Biotechnology
an immediate leakage of about half of the encapsulated vitamin was
observed and a total release was obtained in one day. This could result
from a synergetic effect of sea water ions which destabilize the lipid
membrane by their interaction with phospholipids and of sea water
osmolarity that forces water to flow out of the liposomes simultaneously
dragging thiamine molecules along the membrane.
Several attempts were performed in order to improve thiamine retention
in liposomes especially when dispersed in sea water. Adjustment of
thiamine solution osmolarity, variation of pH and storage temperature
did not significantly reduce vitamin leakage (results not shown). Liposomes can also be coated with a variety of hydrophilic molecules, such
as polyethylene glycol, glycolipids and polysaccharides (Allen, 1994).
In this case, efficient coating of the liposome surface needs the grafting
of a lipid molecule on the polymer that acts as an anchor in the liposome bilayer and so tightly binds the saccharides to the liposome. To
this aim, cholesterol or fatty acid were used but cholesteryl derivatives
of polysaccharides were shown to ensure a better coating of liposomes
than simple monoalkyl or dialkyl chain clerivatives (Kang et al., 1997).
Due to cholesterol grafting, hydrophobized polysaccharides exhibit an
amphiphile character that leads to its partitioning between the liposome membrane phase and the external aqueous phase when in presence of liposome. Hydrophobic attractive forces ensure the coating of
liposome surface when modified polysaccharides are added to preformed
liposomes. This method of coating is usually employed with a variety
of hydrophobized saccharides although incubation time and temperature of the liposome suspension with the modified polymers largely vary
upon the authors, i.e., from 30 minutes to 3 days for the incubation
times and 4°C to 50°C for the incubation temperatures (Kang et al.,
1997; Rosilio et al., 1992). In our case, incubation time lasted overnight, at room temperature, for a 10 mg/ml dextran concentration.
This procedure ensured an efficient coating of the liposome surface
although the exact quantity of polysaccharide bound to the membrane
remains to be determined.
Figures 2a and 2b compare thiamine release from coated and non-coated
liposomes diluted with buffer solution and sea water, respectively. In
both cases, dextran coating significantly improved thiamine retention.
After 50 hours of storage in buffer solution, thiamine release was found
equal to 22/7 and 45/7 for coated and non-coated liposomes, respectively. In the same way, in sea water, after 20 hours of storage, thiamme
leakage decreased from 90/7 for conventional vesicles to 46/7 when
surface was covered with dextran. The exact role of dextran must be
further investigated. It probably acts as a steric barrier hindering liposomes from aggregation, but it could also decrease the osmotic pressure
m proximity of the membrane so that flows of thiamine ancl/or water
should be slowed down. As a whole, these results are in agreement with
studies showing that coated liposomes are structures which gain in
stability in comparison with conventional non-coated liposomes.
217
an immediate leakage of about half of the encapsulated vitamin was
observed and a total release was obtained in one day. This could result
from a synergetic effect of sea water ions which destabilize the lipid
membrane by their interaction with phospholipids and of sea water
osmolarity that forces water to flow out of the liposomes simultaneously
dragging thiamine molecules along the membrane.
Several attempts were performed in order to improve thiamine retention
in liposomes especially when dispersed in sea water. Adjustment of
thiamine solution osmolarity, variation of pH and storage temperature
did not significantly reduce vitamin leakage (results not shown). Liposomes can also be coated with a variety of hydrophilic molecules, such
as polyethylene glycol, glycolipids and polysaccharides (Allen, 1994).
In this case, efficient coating of the liposome surface needs the grafting
of a lipid molecule on the polymer that acts as an anchor in the liposome bilayer and so tightly binds the saccharides to the liposome. To
this aim, cholesterol or fatty acid were used but cholesteryl derivatives
of polysaccharides were shown to ensure a better coating of liposomes
than simple monoalkyl or dialkyl chain clerivatives (Kang et al., 1997).
Due to cholesterol grafting, hydrophobized polysaccharides exhibit an
amphiphile character that leads to its partitioning between the liposome membrane phase and the external aqueous phase when in presence of liposome. Hydrophobic attractive forces ensure the coating of
liposome surface when modified polysaccharides are added to preformed
liposomes. This method of coating is usually employed with a variety
of hydrophobized saccharides although incubation time and temperature of the liposome suspension with the modified polymers largely vary
upon the authors, i.e., from 30 minutes to 3 days for the incubation
times and 4°C to 50°C for the incubation temperatures (Kang et al.,
1997; Rosilio et al., 1992). In our case, incubation time lasted overnight, at room temperature, for a 10 mg/ml dextran concentration.
This procedure ensured an efficient coating of the liposome surface
although the exact quantity of polysaccharide bound to the membrane
remains to be determined.
Figures 2a and 2b compare thiamine release from coated and non-coated
liposomes diluted with buffer solution and sea water, respectively. In
both cases, dextran coating significantly improved thiamine retention.
After 50 hours of storage in buffer solution, thiamine release was found
equal to 22/7 and 45/7 for coated and non-coated liposomes, respectively. In the same way, in sea water, after 20 hours of storage, thiamme
leakage decreased from 90/7 for conventional vesicles to 46/7 when
surface was covered with dextran. The exact role of dextran must be
further investigated. It probably acts as a steric barrier hindering liposomes from aggregation, but it could also decrease the osmotic pressure
m proximity of the membrane so that flows of thiamine ancl/or water
should be slowed down. As a whole, these results are in agreement with
studies showing that coated liposomes are structures which gain in
stability in comparison with conventional non-coated liposomes.
217