Multilayered stable 2D nano-sheets of Ti2NTx MXene: synthesis, characterization, and anticancer activity

Abstract

Background: The biological activity of MXenes has been studied for several years because of their potential biomedical applications; however, investigations have so far been limited to 2D titanium carbides. Although monolayered Ti₂NT_x MXene has been expected to have biological activity, experimental studies revealed significant difficulties due to obstacles to its synthesis, its low stability and its susceptibility to oxidation and decomposition.

Results: In this paper, we report our theoretical calculations showing the higher likelihood of forming multilayered Ti₂NT_x structures during the preparation process in comparison to single-layered structures. As a result of our experimental work, we successfully synthesized multilayered Ti₂NT_x MXene that was suitable for biological studies by the etching of the Ti₂AlN MAX phase and further delamination. The biocompatibility of Ti₂NT_x MXene was evaluated in vitro towards human skin malignant melanoma cells, human immortalized keratinocytes, human breast cancer cells, and normal human mammary epithelial cells. Additionally, the potential mode of action of 2D Ti₂NT_x was investigated using reactive oxygen tests as well as SEM observations. Our results indicated that multilayered 2D sheets of Ti₂NT_x showed higher toxicity towards cancerous cell lines in comparison to normal ones. The decrease in cell viabilities was dose-dependent. The generation of reactive oxygen species as well as the internalization of the 2D sheets play a decisive role in the mechanisms of toxicity.

Conclusions: We have shown that 2D Ti₂NT_x in the form of multilayered nanoflakes exhibits fair stability and can be used for in vitro studies. These results show promise for its future applications in biotechnology and nanomedicine.

Keywords: Anticancer properties; Cytotoxicity in vitro; MXenes; Mammalian cells; Stability; Ti2N.

Fig. 1

Schematic representation of the concept of the present study. The flowchart shows the synthesis process of the 2D Ti₂NT_x MXene for obtaining stable multilayered 2D structures as well as the resulting interactions with human cells

Fig. 2

Characterization of the materials used in the study i.e.: Representative SEM images obtained for a Ti₂AlN starting MAX phase, b Ti₂N MXene after HF etching, and c 2D sheets of Ti₂NT_x MXene after delamination; d Cross-sectional HREM image, e FFT image together with related f IFFT extraction of multilayered structure obtained for 2D sheets of Ti₂NT_x MXene; g The intensity pattern revealing a period related to d-spacing of 0.97 nm; h SADP electron diffraction with indication of the characteristic rings for 2D Ti-N layers; i Comparison of the X-ray diffraction patterns obtained for: Ti₂AlN MAX phase and resulting Ti₂NT_x MXene; j EDX analysis, k UV–VIS spectrum accompanied by Tyndall test, as well as l FTIR spectrum of the Ti₂NT_x MXene after delamination

Fig. 3

The possible arrangements of the oxygen atoms for two adjacent Ti₂N layers. Model 1 is the most stable one from all the configurations considered here

Fig. 4

a The most stable arrangement of the terminated Ti₂NT_x layers by oxygen atoms for two adjacent Ti₂N layers. The solid line denotes the lateral size of the supercell (top view). b Interlayer binding energy curve of Ti₂NO₂ layers. The interlayer binding energy decreases as the number of layers increases, reaching a value of − 16.6 meV/atom for an infinite number of layers. The black line fitted to the data is presented, and the extrapolated value for an infinite number of layers is indicated

Fig. 5

a The results of MTT assay after 24 h exposure of A549, MRC-5, A375, and HaCaT cells to increasing concentrations of multilayered 2D sheets of Ti₂NT_x MXene. b Production of reactive oxygen species (ROS) during incubation with various concentrations of 2D sheets of Ti₂NT_x MXene. Student’s t test, α = 0.005, n = 3; n number of independent experiments

Fig. 6

Distributions of the hydrodynamic diameters obtained for multilayered 2D sheets of Ti₂NT_x MXene suspended in double-distilled water (DDW) as well as cultivation media applied for the in vitro studies, for incubation time t = 0 and t = 24 h

Fig. 7

Representative SEM images obtained for (a, b) reference A375 cell line, and A375 cells treated with multilayered 2D sheets of Ti₂NT_x MXene at a concentration of (c–e) 62.5 mg L⁻¹ and (f–h) 500 mg L⁻¹. SEM analysis was performed with SE mode (a–g) as well as AsB (d, h). 2D Ti₂N sheets internalized by A375 cells were marked with red arrows, whereas those only attached to the cells’ surface were marked with white arrows

Fig. 8

Representative SEM images obtained for (a, b) reference HaCaT cell line, and HaCaT cells treated with multilayered 2D sheets of Ti₂NT_x MXene at a concentration of (c, d, e) 62.5 mg L⁻¹ and (f, g, h) 500 mg L⁻¹. SEM analysis was performed with SE mode (a–d, g) as well as ASB (e, h). 2D Ti₂NT_x sheets attached to the surface of the HaCaT cells were marked with white arrows