Marine lipids
Material and methods
Materials
Marine lipids were obtained from Ifremer (centre de Nantes). Detergent,
n-octyl-β-D-glucopyranoside (OG, mz = 292.4) and thiamine (mz = 337.3)
were purchased from Sigma. Detergent and lipids were used without
peculiar purification. Cholesterol-dextran was synthesized in two steps:
first by grafting amino groups on native dextran (Prigent-Richard et
al., 1998) then cholesterol was grafted according to Akiyoshi et al.
(1991) and characterized as previously described (Cansell et al., in press).
All the liposome preparations were performed in a buffer constituted
of 145 mM NaCl, 10 mM N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic] acid (HEPES, Sigma, purity = 99.5%) adjusted to pH 7.4.
Lipid analysis
Marine lipids were quantified by phosphorus analysis for phospholipids
(Ames, 1966) and enzymatic assays for cholesterol and triglycerides
(Boehringer Mannheim). The following composition was found: 70% of
phospholipids, 15% ofcholesterol and 2% of triglycerides. Fractionation
of the various classes of phospholipids was performed by bidimensional
thin-layer chromatography using chloroform-acetone-methanol-acetic
acid-water (50:20:10:10:5, v/v/v/v/v) as the first elution solvent and
butanol-acetic acid-water (60:20:20, v/v/v) as the second elution solvent
(Wolff et al., 1984). It was found that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) represented 75% of total phospholipids as
already reported (Baudimant et al., 1996). Total lipids or phospholipids
were transmethylated (Morrisson & Smith, 1964), methyl esters were
analysed by gas-liquid chromatography (GLC) using a 0.25 mm internal
diameter - 60 m length BPX 70 column (SGE, France) (Cansell et al.,
1997). As expected for marine lipids, 50% of the total fatty acids were
polyunsaturated with 31% of docosahexaenoic acid (DHA, 22:6 ω3).
Liposome preparation and characterization
Multilamellar liposomes (MLV) were prepared by extrusion of a lipid
suspension according to Olson et al. (1979). Briefly, 30 mg of lipids
were dried by solvent evaporation under nitrogen and the film was
lyophilized overnight. Hydration was performed by addition of HEPES
buffer in order to get a 3 mg/ml lipid dispersion which was extruded
through polycarbonate filters with 5 pm pore size (Isopore, Millipore
Corp., USA). Loss of lipids due to filtration was quantified by phosphorus
assay (Ames, 1966). When thiamine encapsulation was performed, the
vitamin (5 mg/ml) was added to the lipids prior to the extrusion step.
Free non-encapsulated thiamine was separated from liposomes by
steric exclusion chromatography on a Sephacryl S-1000 (Pharmacia)
column (40 cm length, 1 cm internal diameter) using HEPES buffer as
eluent. Thiamine leakage was followed by spectrophotometric absorption
at 266 nm after separation of liposomes from free thiamine either by
214
Material and methods
Materials
Marine lipids were obtained from Ifremer (centre de Nantes). Detergent,
n-octyl-β-D-glucopyranoside (OG, mz = 292.4) and thiamine (mz = 337.3)
were purchased from Sigma. Detergent and lipids were used without
peculiar purification. Cholesterol-dextran was synthesized in two steps:
first by grafting amino groups on native dextran (Prigent-Richard et
al., 1998) then cholesterol was grafted according to Akiyoshi et al.
(1991) and characterized as previously described (Cansell et al., in press).
All the liposome preparations were performed in a buffer constituted
of 145 mM NaCl, 10 mM N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic] acid (HEPES, Sigma, purity = 99.5%) adjusted to pH 7.4.
Lipid analysis
Marine lipids were quantified by phosphorus analysis for phospholipids
(Ames, 1966) and enzymatic assays for cholesterol and triglycerides
(Boehringer Mannheim). The following composition was found: 70% of
phospholipids, 15% ofcholesterol and 2% of triglycerides. Fractionation
of the various classes of phospholipids was performed by bidimensional
thin-layer chromatography using chloroform-acetone-methanol-acetic
acid-water (50:20:10:10:5, v/v/v/v/v) as the first elution solvent and
butanol-acetic acid-water (60:20:20, v/v/v) as the second elution solvent
(Wolff et al., 1984). It was found that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) represented 75% of total phospholipids as
already reported (Baudimant et al., 1996). Total lipids or phospholipids
were transmethylated (Morrisson & Smith, 1964), methyl esters were
analysed by gas-liquid chromatography (GLC) using a 0.25 mm internal
diameter - 60 m length BPX 70 column (SGE, France) (Cansell et al.,
1997). As expected for marine lipids, 50% of the total fatty acids were
polyunsaturated with 31% of docosahexaenoic acid (DHA, 22:6 ω3).
Liposome preparation and characterization
Multilamellar liposomes (MLV) were prepared by extrusion of a lipid
suspension according to Olson et al. (1979). Briefly, 30 mg of lipids
were dried by solvent evaporation under nitrogen and the film was
lyophilized overnight. Hydration was performed by addition of HEPES
buffer in order to get a 3 mg/ml lipid dispersion which was extruded
through polycarbonate filters with 5 pm pore size (Isopore, Millipore
Corp., USA). Loss of lipids due to filtration was quantified by phosphorus
assay (Ames, 1966). When thiamine encapsulation was performed, the
vitamin (5 mg/ml) was added to the lipids prior to the extrusion step.
Free non-encapsulated thiamine was separated from liposomes by
steric exclusion chromatography on a Sephacryl S-1000 (Pharmacia)
column (40 cm length, 1 cm internal diameter) using HEPES buffer as
eluent. Thiamine leakage was followed by spectrophotometric absorption
at 266 nm after separation of liposomes from free thiamine either by
214