For IR780-liposome preparation, DSPC, cholesterol and DSPE-PEG2000 at a molar ratio of 54.8:40:5 were dissolved in chloroform (about 15 mg total lipids/ml); 1 mg/ml IR780 iodide in ethanol then was added to final 0.2% molar ratio of dye to total lipids. The solution was rotary evaporated in the dark to dryness in vacuo. The dried lipid film was hydrated in nitrogen gas-flushed 0.9% saline to a final total lipid concentration of 30 mM, vortexed and ultra-sonicated under a nitrogen atmosphere for final suspension of the lipid particles. The suspension was repeatedly extruded 16 times through a 100 nm Whatman Nucleopore track-etched polycarbonate membrane at 56 °C. The acquired clear bluish suspension was flushed with nitrogen gas, sealed in a glass vial and stored at either 4 or −20 °C in the dark.
To prepare IR780-phospholipid micelles, thin lipid film composed of DSPC and DSPE-PEG2000 containing IR780 (molar ratio, 59.5:40:0.5) was formed in the same way as for the liposomes preparation, then hydrated with nitrogen gas-flushed 0.9% saline to a final total lipid concentration of 15 mM, vortexed and ultra-sonicated at 56 °C for 15 min under a nitrogen gas atmosphere, and extruded through a 100 nm Whatman Nucleopore track-etched polycarbonate membrane. The acquired clear cyan micelle suspension was stored under the same conditions as the liposomes.
To prepare IR780-phospholipid micelles, thin lipid film composed of DSPC and DSPE-PEG2000 containing IR780 (molar ratio, 59.5:40:0.5) was formed in the same way as for the liposomes preparation, then hydrated with nitrogen gas-flushed 0.9% saline to a final total lipid concentration of 15 mM, vortexed and ultra-sonicated at 56 °C for 15 min under a nitrogen gas atmosphere, and extruded through a 100 nm Whatman Nucleopore track-etched polycarbonate membrane. The acquired clear cyan micelle suspension was stored under the same conditions as the liposomes.