Preparation of DRV Liposomes

Dried reconstituted vesicles (DRV) are liposomes that are formulated under mild conditions and have the capability to entrap substantially high amounts of hydrophilic solutes (compared with other types of liposomes). These characteristics make this liposome type ideal for entrapment of labile substances, as peptide, protein or DNA vaccines and sensitive drugs. In this chapter, we initially introduce all possible types of DRV liposomes (in respect to the encapsulated molecule characteristics and/or their applications in therapeutics) and discuss in detail the preparation methodologies for each type. 

Key words DRV, Protein, Peptide, Hydrophilic drug. Encapsulation yield. Vaccine, DNA, Particulate, Bacteria, Cyclodextrin 

DPPE-PEG2000 l,2-Dipalmitoyl-OT-glyceroyl-3-phosphoethanolamine conjugated to 

DRV Dried rehydrated vesicles or Dried Reconstituted vesicles 

DOI 10.1007/978-1 -60327-360-2 3, Humana Press, a part of Springer Science+Business Media, LLC 2010 

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For the preparation of DRV liposomes, empty (see Note 4) SUV liposomes dispersed in d.d. H O with the appropriate lipid composition and concentration are initially prepared (see Note 5). SUV preparation can be performed by several techniques, depending on the specific lipid composition and concentration required, the most convenient and easiest to use being (1) Probe sonication in one step (see Note 6), and (2) Size reduction of MLV liposomes (most applied technique). 

Preparation of DRV Liposomes with Controlled Entrapment Yield and Vesicle Size (as reported in (2))... 

Preparation of Giant Liposome DRVs that Entrap Large Particles, Viruses, or Bacteria (as described in (36-38))... 

For the formation of DRV liposomes entrapping solutes that are not sensitive to the conditions used for MLV and/or SUV preparation, it is possible to prepare drug containing liposomes in the initial step of DRV formation. This is particularly important if amphiphilic/lipophilic or in general substances with low aqueous solubility are to be entrapped. However, when there is interest to have a method that can be easily up-scaled for large batch manufacturing, this approach can be problematic. 

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). 

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. 

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. 

Dried Reconstituted Vesicles (DRV) (see Note 1), were initially developed in 1984 by Kirby and Gregoriadis (1). They are oligo-or multilamellar liposomes with capability of encapsulating high amounts of aqueous soluble molecules. The fact that the DRV technique involves vesicle formation under mild conditions (e.g., conditions that do not cause decomposition or loss of activity of active substances), makes this technique the method of choice for preparation of liposomal formulations of sensitive active substances as peptides, proteins or enzymes. 

As mentioned above, it has been reported that by controlling the sugar/lipid mass ratio, during DRV preparation by the conventional DRV technique (1), the entrapment efficiencies and size distribution of the liposomes produced can be controlled (2). 

Diluted PBS or Phosphate Buffer pH 7.40, for preparation of CF (or calcein) solution. This buffer is prepared by diluting PBS buffer 10 times with d.d. H O (see item 5). This buffer is used for preparation of CF (or calcein) solution, prepared for encapsulation in DRV liposomes. 

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. 

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