Water freeze-dried liposomes

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]. 

Crowe and Crowe [3.39] proved that it is sufficient for certain liposomes, e. g. egg phosphatidyl-choline (DPPC), to be vitrified by trehalose or dextran during freezing and freeze drying. In trehalose the retention rate was almost 100 %, and in dextran more than 80 %. This did not apply to egg PC-liposomes Dextran as CPA alone led to an almost total loss of the CF-indicator, but addition of dextran into a trehalose solution (Fig. 3.20) also reduced the retention rate of CF substantially, e. g. from 90 % in a pure trehalose to approx. 45 % if trehalose and dextran were in equal amounts in the solution. Since T of dextran is approx. -10 °C and Tg- of trehalose is -30 to -32 °C, dextran should form a glass phase at much higher temperatures than trehalose. Therefore the stabilization of egg- PC with trehalose cannot be related with the vitrification. Crowe showd with IR spectroscopy that egg-PC freeze dried with 2 g trehalose/g lipid had almost the identical spectrographic characteristics as the hydrous lipid Trehalose molecules replaced the water molecules, and hydrogen... 

Studies with the freeze dried DPPC liposomes in trehalose solution showed, that not Tg )f the amorphous sugar is the critical temperature during storage, but the bilayer transition emperature Tm. for the lyposomes determines the short term stability of the formulation. With trehalose as lyoprotectant and a low residual water content, Tm proved to be 10 to 30 °C below the onset of T . 30 min heating above Tm but well below T% decreased the retention of CF after rehydration. Tm< after the heating was reduced from 40 to 80 °C to below 25 °C. 

In summary [3.65], with an optimized formulation and freeze drying protocol, liposomes loaded with water-soluble CF or DXR can be freeze dried with a 90 % retention upon rehydration. The cake is stable for at least 6 months at temperatures up to 30 °C. 

Another concern with freeze-drying LEH is the instability of liposome structure upon lyophilization. Vesicle formation occurs in the presence of bulk water and when water is removed, loss of structural integrity is inevitable. Fusion, crystal formation, and phase transition are observed, resulting... 

The long-chain a-amino acid esters (40), (41), and (42) form bilayers on sonication in water under acidic conditions. Liposomes prepared from (40) and (42) precipitate if the aqueous medium is neutralized by titration with NaOH. Only liposomes made from (41) are stable even in basic solutions, as shown by electron microscopy52). Polypeptide formation in oriented spherical vesicles was confirmed by FT-IR spectroscopy. The liposomal solution of (41) was freeze-dried and the spectrum obtained from the residue was comparable with one of the polycondensed monolayers. The formation of polypeptide vesicles is illustrated in Scheme 4. 

A liposomal injection solution of verteporin (Visudyne ) is formulated with egg phosphatidyl-glycerol and dimyristoylphosphatidyl choline. The freeze-dried powder, contains apart from the liposomes, verteporin, lactose as lyoprotector, and osmolality adjusting excipients. By reconstitution with water an opaque dark green injectable solution is generated, which is injected intravenously within the scope of photodynamic therapy. 

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... 

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