Doxorubicin hcl

Doxil (liposomal doxorubicin) was purchased from Avanti Polar Lipids (LipoCure). To prepare Doxil-like liposomes, hydrogenated soybean phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-mPEG2k), and cholesterol were purchased from Avanti Polar Lipids. HEPES buffer, PBS, sodium chloride, ammonium sulfate, doxorubicin HCl, and Sepharose CL-4B were purchased from Sigma-Aldrich.

Doxorubicin dissolution was characterized by measuring the concentration of doxorubicin in the solution (see Supplemental Figure S1). Briefly, doxorubicin crystals were formed by slowly pipetting 50 μL of a 5 mg mL⁻¹ solution of doxorubicin HCl (Sigma) into 800 μL of a solution containing 10 mM HEPES, 140 mM NaCl, 300 mM ammonium sulfate in a microcentrifuge tube. This solution mimics the solution used for remote loading doxorubicin into liposomes [7 (link)]. The solution was left to crystallize for 30 minutes in the dark at room temperature. Dissolution experiments were performed by removing the supernatant and replacing it with 800 μL of 300 mM ammonium sulfate solution pre-incubated to 4, 25, or 37 °C. At the prescribed time points, 400 μL of the ammonium sulfate solution were removed for absorbance measurement, and then replaced in the tube. The concentration of doxorubicin in solution was determined from the absorbance using a calibration curve obtained from known concentrations. The saturation concentration was obtained by allowing the crystals and solution to equilibrate at the given temperature over the course of several days. Volumes and amounts were optimized for UV-vis absorbance measurements.