Biotechnology
dialysis (Spectra-Por cellulose bag, molecular cut-off 14,000 g/mol) or
by centrifugation (Beckman LE-80, 29000 rpm, 25 minutes). Thiamine
encapsulation efficiency was determined after complete solubilization
of the liposomes by the detergent OG and calculated as the ratio of
the amount of encapsulated thiamine to that of thiamme initially added
to the lipids. It was found that, 7% of thiamine was encapsulated.
Liposomes coated with cholesterol-dextran were prepared using the
same procedure as for conventional liposomes except that 10 mg/ml of
the polysaccharide was added to preformed liposomes prior to free
thiamine separation. Coating of the liposome surface was performed by
mixing the liposomes with hydrophobized polysaccharide overnight so
that spontaneous anchoring of the polymer on the liposome outer
bilayer occurred via the cholesterol moiety (Kang et al., 1997).
Size distribution of liposomes was clerermined by granulometry (Malvern, Mastersizer S.Ver. 215) which provided a mean diameter (MD)
associated with a standard deviation (SD). Vesicle suspensions were
observed with a phase-contrast microscope (IM 35 with an oil immersion x 100/1.25 objective, Zeiss). Structural stability of the liposome
preparation was followed by measuring the turbidity at 400 nm as a
function of time.
Results and discussion
In most studies, liposomes are based on phosphatidylcholine mixed
with a small amount of cholesterol and/or charged lipids, so that, few
studies deal with complex mixtures of lipids. Since one purpose of this
work is to validate the potential use of a crude lipid extract directly in a
liposome formulation, loss of lipids due to extrusion was determined. It
was found that phospholipid and cholesterol losses during the fdtration
step were low and in the same order, i.e., 2.7 and 2.1 wt%, for phospholipids and cholesterol, respectively. This indicated that both types
of lipids exhibited the same behaviour during filtration, probably
due to the formation of phospholipid-cholesterol complex. On the
contrary and as expected for this type of lipids, triglycerides were
poorly incorporated in the liposome membrane since 15 wt/% was lost
during filtration.
Liposomes prepared by the extrusion technique on a 5 pm pore diameter
filter exhibited a mean diameter of 5.7 µm but the size distribution was
rather large (5.7 ± 3.7 µm). Optical microscopy observation revealed the
presence of both multilamellar and unilamellar structures. These morphologic characteristics were preserved when thiamine was encapsulated.
Turbidity measurements as a function of time allowed to follow the
physical stability of the liposome preparations. No significant turbidity variation was found over a 2-week period for liposomes prepared
with or without thiamine (result not shown). This was confirmed by
granulometry measurements indicating that size distribution was only
slightly modified after 5 days (5.0 ± 4.6 µm) although a small part (9% )
215
dialysis (Spectra-Por cellulose bag, molecular cut-off 14,000 g/mol) or
by centrifugation (Beckman LE-80, 29000 rpm, 25 minutes). Thiamine
encapsulation efficiency was determined after complete solubilization
of the liposomes by the detergent OG and calculated as the ratio of
the amount of encapsulated thiamine to that of thiamme initially added
to the lipids. It was found that, 7% of thiamine was encapsulated.
Liposomes coated with cholesterol-dextran were prepared using the
same procedure as for conventional liposomes except that 10 mg/ml of
the polysaccharide was added to preformed liposomes prior to free
thiamine separation. Coating of the liposome surface was performed by
mixing the liposomes with hydrophobized polysaccharide overnight so
that spontaneous anchoring of the polymer on the liposome outer
bilayer occurred via the cholesterol moiety (Kang et al., 1997).
Size distribution of liposomes was clerermined by granulometry (Malvern, Mastersizer S.Ver. 215) which provided a mean diameter (MD)
associated with a standard deviation (SD). Vesicle suspensions were
observed with a phase-contrast microscope (IM 35 with an oil immersion x 100/1.25 objective, Zeiss). Structural stability of the liposome
preparation was followed by measuring the turbidity at 400 nm as a
function of time.
Results and discussion
In most studies, liposomes are based on phosphatidylcholine mixed
with a small amount of cholesterol and/or charged lipids, so that, few
studies deal with complex mixtures of lipids. Since one purpose of this
work is to validate the potential use of a crude lipid extract directly in a
liposome formulation, loss of lipids due to extrusion was determined. It
was found that phospholipid and cholesterol losses during the fdtration
step were low and in the same order, i.e., 2.7 and 2.1 wt%, for phospholipids and cholesterol, respectively. This indicated that both types
of lipids exhibited the same behaviour during filtration, probably
due to the formation of phospholipid-cholesterol complex. On the
contrary and as expected for this type of lipids, triglycerides were
poorly incorporated in the liposome membrane since 15 wt/% was lost
during filtration.
Liposomes prepared by the extrusion technique on a 5 pm pore diameter
filter exhibited a mean diameter of 5.7 µm but the size distribution was
rather large (5.7 ± 3.7 µm). Optical microscopy observation revealed the
presence of both multilamellar and unilamellar structures. These morphologic characteristics were preserved when thiamine was encapsulated.
Turbidity measurements as a function of time allowed to follow the
physical stability of the liposome preparations. No significant turbidity variation was found over a 2-week period for liposomes prepared
with or without thiamine (result not shown). This was confirmed by
granulometry measurements indicating that size distribution was only
slightly modified after 5 days (5.0 ± 4.6 µm) although a small part (9% )
215