Cytotoxicity Investigation of Ti3C2Tx MXene toward Human Umbilical Vein Endothelial Cells

MXenes are emerging two-dimensional nanomaterials with promising potential for biomedical applications, including osteogenic differentiation, antibacterial properties, anticancer therapy, and drug delivery systems. However, biocompatibility studies with blood vessels or endothelial cells need more thorough research. In this study, delaminated and multilayered Ti3C2Tix MXenes were synthesized from the Ti3AlC2 MAX phase and characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD) and applying violet, blue, and red side scatter width (SSC-Width) and high (SSC-H) functions of a flow cytometer. MXene-cell association was examined using the red side scatter area (SSC-A) function of the flow cytometer. HUVEC responses following 1- and 3-day exposure to MXenes were evaluated by nuclei count and LDH release. Apoptosis was also evaluated with a flow cytometer and Annexin V/propidium iodide staining. Both types of MXene were associated with HUVECs and showed a concentration-dependent response; however, delaminated 2D-like MXene flakes were significantly more cytotoxic than multilayered 3D-like MXenes: at low concentrations (1-3 μg/mL), their cell number started decreasing, LDH release increased, and late apoptosis and necrosis were induced, while multilayered MXenes showed similar effects at a much higher concentration, at 25 μg/mL. Comparing the influence of two different structures of MXenes suggests that the delaminated MXenes may exhibit enhanced MXene-cell interactions than multilayered MXenes, which is leading to membrane perturbation and subsequent effects on cell viability and apoptosis. Overall, these findings demonstrate that advanced, multiparametric, and high-performance flow cytometry can be used not only for the cell analysis but also for the identification of nanoparticle size, and MXene-cell association. Data also shows that biocompatibility of delaminated and multilayered Ti3C2Tix MXene in HUVECs is a strongly preparation-dependent process, which highlights the importance of evaluation of standardized biocompatibility strategies for the development of safe MXene-based biomedical applications.