Dried reconstituted vesicles

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. 

DRV Dried rehydrated vesicles or Dried Reconstituted vesicles... 

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. 

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. 

Dried-Reconstituted Vesicles (Good for Sterically Stabilised Vesicles)... 

The purple membrane fragments that contained dark-adapted bacteriorhodopsin were used to form reconstituted vesicles with a mixture of phospholipids that contained 80% egg phosphatidyl choline and 20% bovine phosphatidylserine (Lipid Products, Nuttfield, England). The lipids in chloroform and methanol solutions were mixed to the desired composition, dried in a stream of nitrogen, placed in a 0.1-torr... 

Nebulization can cause disruption or processing of multilameller vesicles [52]. Fortunately, these issues can be addressed, and, through manipulation of the composition, buffer and environment liposomes have been aerosolized without causing loss of entrapped drug [53-55]. Liposomes have also been prepared as spray-dried and lyophilized powders [56-59]. The former may be aerosolized directly as a powder, but in both cases reconstitution in an aqueous environment results in liposome formation. However, it is not understood if this is just spontaneous reformation of original liposomes (pre-spray-drying) or the creation of de novo liposomes in an aqueous environment. 

Lipids are mixed together and solvent is removed using freeze-drying. Then an aqueous solution is introduced and the hpid cake around the vessel wall is reconstituted. This method works best for manufacture of neutral liposomes, as the hydro-phobic lipids readily dissolve in solvents such as chloroform and are deposited dry on the wall of the rotavapor vessel. Then the material to be encapsulated is dissolved in an aqueous solution and the dry film on the vessel wall is hydrated with this solution. The exact steps involved in the preparation of hydrogenated soy phosphatidylcholine (PHSPC)- and cholesterol-containing vesicles at a molar ratio of 60 to 40% are as listed here ... 

Figure 2 Light-induced FTIR difference spectra of reaction centers from Rps, viridis reconstituted into lipid vesicles and air-dried onto CaF2 windows. Spectrum a shows changes arising from and spectrum b shows changes due to P IQaQb - Resolution = 4...

The photosynthetic bacteria Rps. sphaeroides R 26 was grown and the chromatophore membranes prepared as described earlier (2, 6). RCs were isolated with the detergent LOAD (2, 6). The LM complex was prepared by precipitation of the H subunit (1, 2). Reconstitution of the RC complexes into phospholipid vesicles was performed as already reported (2, 6). Orientation of the membrane vesicles for IR dichroism analysis was obtained by air-drying the suspension onto CaF discs (4-6). The method for quantitative analysis and calculation ora-helix tilt angles from the IR dichroism spectra and UVCD spectra has been extensively described in our previous papers (4-6). 

FIGURE 1. IR linear dichroism spectra of air-dried LMH and LM reaction centers reconstituted in phosphatidylcholine vesicles. 

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