Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependent drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 degrees C or 30% at 37 degrees C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.     

Introduction of enzymes,by means of liposomes,into non-phagocytic human cells in vitro

Liposomes containing entrapped horsedish peroxidase were incubated with three human cell lines in vitro. Although these cells did not ingest latex particles, and took up less than 1 minut of free peroxidase/5 · 10⁶ from the medium, significant amounts (41–164 munits/5 · 10⁶) of peroxidase became cell-associated by 30 min if the enzyme was presented in negatively charged liposomes (phosphatidylchloline/dicetyl phosphate/cholesterol, 70 : 20 : 10 molar ratio). Uptake of liposome-entrapped peroxidase by lymphocytes or fibroblasts was enhanced 2–5-fold if one molar percent of lysophosphatidylcholine was incorporated as a “fusogen”, and was not appreciately diminished by cytochalasin B, an inhibitor of phagocytosis. Lysophosphatidylcholine containing liposomes did not release trapped peroxidase into the medium during incubation, and studies employing the metallochromic dye, arsenazo III demonstrated lack of access of external Ca²⁺ to the internal, enzyme-laden, aqueous compartments; liposome-liposome fusion was also excluded by similar means. Ultrastructural cytochemstry demonstrated peroxidase within liposomes in the free cytosol of cultured cells 15–90 min after apparent liposome-cell fusion. Data provide evidence that multilamellar liposomes can be as vectors for the introjection of missing enzymes into non-phagocytic human cells.     

Biopolymer excreted by Pseudoalteromonas antarctica NF3, as a coating and protective agent of liposomes against dodecyl maltoside

The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram‐negative species Pseudoalteromonas antarctica NF₃, to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant dodecyl maltoside was investigated. Transmission electron microscopy (TEM) micrographs of freeze fractured liposome/GP aggregates reveal that the addition of the glycoprotein to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that tightly coated PC bilayers. The complete coating was already achieved at a PC : GP weight ratio of about 9:1. Image analysis profiles of digitalized TEM micrographs (PC : GP weight ratio 8:2) show that this film was formed by a multilayer structure. The periods of the average distance of the pattern ordering in layer structures (9–10 layers) were of about 2–3 nm and the thickness of the complete film was of about 25 nm. Higher amounts of glycoprotein resulted in a growth of this film, which exhibited at the highest proportion of this compound (50% in weight) a multifilm structure. An increasing resistance of liposomes to be affected by dodecyl maltoside both at subsolubilizing and solubilizing levels occurred as the proportion of the glycoprotein in the system rose, although this protective effect was more effective at low proportions of this compound (PC : GP weight ratios from 9:1 to 8:2). Thus, although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactant, the more effective protection occurred when this structure was a thin film formed by the assembly of various layers of GP of about 2–3 nm. © 1999 John Wiley & Sons, Inc. Biopoly 50: 579–588, 1999