyme catalyzes a dehydration-rehydration reaction interconverting citrate and 2R,3S-isocitrate via the allylic intermediate ds-aconitate. 

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

Worthy of note in this reaction is that citrate displays prochirality (see Section 3.4.7). The methylene carbons may be considered prochiral, in that enzymic elimination of a proton is likely to be entirely stereospecific. In addition, the apparently equivalent side-chains on the central carbon are also prochiral and going to be positioned quite differently on the enzyme. This means that only one of these side-chains is involved in the dehydration-rehydration... 

The role of water with protein molecules, stated in (ii) above, can be illustrated by some dehydration-rehydration studies of lysozyme reviewed by Finney (1986). The interpretation of the experimental data leads to several conclusions. A certain amount of water is essential at key sites in the enzyme the water facilitates a flexibility that is critical for the enzyme function and finally, the water permits a proton redistribution as the polar and charged groups revert to the aqueous solution order. 

The freeze-drying (or dehydration-rehydration) method was developed by Ohsawa et al. (1984) and Kirby and Gregoriadis (1984). This method is used to prepare MLVs with high entrapment... 

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