Multilayered stable 2D nano-sheets of Ti2NTx MXene: synthesis, characterization, and anticancer activity
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 Ti2NTx 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.
In this paper, we report our theoretical calculations showing the higher likelihood of forming multilayered Ti2NTx structures during the preparation process in comparison to single-layered structures. As a result of our experimental work, we successfully synthesized multilayered Ti2NTx MXene that was suitable for biological studies by the etching of the Ti2AlN MAX phase and further delamination. The biocompatibility of Ti2NTx 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 Ti2NTx was investigated using reactive oxygen tests as well as SEM observations. Our results indicated that multilayered 2D sheets of Ti2NTx 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.
We have shown that 2D Ti2NTx 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.
KeywordsMXenes Ti2N Cytotoxicity in vitro Stability Mammalian cells Anticancer properties
2D multilayered nano-sheets of Ti2NTx MXene were successfully obtained using classic etching and delamination
2D Ti2NTx showed higher toxicity towards cancerous cell lines (MCF-7, A365)
The decrease in cells viability was dose-dependent
2D Ti2NTx was not toxic towards non-malignant cells (MCF-10A, HaCaT)
ROS generation and internalization were identified mechanisms of toxicity
The discovery of graphene shifted the research on nanomaterials towards their two-dimensional (2D) homologs . Since then, the dynamic development of this field of nanotechnology has progressed, and new members have joined the family of 2D materials. Within this family, several smaller groups can be distinguished, such as transition metal dichalcogenides , nitrides , di-transition metal sulfides, selenides and phosphides [4, 5], 2D oxides and hydroxides [6, 7], 2D metal–organic covalent frameworks , as well as Xenes . The most recent members of this group are early transition metal carbides, nitrides, and carbonitrides called MXene phases. They were first discovered in 2011 during studies on the application of MAX phases in supercapacitors by Naguib, Barsoum, and Gogotsi from Drexel University, USA . This new class of nanomaterials has so far demonstrated significant potential in many applications, such as energy storage , ceramic matrix composites [12, 13, 14], and macromolecules’ adsorption . Other studies also revealed possibilities of their usage as biocides [16, 17, 18], in nanomedicine , or environmental remediation . Due to the fact that the MXenes are developing much more dynamically in comparison to other 2D materials apart from graphene, they were considered as the most interesting and bringing the greatest innovation potential among their analogs .
At the same time, there is concern about the stability of some members of the MXene family, which can significantly affect their future use. The reason is that they are variable non-stoichiometric phases of their thermodynamically stable macroscopic counterparts (MAX phases). Their formulae are also written with a ‘Tx’ termination, the concept of which is to indicate the presence of various functional groups present on their surface (i.e. –OH, –F, or =O) [22, 23]. In the great majority, the presence of OH groups indicates not only the hydrophilic properties of the MXenes’ surface, but also the process of surface oxidation . Nevertheless, from the large number of MXenes phases, Ti3C2Tx and Ti2CTx have so far proven to have relative stability in a number of applications . There are also high hopes for the phases from the Nb-C or Mo-C  systems in relation to many applications. These phases are relatively easy to manufacture and to delaminate into 2D flakes.
On the other hand, there are MXene phases that are considered relatively unstable, such as Ti2NTx  or Ti4N3Tx MXene . The single-layered Ti2NTx with ferromagnetic and partially metallic properties has been outlined as a result of theoretical research , whereas Ti4N3Tx has been recently successfully synthesized using molten fluoride salts . Theoretical calculations, however, indicated that aqueous hydrofluoric acid (HF) solution cannot be successfully used for the aluminum etching and delamination of the precursor Ti2AlN MAX phase , due to the fact that single layered nitride MXenes possess poor stability in this solution. They should also possess very low values for the calculated cohesive energies of the hexagonal crystal structure, together with a stronger bonding of Al atoms [29, 30]. These values indicate a low stability for their single-layered structure, which is present only in some theoretical calculations. On the other hand, the Ti2N phase is expected to have intriguing properties as a half-metal and spin-gapless semiconductor in spintronic applications . Researchers who were interested in the verification of these theories in an experimental manner have attempted to conduct syntheses of these materials. The first success in this endeavor belonged to Soundiraraju et al. , who carried out the process of KF/HCl etching of the Ti2AlN phase into Ti2NTx. Also, nitridation of the Ti3C2Tx MXene by Yang et al.  allowed N-doped 2D Ti2NTx to be obtained. It should be noted that the desirable phase was present in up to 20.7 at.% in the final product. However, the Ti2NTx obtained by nitridation was not assessed for potential cytotoxicity. In the next stage of the development of Ti2NTx technology, the possibility of the preparation of 2D monolayers was explored by Tsai et al. . Non-layered Ti2N with a work function of ∼ 4.75 eV was synthesized by N2 plasma immersion and was successfully applied as the anode material of lithium-ion batteries.
