1 stearoyl 2 hydroxy sn glycero 3 phosphocholine

All lipids, dipalmitoylphosphatidylcholine
(DPPC), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine
(MSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), cholesterol,
and hydrogenated-l-α-phosphatidylcholine (HSPC) were
purchased from Avanti Polar Lipid. Doxorubicin hydrochloride was purchased
from Fisher Scientific.
LTSLs and NTSLs were synthesized by
a reverse-phase evaporation method.^{56 (link)−58 (link)} Briefly, 5 mg of lipid
(for LTSLs: DPPC–MSPC–DSPE-PEG2000 = 90:10:4; for NTSLs:
HSPC–CHOL–DSPE-PEG2000 = 75:50:3 in molar ratio)^{59 (link),60 (link),64 (link),65 (link)} was dissolved in 1 mL of chloroform, then dried using nitrogen air
and subsequent vacuum. To form the liposomes, the lipid film was hydrated
with a 300 mM citrate buffer (pH 4.0) for 60 min (at 55 °C for
LTSLs and 60 °C for NTSLs). The liposomes were then extruded
10 times through a 400 nm and a 100 nm polycarbonate membrane to obtain
the desired size. The outside pH of the liposome solution was titrated
to pH 7.5 using 0.5 M sodium carbonate. As a result, a pH gradient
was generated across the lipid bilayer.^{61 (link)−63 (link)} DOX was then added to
the liposome solution at a 1:20 DOX/lipid weight ratio and mixed for
60 min (at 37 °C for LTSLs and 60 °C for NTSLs). The final
product was passed through a PD10 column to remove excess DOX.