Multifunctional microplate reader

The in vitro DOX release from the copolymer micelles was studied using a dialysis bag (molecular weight cutoff size 3,500 Da) under sink conditions. Aliquots of DOX-incorporated micelles (3.0 mL) were transferred into the dialysis bags and dialyzed against 80 mL of phosphate buffers (pH 7.4 and 5.5) containing 0.5% (v/v) Tween-80 at 37°C, respectively. At predetermined time intervals, 0.2 mL aliquots were withdrawn and replaced with an equal volume of fresh medium. The concentration of DOX was measured using a multifunctional microplate reader (Tecan) with the wavelength set at 480 nm. The in vitro release experiments were conducted in triplicate at each pH value. The in vitro DOX release profiles were plotted with cumulative drug release as a function of time.

In vitro cytotoxicity against MCF-7 and MCF-7/ADR cells of DOX-incorporated micelles was assessed by a standard MTT assay.32 MCF-7 cells and MCF-7/ADR cells were seeded in 96-well plates at a density of 5×10³ cells per well and incubated for 24 h, respectively. The growth medium was replaced with fresh medium containing an indicated concentration (0.5, 1, 2, 4, 6, 8, 10 and 12 µg/mL for MCF-7 cancer cells; 1, 5, 10, 50, 100, 150, 200 and 300 µg/mL for MCF-7/ADR cancer cells) of the tested formulations (AT-M/DOX, _pHT-M/DOX, _endoE-M/DOX, PT-M/DOX and DOX solution). Control wells were treated with an equivalent volume of DOX-free medium. The cells were incubated at 37°C for 24 h. After incubation, the wells were rinsed with PBS, which was followed by addition of MTT solution (5 mg/mL) to each well. The plates were further incubated for 4 h, allowing the viable cells to reduce the yellow MTT into purple formazan crystals. After incubation, the medium was removed completely and the purple formazan crystals were dissolved by adding 150 µL of dimethyl sulfoxide. The absorbance was measured at 570 nm using a multifunctional microplate reader (Tecan). The half maximal inhibitory concentration (IC₅₀) values were calculated using nonlinear regression analysis, and the MDR reversal effect was assessed by quantifying the IC₅₀ values of the tested formulations.

Fresh hRBCs were washed with PBS (pH 7.4) three times, centrifuged at 1,000 g for 5 min, and resuspended in PBS to attain a dilution of ~1% (v/v) of the erythrocyte. Then, 100 µL hRBCs solution was placed in each well of 96-well plates, and 100 µL of serial dilution of nano-BAs was added to each well and incubated for 2 h at 37°C. Then, the mixture was centrifuged at 1,000 g for 5 min at 4°C, aliquots (100 µL) of the supernatant were transferred to 96-well plates, and the release of hemoglobin was measured by monitoring the OD at 576 nm using a multifunctional microplate reader (Tecan, Austria). The hRBCs in PBS and 0.5% Triton X-100 were used as negative and positive controls, respectively. The tests were repeated six times, and the data are expressed as the mean and standard deviation of six replicates. The percentage of hemolysis was calculated using the following equation:
$\text{Hemolysis}(\%)=\frac{O{D}_t-O{D}_0}{O{D}_{100}-O{D}_0}\times 100\%$ where OD_t is the observed fluorescence at a given nano-BA concentration, OD₀ is the observed fluorescence in PBS, and OD₁₀₀ is the observed fluorescence in 0.5% Triton X-100.

A thiazolyl blue tetrazolium bromide (MTT) assay was used to assess the in vitro cytotoxicity of nano-BAs against HK-2 cells. Briefly, HK-2 cells were seeded at a density of 5×10³ cells per well in 96-well plates and then incubated for 24 h. Then, the growth medium was replaced with fresh medium containing an indicated concentration of the tested formulations (Nano-BA_3K, Nano-BA_5K, and BA). Control wells were treated with an equivalent volume of BA-free medium. The cells were incubated at 37°C for 48 h. After incubation, the wells were rinsed with PBS, and MTT solution (5 mg/mL) was added to each well, and the plate was incubated for 4 h, thus allowing the viable cells to decrease the yellow MTT into purple formazan crystals. Finally, the medium was completely removed, and 150 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the purple formazan crystals. The absorbance was measured at 570 nm using a multifunctional microplate reader (Tecan, Austria). The IC₅₀ values were calculated using nonlinear regression analysis, and cell cytotoxicity was assessed by quantifying the IC₅₀ values of the tested formulations.