To sum up, while 2D sheets of Ti3C2Tx or Ti2CTx MXenes have been already widely studied with respect to their cytotoxicity, investigations on 2D Ti2NTx have not been focused on biologically-related studies. As a result, up to now 2D Ti2NTx has been an ‘enigma’ in terms of its potential cytotoxicity.
Materials and methods
The MAX product was then initially crushed and then milled with the use of rotary-vibratory mill in isopropanol environment for 12 h. The ‘widia’ (WC–Co) balls were used as milling media.
The obtained powdered MAX phase was immersed for etching in concentrated 48% (v/v) hydrofluoric acid (HF, Sigma-Aldrich) for 24 h with continuous stirring at room temperature using a ratio of acid: powder of 10 cm3: 1 g. In the next step, the obtained material was subjected to sedimentation, washed 5× with distilled water and 5x with ethanol. The washed material was dried overnight at room temperature.
Delaminated 2D sheets of the Ti2NTx were obtained using the protocol previously developed by us for obtaining multilayered 2D Ti3C2Tx and Ti2CTx MXenes . Briefly, it was a two-step process in which non-polar (hexane) and polar (isopropyl alcohol) solvents were used one after another with a ratio of 50 cm3 per 1 g of MXene powder. In the first step, the non-polar solvent hexane was used together with high-energy probe sonication (Vibra-Cell VCX750, 750Watt, 20 kHz, Sonics & Materials Inc.) on the prepared suspension for a total of 0.5 h. Subsequently, the powder was easily decanted, removed, and dried in the air. The next step involved tip sonication in isopropyl alcohol for a total of 15 min. The obtained suspension was centrifuged at 2500 rpm for 2 min. The obtained supernatant was removed, and the solid was dried for 6 h at room temperature and stored in argon at 5 °C for further use.
A scanning electron microscope (SEM-LEO 1530, Zeiss, USA) was employed for the morphology analysis of the Ti2AlN MAX phase, Ti2NTx MXene after etching, as well as the multilayered 2D sheets of Ti2NTx at an accelerating voltage of 5.0 kV. The sample was directly placed on sticky carbon tape and coated with a thin carbon layer. The multilayered 2D structure of Ti2NTx MXene (placed on Cu-C mesh) was also examined in cross-section using a high-resolution transmission electron microscope (HREM, Philips CM 20). The atomic-scale of the Ti2N layers was revealed using Fourier transformation (FFT) together with subsequent reversed Fourier transformation (IFFT). The layered structure was also analyzed quantitatively by an intensity pattern taken perpendicularly to the plane formed by the individual Ti2N layers. This enabled analysis of the spacing between the brightest periods (i.e., d-spacing in nm). The selected area diffraction pattern (SADP) of the electrons was used for indication of the characteristic rings and identification of the measured interplanar distances for each Ti2N monolayer. The obtained results were analyzed for their similarity to the possible lattices of the space groups. The elemental composition of the multilayered 2D sheets of Ti2NTx was checked using an energy-dispersive X-ray spectroscopy (EDX) unit coupled with a transmission electron microscope.
