Understanding the Mechanism of DNA Fragmentation by MXene in DNA Comet Assay through In-vitro Electrophoresis

MXenes, a novel family of 2D materials, are considered highly promising for a wide array of application due to their extraordinary properties and structure. Studies have demonstrated their minimal toxicity, excellent biocompatibility and favorable interactions with biological systems. It is becoming increasingly clear that MXenes will soon find their usage in many biomedical applications.

Despite being actively investigated, the genotoxic properties of MXenes are yet to be thoroughly examined. Our previous research employing DNA comet assay unveiled the substantial occurrence of DNA comets in diverse cell cultures following the exposure to Ti3C2 and Nb4C3 MXenes. The precise source of the observed DNA fragmentation remains elusive. We hypothesized that the sharp edges of MXenes may instigate DNA breaks when subjected to an electric field during the electrophoresis step in the DNA comet assay. 

In order to test this hypothesis, we devised the technique of in-vitro electrophoresis. For this, cells were cultured in 6-well plates and loaded with MXenes. Two platinum electrodes were positioned at opposite sides of the cell culture wells using 3D printed polymer inserts, and an electric field was applied, mirroring the conditions employed in the DNA comet assay. Cell viability was monitored by resazurin reduction assay. 

It was anticipated that if the MXenes indeed could fragment cellular chromosomal DNA while moving in the electric field, the MXene loaded cell would undergo apoptosis or necrosis when exposed to the electric field. We found that the electrophoresis conditions indeed compromised cell viability, potentially attributable to pH alterations. However, reliable detection of the increased cell death in the presence of MXenes under electrophoresis conditions could not be consistently achieved. Moreover, we did not observe any effect of MXenes on cell viability when we placed the whole cell culture plate with the MXene loaded cells in a strong electric field.  

The outcomes of the in-vitro electrophoresis and the molecular mechanisms of the DNA comets in the presence of MXenes continue to be the subject of ongoing debates. However, it is becoming increasingly clear that the observed DNA fragmentation manifested in the DNA comets were the result of the artefact of the DNA assay which could not be verified in the independent assays. Further studies are required to uncover the mechanisms of the DNA comets, while the nature of MXene genotoxicity, suggested by the DNA comet assay, remains elusive.