The Modification Of MTT Assay To Evaluate The Cytotoxicity Of Dextran-coated Superparamagnetic Iron Oxide Nanoparticles And Its Mechanism Study

Magnetic nanoparticles have become the hotspots because of their advantageous properties both as magnetic materials and nanomaterials. Among all the magnetic nanoparticles, functionalized magnetic nanomaterials have been widely used in new materials, catalysis, food testing as well as biomedicine. Owing to their superparamagnetism, surface modification and biocompatibility, dextran superparamagnetic iron oxide nanoparticles (DSPIONs) have been applied in drug delivery, magnetically mediated hyperthermia and magnetic resonance imaging, which receive considerable attention. Herein, we synthesized a kind of DSPIONs independently and its cytotoxicity was also tested by MTT assay, WST-8 assay and trypan blue assay. However, the MTT assay overestimated the cell viability and gave a false negative results in a higher dosage of DSPIONs. Thus, we proposed a modified MTT assay, which was comparable with the other assays. Finally, the mechanism of MTT reduction in DSPIONs and RPMI-1640 medium was also studied. The detailed contents are as follows:(1) MTT assay, WST-8 assay and trypan blue assay were adopted to study the cytotoxicity of DSPIONs in human liver cancer cells (HepG2). The cell viability determined by MTT assay exceeded 100% in all DSPIONs concentration, which showed false positive result. The relationship between cell viability and nanoparticles was not a linear relationship between dose and concentration. Besides, there is a poor repeatability in repeated experiments. The results from WST-8 assay and trypan blue assay were found reasonable and had a good linear relationship.(2) Analysis given by MTT assay suggested that the cell viability increased with the DSPIONs concentration. In order to eliminate the interference from DSPIONs, a modified MTT assay was established by adding the background group with DSPIONs but without cells and subtracting the absorbance of the background group. Moreover, the results in modified MTT assay were comparable with that in WST-8 assay and trypan blue assay, verifying that a logical and scientific method was built.(3) To illustrate a possible reason for the overestimated results from the MTT assay, the detection of ·O2- in DSPIONs system was carried out using hydroxyl amine oxidation method and the absorbance of azoic compound at 530 nm was recorded. The results showed that the produced ·O2- by DSPIONs played a leading role in the reduction of MTT, which consists with the SOD quench experiment. The medium experiment indicated that three ingredients contributed to the production of formazan-much more ·O2- was generated when L-cystine dihydrochloride promoted was added, some reductive substance may also react with MTT, including L-glutathione reduced and pyridoxine hydrochloride. Moreover, the kinetics of formazan formation conformed to a zero-order reaction.