X-ray diffraction (XRD) was employed for the analysis of both the Ti2AlN MAX phase and the resulting Ti2NTx MXene. The phase composition was examined in an X-ray diffractometer (Bruker D8 Advanced). The results were recorded within the 2ϴ angle range from 15 to 120° using CuKα radiation (λ = 1.54 Å). The measuring step was Δ2θ = 0.025° and the computation time was 3 s/step. The transmittance spectrum of the multilayered 2D sheets of Ti2NTx was obtained using a UV–VIS spectrometer (Evolution 220 from Thermo Scientific) equipped with an integrating sphere (0.3 s of integration), in a range from 220 to 1100 nm, with a resolution of 1 nm, and a scanning speed of 200 nm min−1. The measurements were accompanied by a Tyndall test employing a sample suspended in double-distilled (DDW) water using red laser light.
Qualitative analysis of the presence of oxygen-containing functional groups on the surface of multilayered 2D sheets of Ti2NTx was carried out using diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS-FTIR, Nicolet iS5 Spectrometer from Thermo Scientific). The sample was mixed with dried KBr at a concentration of 2.5 wt %. Each spectrum was recorded with 60 scans, and OMNIC software from Thermo Fisher was used to analyze the obtained data.
The stability of the multilayered 2D sheets of Ti2NTx was analyzed by dynamic light scattering (DLS) and zeta potential measurements (with the Smoluchowski model) using a NANO ZS ZEN 3500 analyzer (Malvern) equipped with a green laser light scattering detector, operating at an angle of 173º. All investigations were carried out in a double distilled water (DDW) suspension of multilayered 2D sheets of Ti2NTx MXene phase at a concentration of 3.3∙10−5 g ml−1, at 25 °C. For the assessment of the stability of 2D sheets of Ti2NTx in the most widely used in vitro cell culture media, the DDW was changed for the appropriate amounts of the culture media and incubated for t = 0 and t = 24 h. The analyzed media were Dulbecco’s Modified Eagle Media (DMEM), Roswell Park Memorial Institute (RPMI), and Minimum Essential Medium Eagle (MEME). The obtained results were presented as the mean value of zeta potential and hydrodynamic diameter, based on three independent experiments, respectively, with standard deviation.
Calculations were performed using spin polarized density functional theory (DFT) [34, 35] with the PBE exchange–correlation functional  as implemented in the VASP package [37, 38]. The electron–ion interaction was modeled by using projector augmented wave (PAW) pseudopotentials [39, 40], and a plane wave basis was set with a cutoff energy of 500 eV. The weak van der Waals forces were included by using the Grimme corrected approach (DFT + D2) . The Brillouin zone integration of a k-point mesh of (24 × 24 × 2) in the Monkhorst–Pack sampling scheme  was used for the lateral (1 × 1) supercell and at least 17 Å of vacuum thickness. For the strongly localized 3d states of titanium, the DFT + U approach introduced by Dudarev et al.  was used. The effective on-site Coulomb and exchange parameters for the 3d states were set to U = 5 eV and J = 1 eV, respectively. All the structures were fully structurally optimized until the force on each atom and each component of the stress matrix was below 0.005 eV/Å and 2 kbar, respectively.
Analysis of cytotoxicity in vitro
The biocompatibility of 2D Ti2NTx MXene was evaluated towards human skin malignant melanoma cells (A375, ATCC), human immortalized keratinocytes (HaCaT, ThermoFisher), human breast cancer cells (MCF-7, ATCC), and normal human mammary epithelial cells (MCF-10A, ATCC). The cells’ viabilities were monitored after the incubation of cultures with increasing concentrations (0–500 mg L−1) of the studied material. The A375, HaCaT, and MCF-7 cell lines were cultured using complete Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) of penicillin and streptomycin, and 1% (v/v) of l-glutamine. The MCF-10A line was grown in F-12 DMEM containing 5% (v/v) horse serum, 10 μg mL−1 human insulin, 10 ng mL−1 epithelial growth factor, and 5 μg mL−1 hydrocortisone (Sigma-Aldrich). The cells were cultured under 5% CO2 at 37 °C and 95% humidity.
The tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied as a tool for studying the influence of the tested material on cell cultures. All cell lines were seeded at a density of 1 × 104 cells per well. Subsequently, the cells were incubated to assure their adhesion to the surface. The supernatant was then replaced with a series of dilutions containing various concentrations of MXene (100 μL per well), and the resulting mixtures were incubated for 24 h. Controls were run in the absence of MXene (the cell culture was incubated with the appropriate fresh medium only). Each experiment was conducted at least three times independently. After exposure to 2D Ti2NTx, the cells were washed three times with phosphate-buffered saline (PBS, Sigma-Aldrich) and treated with MTT (Sigma-Aldrich) solution (0.5 mg mL−1 in PBS; 100 μL per well). The cells were incubated with MTT for the next 4 h and protected from light. The supernatant was then carefully removed, and the formed violet formazan crystals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich; 100 μL per well). The absorbance was measured at 570 nm, and the results were expressed as the percentage viability comparing to the controls.
Analysis of the mechanism of action
In order to verify if the potential cytotoxicity of Ti2NTx is the result of oxidative stress, the level of intracellular reactive oxygen species (ROS) was measured in the presence of the non-specific fluorescent dye, 2′,7′–dichlorofluorescein diacetate (DCF–DA, Sigma–Aldrich). After 24 h of exposure to MXene, the cells were washed three times with PBS and subsequently maintained in DCF–DA solution (20 μM in PBS; 100 μL per well) for 30 min in the dark. The supernatant was then replaced with 0.1% Triton-X solution (Sigma-Aldrich) (v/v) (100 μL per well). Cells were incubated with a solution of surfactant until lysis was observed (1 h). The fluorescence intensity was measured at 530 nm (excitation wavelength: 495 nm), and the results were expressed as a percentage of ROS compared to the controls.
Analysis of the cellular uptake of 2D Ti2NTx sheets was performed using a scanning electron microscope (SEM) combined with an energy-selective backscattered (ESB) detector for the imaging of their presence inside or outside the cells. The cells derived from skin were seeded on the surface of Muscovite Mica rings (V1, Science Service) suitable for SEM observations and cultured under physiological conditions until cellular attachment was observed. Subsequently, the culture media were replaced with MXene suspensions in concentrations of either 62.5 μg mL−1 or 500 μg mL−1. Controls were run in the absence of MXene (the cell culture was incubated with fresh DMEM only). After 24 h of exposure to the tested nanomaterials, the cells were fixed in the presence of 3% (v/v) glutaraldehyde (Sigma-Aldrich) in PBS for 4 h at 0 °C. The cells were then washed three times with PBS (3 × 10 min) and dehydrated using increasing concentrations of ethyl alcohol (Sigma-Aldrich), 50%–100% (v/v), for 5 min at each concentration. The samples were next coated with a thin carbon layer and were observed by SEM (LEO 1530, Zeiss, USA). The SEM observations were conducted at 2.0 kV, while ESB imaging was conducted at 12.0 kV.
Results and discussion
Results of characterization
Theoretical calculations results
It has been theoretically shown that functionalization of MXene monolayers stabilizes them with respect to their pristine Tin+1NnTx counterparts. In particular, oxygen functionalization exhibits the lowest cohesive energy compared to –F or –OH functionalized groups . It is worth noting that stable structures after functionalization were also predicted theoretically for many other materials, e.g. carbon-like systems [45, 46].
Structural parameters of n-layers of Ti2NO2. Methods of computation: GGA + U(U = 5 eV, J = 1 eV), Grimme correction (DFT + D2)
Structural parameters of n-layers of Ti2NO2. Methods of computation: GGA + U(U = 5 eV, J = 1 eV), Grimme correction (DFT + D2)
Structural parameters of n-layered Ti2NO2
Our results showed that the stabilization of the TiN2O2 can be enhanced by adding layers to the structure, and the interlayer binding energy can reach a limit of − 16.6 meV per atom for an infinite number of layers. In other words, the weak van der Waals forces stabilize TiN2O2 material containing a few layers. This result indicates that it is experimentally easier to obtain thicker samples than monolayer ones.
