*2.13. Drug Loading E*ffi*ciency*

The supernatant was diluted with PBS at ratio of 1 (supernatant): 20 (PBS). Then the drug concentration in supernatant was determined by MTX Elisa kit (ENZ-KIT 142-0001, Enzo Life Science, Farmingdale, NY, USA). Experiment procedure followed the manufactory product manual. First, MTX standard (S1-S6) was prepared by diluting the company supplied lyophilized MTX to suggested concentrations (1000 ng/mL, 166.7 ng/mL, 27.8 ng/mL, 4.6 ng/mL, 0.77 ng/mL, 0.13 ng/mL). Then, 100 μL of standard solution and samples were added into 96 wells, 100 μL of assay buffer 13 was added to one well and set as S0 (0 ng/mL standard), and 150 μL of assay buffer 13 was added to another well set as NSB well. 50 μL of MTX antibody was added to all wells except for the NSB and blank, and this 96 well plat was sealed and incubated at room temperature on a plate shaker (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at ~500 rpm.

Then, all the wells were washed by wash buffer for 3 times. Followed with addition of 100 μL of MTX conjugated solution to all the wells except the blank, this plate was incubated at room temperature as before. After another 3 times wash, 100 μL of substrate solution was added to all wells and went through the final incubation as same as above. Finally, add 100 μL of stop solution to all wells and read the plate at 450 nm. According to the standard curve to calculate the MTX concentration of supernatant.

The drug loading efficiency was determined by following equation:

Loading Efficiency = (Total amount of MTX − supernatant MTX)/Total amount of MTX × 100% (1)
