301
2. Dissolve the weighted amounts of the lipid and the examined
guest molecule in organic solvent, for example chloroform or
chloroform/methanol (9:1 v/v) (see Note 2), transfer into a
round flask, and connect it to a rotary evaporator.
3. Apply vacuum, until a mixed thin film is formed by slow removal
of the solvent at 50 °C for 2 h.
4. The mixed lipidic films should be maintained under vacuum for
at least 24 h in a desiccator to remove traces of the solvent.
5. Hydrate the mixed films in water solvent (see Note 3), by slowly
stirring for 1 h in a water bath and by heating above the phase
transition of the used lipid. The concentration of the lipid
should be 30 mg mL
−1
, in order to achieve liposomal bilayers.
In the cases of lipidic bilayers with incorporated guest molecules, follow only steps 1–4.
6. The resultant formulations (multilamellar vesicles, MLVs) can
be subjected to sonication cycles before their use, until unilamellar vesicles (SUVs) be achieved (see Note 4). After the size
reduction, liposomes should be allowed to anneal for 30 min
before their use.
3 Methods
1. Use sealed stainless steel crucibles (for example aluminum) with
O-ring as sample holders, as well as a crucible with water or buffer as reference.
2. Prepare the samples into the crucibles:
(a) In the case of pre-prepared liposomes with incorporated
guest molecules: taking into consideration the lipid concentration of the liposomal dispersion (30 mg mL
−1
), use
directly the appropriate volume of the dispersion, in order
to achieve in the crucible a final quantity of 4–7 mg lipid.
Seal the crucible.
(b) In the case of lipidic bilayers with incorporated guest molecules: weight 4–7 mg of the dry lipidic bilayer directly in
the crucible and hydrate/disperse it with the chosen
hydration medium, in the appropriate concentration
(see Notes 3, 5 and 6). Seal the crucible and subsequently
vortex for 5 min, so MLVs can be achieved. The sample
should be prepared 30 min before the DSC measurement,
in order to be efficiently equilibrated before the
measurement.
3. Calibrate the DSC instrument. Pure indium with main transition temperature (T m ) at 156.6 °C is usually used as a standard
sample for the calibration of the instrument.
3.1 Differential
Scanning Calorimetry
(DSC) Measurements
DSC on Liposomes and Bilayers Incorporating Drugs and Biomaterials
2. Dissolve the weighted amounts of the lipid and the examined
guest molecule in organic solvent, for example chloroform or
chloroform/methanol (9:1 v/v) (see Note 2), transfer into a
round flask, and connect it to a rotary evaporator.
3. Apply vacuum, until a mixed thin film is formed by slow removal
of the solvent at 50 °C for 2 h.
4. The mixed lipidic films should be maintained under vacuum for
at least 24 h in a desiccator to remove traces of the solvent.
5. Hydrate the mixed films in water solvent (see Note 3), by slowly
stirring for 1 h in a water bath and by heating above the phase
transition of the used lipid. The concentration of the lipid
should be 30 mg mL
−1
, in order to achieve liposomal bilayers.
In the cases of lipidic bilayers with incorporated guest molecules, follow only steps 1–4.
6. The resultant formulations (multilamellar vesicles, MLVs) can
be subjected to sonication cycles before their use, until unilamellar vesicles (SUVs) be achieved (see Note 4). After the size
reduction, liposomes should be allowed to anneal for 30 min
before their use.
3 Methods
1. Use sealed stainless steel crucibles (for example aluminum) with
O-ring as sample holders, as well as a crucible with water or buffer as reference.
2. Prepare the samples into the crucibles:
(a) In the case of pre-prepared liposomes with incorporated
guest molecules: taking into consideration the lipid concentration of the liposomal dispersion (30 mg mL
−1
), use
directly the appropriate volume of the dispersion, in order
to achieve in the crucible a final quantity of 4–7 mg lipid.
Seal the crucible.
(b) In the case of lipidic bilayers with incorporated guest molecules: weight 4–7 mg of the dry lipidic bilayer directly in
the crucible and hydrate/disperse it with the chosen
hydration medium, in the appropriate concentration
(see Notes 3, 5 and 6). Seal the crucible and subsequently
vortex for 5 min, so MLVs can be achieved. The sample
should be prepared 30 min before the DSC measurement,
in order to be efficiently equilibrated before the
measurement.
3. Calibrate the DSC instrument. Pure indium with main transition temperature (T m ) at 156.6 °C is usually used as a standard
sample for the calibration of the instrument.
3.1 Differential
Scanning Calorimetry
(DSC) Measurements
DSC on Liposomes and Bilayers Incorporating Drugs and Biomaterials