R.A. Schwendener

The preparation of large single bilayer liposomes by a fast and controlled dialysis

A new method is described for the preparation of large, homogeneously sized, single bilayer phospholipid vesicles. This method, based on a fast and controlled dialysis of sodium cholate from phosphatidylcholine/cholate mixed micelles, has the advantage of high yield of homogeneous vesicles avoiding any dilution and mechanical stress during preparation. Physicochemical properties of these vesicles are examined by several techniques and compared with those prepared with other methods.

n-alkyl-glucosides as detergents for the preparation of highly homogeneous bilayer liposomes of variable sizes (60–240 nm φ) applying defined rates of detergent removal by dialysis

Abstract The variation of the sidechain length of n-alkyl-β-D-glucopyranosides and the use of mixtures of these detergents for the preparation of lipid/detergent mixed micelles followed by controlled detergent removal applying defined dialysis rates yields best characterized bilayer liposomes in the size range of 60 to 240 nanometers. Further advantages of the liposomes prepared with the described method are the extremely narrow volume distribution and the wide variation possibility of size.

5′-O-palmitoyl- and 3′,5′-O-dipalmitoyl-5-fluoro-2′-deoxyuridine - novel lipophilic analogues of 5′-fluoro-2′-deoxyuridine: Synthesis, incorporation into liposomes and preliminary biological results

5-O-palmitoyl- and 3,5-O-dipalmitoyl-5-fluoro-2-deoxyuridine were prepared by the reaction of 5-fluoro-2-deoxyuridine in dimethylacetamide with palmitic acid chloride. The incorporation of the synthesized prodrugs into liposomes composed of egg phosphatidylcholine/stearylamine/cholesterol/alpha-tocopherol at a molar ratio of 10:1:2:0.05 was nearly quantitative; homogeneous bilayer vesicles (75 nm diameter) were obtained. Preliminary tolerance studies revealed that the prodrug-liposome preparations are about 20-60 times more toxic than the parent drug. The prodrugs incorporated into liposomes were 10 to 30 times more active against murine colon 38 carcinoma compared to the free drug. In comparison to the administration of the prodrugs in peanut oil the liposomal preparations seem to exert improved effects and represent a valuable drug delivery system for parenteral applications.

The binding of chlorpromazine to bilayer liposomes. Evaluation of stoichiometric constants from equilibrium and steady state studies

Abstract The binding of chlorpromazine to egg yolk lecithin bilayer liposomes has been studied with equilibrium dialysis as well as with a new steady state method. The influence of the cholesterol amount previously incorporated into the liposomes to the binding of chlorpromazine was examined. The binding of chlorpromazine was evaluated in terms of stoichiometric constants and shows a strong positive cooperative binding step, followed by an equally strong negative cooperative step. The first step, which is only detectable with the steady state method may be related to the partition of chlorpromazine into the lipophilic bilayer core, whereas the second and the following steps describe the overall saturation of the liposome membrane. The binding of chlorpromazine is attenuated by increasing amounts of cholesterol present in the bilayer liposomes. Investigating the binding to liposomes without cholesterol a saturation up to 75 per cent can be reached experimentally.

Palmitoyl derivatives of L-cysteine, cysteamine, L-cystine, cystamine and their incorporation into the bilayers of unilamellar liposomes

The amino groups of the amino acids L-cysteine and L-cystine as well as their biogene amines cysteamine and cystamine were derivatized with palmitoyl residues. The obtained lipophilic R-SH and R-S-S-R components were incorporated into the bilayers of unilamellar liposomes. The resulting liposomes carrying about 2000 functional groups each remained stable and homogeneous during 60 days after incorporation of N-palmitoyl cysteamine and N,N-dipalmitoyl cystamine. The incorporation of the lipophilic amino acid derivatives, however, destabilized the resulting liposomes. Via the thiol residues of the functionalized liposomes activated molecules can be linked to the liposomal surface by disulfide bonds.

Synthesis of lipophilic amino and carboxyl components for the functionalization of liposomes

Abstract l -Lysine, 2-hydroxyethylpalmitoylamide and dl -glyceric acid were used as starting for the synthesis of N 2 - palmitoyl- l -lysine methyl ester ; N 2 - palmitoyl -N 6 - succinoyl- l -lysine ; N 2,6 - dipalmitoyl- l -lysine ; N-(2-succinoylethyl)palmitoylamide and dl -2,3-dipalmitoylglyceric acid. By means of the detergent dialysis method, the lipophilic amino-acid and carboxylate components were incorporated into liposomal membranes in such a way that one liposome carried 500–2000 functional residues on its outer side. The resulting unilamellar liposomes had hydrodynamic diameters of 60–80 nm and a population homogeneity of 22–44%. Both parameters remained nearly constant for 60 days. Being polycations, liposomes functionalized with different amounts of amino components exhibited distinct mobilities in free-flow electrophoresis. The amino functions of the liposomes were derivatized with maleinimido residues to which sulfhydryl components like 2-mercaptoethanol as a model compound were covalently linked. Preliminary results showed that liposomes functionalized with carboxylate components, could be coupled to antibodies in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.