Synthesis of gadolinium lipid (GdL)

The synthesis of Gadolinium ion (Gd³⁺)-chelated DSPE-DOTA was performed using a two-step method as previously described with some modifications ^{41, 57, 58}. Briefly, 75 mg of DSPE was first dissolved in 10 mL chloroform containing 2% (v/v) triethylamine (TEA), then 86 mg DOTA-NHS ester was added, and the mixture was incubated 3 h at 40 ºC. The product was concentrated under vacuum. Purification of the conjugating DSPE-DOTA lipid was achieved by freeze-thaw cycles, followed by centrifugation at 4500 g for 10 min at room temperature to precipitate by-products. The supernatant was then sterile filtered (0.22 µm pore size) and lyophilized. DSPE-DOTA-Gadolinium (GdL) was prepared by adding 10 mL of acetate buffer (pH: 5.5) to 50 mg DSPE-DOTA (0.05 mmole). The suspension was then treated with 0.5 mmole of Gd(OAc)₃ at 50 ºC for 12 h. After incubation, the Gd-DOTA-DSPE was purified by centrifugation at 4500 g for 10 min at room temperature. The obtained product was washed 3 times with acetate buffer (pH: 5.5) and 3 times with distilled water to remove non-chelated Gd³⁺ and then lyophilized.