For MXene preparation, aluminum titanium carbide powder (Ti₃AlC₂ or MAX phase) was purchased from Carbon-Ukraine ltd. Hydrofluoric acid (HF, 48%) was supplied by Merck Schuchardt OHG, Germany. Dimethyl sulfoxide (DMSO) was obtained from Honeywell Riedel-de Haën®, Germany.

MXene was synthesized by following the protocol from Dr. Yury Gogotsi (Drexel University) who produced MXene for the first time (Alhabeb et al., 2017). In brief, 10 ml of 48% hydrofluoric acid was added to 1 g of Ti₃AlC₂ MAX phase with overnight stirring to allow for chemical etching of aluminum (Al) from Ti₃AlC₂ MAX phase. This was followed by water washes until a safe pH was achieved (pH = 5–6). After that, MXene was intercalated with DMSO and then delaminated using a probe sonicator with argon gas flowing, followed by 1-h centrifugation at 4°C.

The produced MXene sheets were characterized by X-Ray diffraction (XRD) to reveal the crystal structure using PAN analytical X-ray diffractometer. XRD was performed at 45 kV and 20 mA. The results were acquired from 2θ = 5° to 100°. Surface morphology and elemental analysis were assessed using NOVA NANOSEM 450 (N-SEM) and energy dispersive spectrometer (EDS), respectively. A voltage between 500 V and 30 kV was applied with a 10 mm distance between the sample and electron source, which is satisfactory to obtain good images. Talos transmission electron microscope (TEM) was used to further confirm the morphology of the produced MXene sheets. For image acquisition, the microscope was operated at 200 kV. A very thin layer of the sample was placed in a carbon-coated grid and then positioned on an electron beam produced by the electron gun. ZetaSIZER NANO-Malvern hosted at CAM was used to assess the surface charge of MXene. The device can find the zeta potential by using electrophoretic light scattering. MXene absorbance and the refractive index were collected from the literature of Berdiyorov (2016) and Li et al. (2017). MXene solution was diluted to 5 µg/ml before obtaining the measurements, and it was placed in a disposable folded capillary cell to be processed by the device.

To assess the light to the heat conversion efficiency of the produced nanosheets, FLIR C3 thermal camera was used to assess the temperature of MXene samples upon laser irradiation. MXene samples were exposed to laser at different power densities (PD = 1, 2, 3, and 5 W/cm²) for 5 min.

In this study, we used MDA-231 human breast cancer cells, from ATCC® (ATCC® HTB-26™). Cells were cultured in a complete media consisting of RPMI 1640 media (61870143-Thermofisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin according to the supplier’s protocol.

Following the harvesting of 80% confluent cells in a T25 culture flask, they are washed twice in phosphate buffer saline (PBS) followed by trypsinization for 5 min using 0.25% trypsin. Then a complete cell media and antifungal solution were added to the trypsinized cells to stop the action of trypsin, which was followed by 5 min centrifugation at 1,000 rpm. Thereafter, the supernatant was removed, and 1 ml of complete media is added to the cell pellet and mixed thoroughly. A final density of 10,000 cells (375 cell/cm²) in complete media was seeded in 40-mm-diameter circular coverslips placed inside 50-mm-diameter Petri dishes, which were then incubated in a cell incubator at optimum conditions (37°C, 5% Co₂, and 95% air).

A parallel plate flow chamber (PPFC) was used to create dynamic conditions for cultured cells. Here, we used the FCS2 system from Bioptechs. A microperfusion peristaltic pump was used to generate flow over cultured cells. The flow rate was calculated using the HagenPoiseuille equation for a Newtonian fluid. A shear rate of 0.1 Dyn/cm² was used to model the interstitial flow fluid shear stress (FSS) over cultured cells. The flow of interstitial fluid was shown to induce FSS to the cancer cells within the localized tumor (Yao et al., 2012) with a shear rate of 0.1 (Dyn/cm²) (Kang et al., 2016). After pump calibration and speed calculation, the pump was connected to Tygon tubing connecting the pump to the flow chamber in a closed circuit (Figure 1) (Shurbaji et al., 2020b). MXene was sonicated and then diluted in cell media to the desired concentration. 10,000 cells were seeded in a 40-mm-diameter circular coverslip which was then assembled in the chamber as in Figure 1B. After that, the whole setup was incubated in a cell incubator under optimum conditions for the desired incubation time (4 or 8 h). We tested two MXene concentrations of 100 and 200 µg/ml as based on previous work in our laboratory, 50 and below did not lead to internalization, and above 200 caused toxicity.

VENUS series high stability benchtop laser was used to expose the cells to laser at a wavelength of 808 nm with different PDs (1, 3, or 5 W/cm²) for different durations (5, 10, or 15 min).

