Evaluating invitro cytotoxicity effect of nanoparticles using MTT

In the present study, we adopted the MTT colorimetric test to evaluate cytotoxicity. In this method, the yellow tetrazolium salt is converted into purple formazan crystals by mitochondrial enzymes in living cells⁴⁰. To determine the cytotoxicity of two nanoparticles, as well as vincristine administered in isolation, we used the MTT assay, which involved seeding of Y79 and ARPE-19 cells at a cellular density of 10⁴ cells per well in 96-well plates, followed by incubation for a period of 24 h. Subsequently, we subjected our cellular specimens to varying concentrations of vincristine in combination with equivalent dosages of empty and substance-incorporated nanoparticles, ranging from 0.6 to 80 micromoles, over a period of 48 h. In the case of adherent cells, the culture medium was removed by aspiration. To process suspended cells, we subjected 96-well plate to centrifugation at a force of 1000×g and maintained a temperature of 4 °C, utilizing a microplate-compatible centrifuge for a duration of 5 min, followed by the careful aspiration of the media. Subsequently, the cells were subjected to a treatment regimen involving the application of 5 mg/mL of MTT, followed by the addition of 100 μL of solvent for MTT to each individual well. This incubation took place in a dark environment at a temperature of 37 °C for four hours. In order to process cells in suspension, it is recommended to subject the 96-well plate to centrifugation in a centrifuge compatible with the plate at 1000×g and 4 °C for a duration of 5 min. The medium should then be carefully aspirated followed by complete aspiration of the MTT solvent. Subsequently, a volume of 200 µL of dimethyl sulfoxide (DMSO) solvent was introduced into each well of the receptacle. The aforementioned dish was subsequently enveloped with aluminum foil and subjected to 15 min of agitation utilizing an orbital shaker. The measurement of readout absorbance at a wavelength of 590 nm was conducted through the utilization of a microplate reader (BioTek, Winooski, VT). The quantification of the absorbance that is proportionate to cell number has been ascertained by deducting the absorbance detected in the control or background sample from the obtained experimental results. Subsequently, the aforementioned entities were employed to determine the concentration of both vincristine and vincristine-loaded nanoparticles necessary to attain 50% inhibition of Y79 and ARPE-19 cells, denoted as IC50. Cytotoxicity was assessed as a percentage and was determined via the utilization of Eq. (5) by using the corrected absorbance.