Liposomes temperatures

As for conventional liposomes, temperature and time of incubation are important factors for PEG-lipid insertion into cationic bilayers (48). Transition temperature of cationic lipids has not always been determined, although it would be interesting data to have. The incorporation of PEG-lipid into the film before hydration is usually more efficient than its postinsertion into the particles. However, postincorporation allows to work with limited amounts of materials, and to test more easily multiple conditions. 

For liposomal loading and storage of the liposomes, temperatures less than 2 °C must be avoided to prevent lower encapsulation efficiencies and leakage. 

Abbreviations DMPC, DPPC, DSPC, dimyristoyl-, dipalmitoyl-,distearoyl-phosphatidylcholine DPPA, dipalmitoylphosphatidic acid PE, phosphatidylethanolamine PS, phosphatidylserine (U) and (M), unilamellar and multilamellar liposomes temperature of crystal liquid-crystal transition Diff. spectr., difference spectroscopy. 

The development of monoalkyl phosphate as a low skin irritating anionic surfactant is accented in a review with 30 references on monoalkyl phosphate salts, including surface-active properties, cutaneous effects, and applications to paste and liquid-type skin cleansers, and also phosphorylation reactions from the viewpoint of industrial production [26]. Amine salts of acrylate ester polymers, which are physiologically acceptable and useful as surfactants, are prepared by transesterification of alkyl acrylate polymers with 4-morpholinethanol or the alkanolamines and fatty alcohols or alkoxylated alkylphenols, and neutralizing with carboxylic or phosphoric acid. The polymer salt was used as an emulsifying agent for oils and waxes [70]. Preparation of pharmaceutical liposomes with surfactants derived from phosphoric acid is described in [279]. Lipid bilayer vesicles comprise an anionic or zwitterionic surfactant which when dispersed in H20 at a temperature above the phase transition temperature is in a micellar phase and a second lipid which is a single-chain fatty acid, fatty acid ester, or fatty alcohol which is in an emulsion phase, and cholesterol or a derivative. 

Fr kjaer et al., 1984 Grit et al., 1989). An example of the pH dependency on the hydrolysis rate of liposomes consisting of soybean phosphatidylcholine is presented in Fig. 6. Hydrolysis kinetics changed rather abruptly around the phase transition temperature. 

Stndies of the antoxidation of carotenoids in liposomal suspensions have also been performed since liposomes can mimic the environment of carotenoids in vivo. Kim et al. stndied the antoxidation of lycopene," P-carotene," and phytofluene" " in liposomal snspensions and identified oxidative cleavage compounds. Stabilities to oxidation at room temperature of various carotenoids incorporated in pig liver microsomes have also been studied." The model took into account membrane dynamics. After 3 hr of reactions, P-carotene and lycopene had completely degraded, whereas xanthophylls tested were shown to be more stable. 

Figure 2. Liposomes with a specific character a), temperature-sensitive liposome b). target-sensitive liposome c). pH-sensitive liposome. Closed triangles and rectangles in lipid bilayer indicate amphiphiles which change the liposome s hydration with pH changes.

The binding of carotenoids within the lipid membranes has two important aspects the incorporation rate into the lipid phase and the carotenoid-lipid miscibility or rather pigment solubility in the lipid matrix. The actual incorporation rates of carotenoids into model lipid membranes depend on several factors, such as, the kind of lipid used to form the membranes, the identity of the carotenoid to be incorporated, initial carotenoid concentration, temperature of the experiment, and to a lesser extent, the technique applied to form model lipid membranes (planar lipid bilayers, liposomes obtained by vortexing, sonication, or extrusion, etc.). For example, the presence of 5 mol% of carotenoid with respect to DPPC, during the formation of multilamellar liposomes, resulted in incorporation of only 72% of the pigment, in the case of zeaxanthin, and 52% in the case of (1-carotene (Socaciu et al., 2000). A decrease in the fluidity of the liposome membranes, by addition of other... 

FIGURE 2.3 Temperature dependency of the absorption spectra of zeaxanthin incorporated into the liposomes formed with DPPC. The initial concentration of zeaxanthin in the medium used to prepare liposomes was 5 mol% with respect to lipid. (Based on the results presented in Sujak, A. et al., Biochim. Biophys. Acta, 1509, 255, 2000.)... 

Cholesterol s presence in liposome membranes has the effect of decreasing or even abolishing (at high cholesterol concentrations) the phase transition from the gel state to the fluid or liquid crystal state that occurs with increasing temperature. It also can modulate the permeability and fluidity of the associated membrane—increasing both parameters at temperatures below the phase transition point and decreasing both above the phase transition temperature. Most liposomal recipes include cholesterol as an integral component in membrane construction. 

React for 2 hours at room temperature. If liposome aggregation or protein precipitation occurs during the crosslinking process, scale back the amount of EDC added to the reaction. 

Figure 5.28 Circular dichroism spectra of DC8 9PC tubules prepared in (a) ethanol-water (7 3), (b) methanol-ethanol-water (35 35 20), and (c) methanol-water (70 30) and (d) DCj PC liposomes above melting temperature. All samples were prepared at lipid concentration of 2.0 mg/ml and spectra for tubules were recorded at 25°C. Liposome spectrum was recorded at 40°C and peak intensity is about 104 smaller than that from tubules.

Figure 5.30 Temperature dependence of molar ellipticity at 218 nm for liposomes prepared from L-DMPC, L-DPPC (39), and L-POPC (40). Reprinted with permission from Ref. 134. Copyright 1997 by the American Chemical Society.

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. 

Freeze dried liposomes loaded with doxorubicin (DXR) have been stored for 6 months at temperatures between -20 and +50 °C. Up to 30 °C, no sign of degradation was found, but at 40 to 50 °C - well below T of the dried cake - the total DXR content and the retention of the drug after dehydration decreased, while the size of the liposomes increased jo a certain extent. The stability with RM below 1 % has been better than with 2.5-3.5 %. 

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