After exposing the cells to MXene and laser, cell viability was assessed using an live/dead stain from ThermoFisher (Cat. No L3224), which includes calcein and ethidium homodimer-1. In brief, 5 μl calcein and 5 μl ethidium homodimer-1 were mixed with 10 ml serum-free media to make the stain at a final working concentration of 1 μM. The cells were incubated with the stain for 10 min at room temperature followed by fluorescent imaging using Olympus microscope IX73. Live images were taken by the GFP green filter, whereas dead images were taken by the C3Y red filter. Ten representative images were taken for live and dead cells at ×10 magnification. The images were then merged in ImageJ, and the live and dead cells were quantified to determine the viability percentage using the following formula:

Viability percentage = (Number of live cells)/(Total number of cells)×100%

MXene uptake by cells was assessed by the following methods: Confocal microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive spectrometer (EDS).

Zeiss confocal LSM microscope equipped with the ARIES imaging program was accessed from the laboratory in Weill Cornell Medical College in Qatar. The cells embedded on the slides were stained with the cytoplasmic marker (Calcein AM) and the nuclear marker (DAPI, D130 Thermofisher scientific). Then the cells were fixed in 4% paraformaldehyde for 15 min, washed twice in PBS, mounted, and coverslipped. The stained samples were visualized at ×63 magnification, and 2D and stacked images were obtained for the cytoplasm (Calcein AM, green channel), nucleus (DAPI, blue channel), and nanoparticles (phase-contrast). Images were analyzed using ImageJ software to produce 3D projections, 3D volumes, and sliced 3D volumes.

The following protocol was used to prepare the samples for TEM imaging. Samples were fixed for one hour in a mixture of 2% paraformaldehyde and 2% glutaraldehyde (G5882-100, Sigma) followed by overnight fixation in 2% glutaraldehyde at 4°C. Samples were then washed twice in 0.1 M phosphate buffer and embedded in 2% agarose followed by 15 min centrifugation at 3,000 rpm to solidify. The agarose pellet was trimmed to <0.5 mm and post-fixed in 2% osmium tetroxide for one hour followed by 3–5 washes in distilled water. In contrast, samples were incubated in 2% uranyl acetate (22,400–4, EMS) for 30 min and then washed 3–5 times in distilled water. This was followed by serial dehydration in 30, 50, 70, and 90% acetone (for 15 min each) and three changes in 100% acetone (for 30 min each). For embedding, a medium formulation of Agar 100 resin (AGR1031, Agar Scientific) was prepared according to the manufacturer’s protocol. Samples were gradually infiltrated in a 2:1 mix of acetone: resin, then in 1:1 mix and 1:2 mix of acetone: resin (for 30 min each) followed by overnight infiltration in 100% resin. This was then replaced with a fresh 100% resin for five hours infiltration before placing the samples in embedding molds that were filled carefully with a fresh 100% resin and cured in an oven at 60°C for 1–2 days. Ultra-thin sections (approximately 100 nm) were cut using ultra-microtome (Leica EM UC7), and sections were mounted on 200 mesh copper grids and coated in carbon before imaging in a transmission electron microscope (Talos F200C, ThermoFisher Scientific).

The cells embedded on slides were fixed with 2% glutaraldehyde for 24 h at 4°C. The slides were then washed twice in PBS followed by a series of dehydration steps in ethanol (50, 70, 80, 90, 95, and 100%) and allowed to air-dry. All slides were coated with gold (Au) to be conductive and allow for SEM and EDS analysis.

Changes in cytoskeletal structure have been shown in some studies to affect the cellular uptake of nanomaterials (Panariti et al., 2012). To reveal if the cytoskeletal structure is altered under fluid flow, cells were grown at 50% confluency and were either cultured in static or in dynamic conditions then fixed with 4% paraformaldehyde for 15 min. The samples were permeabilized using Triton X-100 solution, and then bovine serum albumin was added to block the unspecific bindings. The samples were then incubated overnight at 4°C with Alexa-488 actin labeled phalloidin antibody (A12379- Thermofisher scientific), which binds specifically to actin fibers in the cellular cytoskeleton. Following that, samples were mounted and coverslipped and were imaged using Olympus fluorescent microscope at ×60 magnification using the GFP filter.

ANOVA test was performed to find statistical significance followed by the Tukey’s test to compare the significance between the sample means (n ≥ 3).

SEM images are represented in Figure 2. The MAX phase has a closely packed layered structure, which spread out after the removal of Al resembling an accordion (Tang et al., 2018) (Figure 2B). Delaminated single MXene sheets are represented in Figure 2C. Using SEM measurements, the average sheet size was found to be 190 ± 35 nm. The TEM image in Figure 2B further confirms the presence of 2D sheets as well as the sheet size. XRD was employed for the MAX phase, etched MAX, and MXene, and the results are represented in Figure 2C. Successful etching can be observed by the loss of Al peak from MAX pre-cursor at 2θ ∼ 39° in both etched MAX and MXene. Comparing the MAX phase to etched MAX and MXene, it can be observed that the two peaks (002) and (004) become wider and less intense with a slight shift to lower angles. Diffraction peaks from 2θ = 35° to 45° were lost in MXene XRD which indicates delamination and loss of stacking. Furthermore, MXene surface charged was assessed and found to be −21.3 ± 0.643.

