Safire fluorescence microplate reader

Doxorubicin (Dox, LC Labs # D-4000) was loaded via the ammonium sulfate gradient method^{[70 (link)]}. Dox with a drug to lipid molar ratio of 1:8 was added into the lipid solutions and incubated at 60 °C for 1 h. Liposomes sizes and polydispersity index were determined by dynamic light scattering in PBS. Dox loading efficiency were determined by running 20 µL of liposomes (20 mg/ml lipids) diluted in 1 mL of PBS over a Sephadex G-75 column. 24×1mL fractions were collected and Dox fluorescence in each fraction was measured using a TECAN Safire fluorescence microplate reader (excitation and emission wavelengths of 480 nm and 590 nm, respectively). Loading efficiency was determined as the percentage of drug in the liposome-containing fractions (first 3–8 fractions). Negative stained transmission electron microscopy (TEM) was performed using a JEM-2010 electron microscope with 1% uranyl acetate staining. Serum stability was performed by incubating PoP liposomes (20 mg/mL lipids) diluted 200 times in 50% sterile bovine serum (Pel-Freeze) at 37 °C for the indicated times. 0.25 % Triton X-100 was added to read the total Dox fluorescence. Dox release was measured by florescence using the formula: % Dox release= (F_final−F_initial)/(F_x-100−F_initial) × 100%.

10 µL of empty PoP liposomes (20 mg/mL lipids) were diluted 20 times in PBS in a microplate well. Laser irradiation was performed at 665 nm and 250 mW/cm² for 3 min at room temperature. Calcein (50 mM) was added before or after irradiation as indicated. Liposome samples were loaded onto a Sephadex G-75 column immediately after treatment (Fig. 5C). For the kinetics of calcein encapsulation into pre-irradiated empty PoP liposomes, calcein was added immediately after, 1 min, 10 min, 30 min or 60 min after irradiation. Samples were then added to G-75 columns after incubation at room temperature for 3min (Fig. 5D). Alternatively, calcein was added before irradiation (250 mW/cm² for 3 min) and incubated at room temperature for 0 min, 1 min, 10 min, 30 min and 60 min (Fig. 5E). Calcein encapsulation efficiency was determined by gel filtration with a Sephadex G-75 column. 16×0.5 mL fractions were collected, calcein (485 nm/525 nm) and PoP (420 nm/670 nm) fluorescence were read with a TECAN Safire fluorescence microplate reader. Calcein/PoP ratios in the liposomal fractions (6–9 fractions) were calculated by simple division.