Results of in vitro studies, mode of action analysis, and stability features
Stability analysis results obtained for multilayered 2D sheets of Ti2NTx MXene suspended in double distilled water (DDW) water as well as cultivation media applied for the in vitro studies, for incubation time t = 0 and t = 24 h
Zeta potential after t = 0
− 21.3 ± 0.1
− 7.6 ± 0,3
− 8.4 ± 0.6
− 8.3 ± 0.3
− 8.0 ± 0.6
Zeta potential after t = 24
− 21.4 ± 0.2
− 8.0 ± 0.2
− 8.6 ± 1.0
− 7.9 ± 0,1
− 9.1 ± 0.8
after t = 0
564 ± 24
611 ± 21
542 ± 12
595 ± 16
518 ± 13
after t = 24
383 ± 44
293 ± 11
277 ± 54
325 ± 86
177 ± 25
Theoretical calculations show the possibility of bypassing the problem of instability of single-layered 2D Ti2NTx MXene i.e., higher likelihood of forming multilayered Ti2NTx structures during the preparation process in comparison to single-layered structures,
2D multilayered sheets of Ti2NTx MXene can be thus successfully obtained using classic etching and delamination,
The multilayered 2D sheets of Ti2NTx show higher toxicity towards cancerous cell lines (MCF-7and A365) in comparison to normal ones,
The decrease in the cells’ viability is dose-dependent,
2D Ti2NTx is not toxic towards non-malignant cells (MCF-10A and HaCaT),
The identified mechanisms of toxicity are the generation of reactive oxygen species as well as the 2D sheets’ internalization.
The results of the present study provide the principal knowledge to date regarding the toxicity and potential anticancer properties of the delaminated 2D multilayered sheets of Ti2N MXene. We also reveal that the 2D Ti2N in its multilayered form exhibits fair stability, so can be applied in in vitro studies. These results show promise for its future application in biotechnology and nanomedicine.
Access to computing facilities of PL-Grid Polish Infrastructure for Supporting Computational Science in the European Research Space and of the Interdisciplinary Center of Modeling (ICM), University of Warsaw is gratefully acknowledged.
AS analysed cytotoxicity of the 2D Ti2NTx as well as carried out samples preparation for SEM; AR-W characterized 2D Ti2N using FTIR and UV–VIS; SP performed analysis of zeta potential and DLS method; TW carried out acidic etching of the Ti2NTx MXene; MB and MP carried out theoretical calculations; MC supervised MTT and ROS analyses; WZ supervised MXene synthesis; LC prepared the starting MAX phase; DM performed XRD study; AO revised the prepared manuscript. JAM supervised theoretical calculations; AMJ designed the experiment and supervised the whole research as a project leader, collected and analyzed the obtained results as well as coordinated the preparation of the manuscript. All authors read and approved the final manuscript.
The study was accomplished thanks to the funds allotted by the National Science Centre on the basis of the decision no. DEC-2017/26/E/ST8/01073, within the framework of the research project ‘SONATA BIS 7’ No. UMO-2017/26/E/ST8/01073. M.B. is funded by the National Science Centre Grant No. UMO-2016/23/D/ST3/03446.
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Authors declare consent for publication.
The authors declare that they have no competing interests.
- 5.Yin J, Wu B, Wang Y, Li Z, Yao Y, Jiang Y, Ding Y, Xu F, Zhang P. Novel elastic, lattice dynamics and thermodynamic properties of metallic single-layer transition metal phosphides: 2H-M 2P (Mo2P, W2P, Nb2P and Ta2P). J Phys. 2018;30(13):135701. https://doi.org/10.1088/1361-648x/aaaf3c.CrossRefGoogle Scholar
- 16.Jastrzębska AM, Karwowska E, Wojciechowski T, Ziemkowska W, Rozmysłowska A, Chlubny L, Olszyna A. The atomic structure of Ti2C and Ti3C2 MXenes is responsible for their antibacterial activity toward E. coli bacteria. J Mater Eng Perform. 2018. https://doi.org/10.1007/s11665-018-3223-z.CrossRefGoogle Scholar
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