The thermal camera was used to assess the rise in MXene temperature after laser exposure. Here, MXene was dispersed in cell media and sonicated for 10 min at room temperature. After that, 100 ul of the solution was placed on a 96-well plate for laser exposure. Different laser power densities were applied for two MXene concentrations (100 and 200 ug/ml). For all the cases, it took the MXene one min to reach the maximum temperature which stays constant for the rest of the 5 min. After removing the incident laser light, it took ∼4 min for the solution to reach 37°C.

The results in Figure 3 show a trend, in which increasing the power densities in MXene concentrations results in increasing the temperature. However, similar temperature values were obtained for power densities of 3 and 5 W/cm², which might be because the temperature has reached the maximum value at ∼99°C.

Confocal microscopy analysis was performed as a preliminary test to confirm that the material can be internalized by cancer cells, however MXene quantification was not possible as MXene was not fluorescently labeled. Figure 4 represents MDA-231 cells treated with 100 µg/ml MXene under dynamic and static conditions for 4 h. The 3D crosssections show a few material uptakes without any difference between the two conditions.

To visualize the material clearly inside cancer cells, TEM was performed. Figure 5 represents different TEM images for cells treated under different conditions. For the static and dynamic conditions, cells were treated with 100 or 200 µg/ml MXene and were exposed for four or 8 h. Material uptake was considered to occur if the material is attached to the cell’s surface (association) or present inside the cell (internalization). As shown in Figure 5, few MXene particles were observed inside the cells in all conditions (indicated as red circle), and a slightly higher number of particles are presented when increasing the concentration. Nevertheless, the duration of exposure did not show any significant difference in MXene cellular uptake. Overall, the material uptake under all conditions is limited with only slight differences between all groups. Figure 6 represents EDS results and shows the weight percentage of Ti inside MDA-231 cells in eight different conditions (as in TEM). The results show a non-significant difference between all groups. However, for the dynamic groups treated for 8 h duration, the uptake was slightly higher than the other groups, though the difference was not significant.

Cell viability after laser exposure was also assessed under different conditions to find out if the heat generated due to material internalization was sufficient to induce cell death. Figure 7 shows bar charts for the viability of cells treated with 100 µg/ml MXene under fluid flow and then exposed to laser at different power densities (1, 3, and 5 W/cm²) for different durations (5, 10, and 15 min). The viabilities for all the studied groups were similar to that of control groups, which indicates no significant effect of the applied power densities and durations on cells viability.

The incubation time might affect the material and cell interaction, as there will be more time for the particles to be internalized by the cells. Safi et al. showed that the cellular uptake of quantum dots was directly proportional to increasing the incubation time (Safi et al., 2016). To test that in our study, 200 µg/ml MXene was incubated with the cells under fluid flow for 8 h (doubling the incubation time) and then exposed to laser at PD = 5 for 15 min. Figure 8A shows no difference in cell viability in control groups compared to the treated groups.

The concentration of nanomaterials plays a role in material uptake by cells, i.e., changing the concentration will influence the way that the cells uptake the materials. As the concentration increases, there is a higher tendency that the material will form aggregates. These aggregates may affect the size of the material; thus, the uptake route might be altered (Mustafa, 2011). Figure 8B represents a bar chart for cells treated with 200 µg/ml MXene for 4 h and then exposed to laser at PD = 5 for 15 min. The results indicate that doubling the concentration of MXene did not affect cell viability, i.e., no cell death was induced.

In this study, we are examining the MXene uptake by cancer cells under dynamic culture and static culture. Previous studies reported that fluid flow can increase particle uptake due to the formation of cytoskeletal stress fibers and membrane ruffles (Ispanixtlahuatl-meráz et al., 2017). To examine if cells under dynamic conditions would have stress fibers, we stained actin cytoskeletal for the cells under static and dynamic conditions. Consistent with previous studies, the stress fibers for the cells exposed to flow were clear. Interestingly, MXene uptake showed no difference under fluid flow in our experiments (Figure 9).

In an attempt to quantify the concentration of internalized MXene, we first cultured cells with 200 µg/ml MXene for 4 h under static conditions, then exposed them to a photothermal laser for 15 min, and measured the temperature of cells with the thermal camera. Cell temperature for PD 1 W/cm² was 33.9°C, for PD 3 W/cm² was 38.7°C, and for PD 5 W/cm² was 42.8°C. To identify what concentrations of MXene these temperatures are matching, we exposed different concentrations of MXene alone at different PDs and match those with temperature results obtained from culture experiments. Using the thermal camera, we tried to find the maximum temperature that can be achieved when treating the cells with 200 µg/ml MXene for 15 min at different power densities. The MXene concentration that gives approximately the same temperatures for MXene/cell culture and MXene alone measurements was 3 µg/ml, suggesting internalized MXene concentration is about 3 µg/ml (Figures 10, 11).