Avanti s mini extruder

Liposomes were composed of
dipalmitoylphosphatidylcholine (DPPC), DSPE-PEG
(2000) maleimide, cholesterol, 1.2-distearoyl-snglycero-
3-phosphoethanolamine-N-(amino(polyethylene
glycol)-2000) (DSPE-PEG2000) with respectively 7,
0.1, 2.5 and 0.4 µmol volume, obtaining from Avanti
Polar Lipids (USA). After dissolving in chloroform and
methanol solutions (rate of 9:1 v/v, both from Sigma-
Aldrich, USA), thin biofilm was formed in a round-bottom
flask. After evaporation of the resulting suspension, a
rotary evaporator under low pressure (45°C, 70 rpm) was
used up to completely removing the solvents. In continue,
the produced biofilm was hydrated in 1.2 ml sodium
phosphate buffer (including 50 mM NaH₂PO₄, 0.15 mM
NaCl and 1 mM EDTA, pH=7.0) at 70°C resulting in
spontaneously organized multi-lamellar vesicles (MLVs).
Finally, the MLVs were extruded 21 times at 65°C through
0.1 µm pore sized polycarbonate membranes (Avanti Polar
Lipids, USA) using an Avanti’s mini-extruder (Avanti
Polar Lipids) to form small uni-lamellar vesicles. After
incubation of the liposomes at RT to cool-down, they
were stored at 4°C. Produced liposome diameters were
defined by a Zetasizer Nano APS (Malvern Instruments
Ltd, UK) at 25°C following the appropriate dilution with
phosphate buffered saline (PBS).