Engineered Ti₃C₂(O,Cl) MXenes with dual functionalization: a new Frontier in targeted head and neck squamous cell carcinoma and breast adenocarcinoma

Abstract

Ti₃C₂T_x MXenes have attracted significant attention in the realm of anticancer therapeutics owing to their remarkable properties, including cyto-compatibility and targeted drug delivery capabilities. In this study, Ti₃C₂ was intentionally modified with both chlorine and oxygen surface groups, as each of these functional groups have individually demonstrated promising anticancer properties. Our aim was to combine them in a single compound to explore how this dual-functionalized material might perform in a therapeutic context. This study synthesizes Ti₃C₂(O,Cl) MXenes using a novel electrochemical etching technique that allows for precise tailoring of the surface terminations with O and Cl groups. The synthesised Ti₃C₂(O,Cl) has biological activity in two cancerous (FaDu and MCF-7) and two normal (H9C2 and HEK-293) cell lines. The results of cytotoxicity data showed that the observed toxic effects were higher against cancerous cells (∼91%) than normal cells (∼40%). The mechanisms of potential toxicity were also elucidated. The synthesized Ti₃C₂(O,Cl) MXene has an effect on oxidative stress, resulting in an increase of more than 91.44% in reactive oxygen species (ROS) production in malignant cells. The results of this study provide major insights to date into the biological activity of Ti₃C₂(O,Cl) MXenes and develop their application in anticancer treatments.

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1. Introduction

Cancer (karkinos) has a profound and far-reaching impact on human life, affecting individuals, families, and society worldwide. Countless chronic novel instances of cancer are discovered each year, and the disease affects millions of lives globally, resulting in a significant cause of illness and mortality. With 8.97 million deaths annually, cancer is currently the second leading cause of death worldwide.¹ It is projected to overtake all other causes of death by 2060 (about 18.63 million deaths annually).² Multiple methodologies are available for the management of carcinoma, including interventions such as chemotherapy, radiotherapy, immunotherapy, hormone therapy, and targeted medication therapy.³ Nonetheless, each technique has limitations and detrimental effects. Therefore, it is essential to identify a reliable and effective method that can prolong life expectancy while improving health. A versatile framework of biocompatible and biodegradable systems is made possible by nanomedicine, which can be employed to deliver typical chemotherapeutic drugs in vivo, boost their bioavailability and concentration amid tumor tissue, and improve their release profile. Among them, a few nanoparticles including gold nanoparticles,⁴ molybdenum oxide (MoO_x),⁵ silver nanoparticles,⁶ copper selenide (CuSe),⁷ tungsten disulfide (WS₂),⁸ self-assembled organic polyamic materials,⁹ dyes,¹⁰etc. have demonstrated excellent cytotoxicity towards cancer cell lines.

In recent years, MXenes, a family of two-dimensional (2D) nanomaterials that were discovered in 2011, have attracted significant attention due to their intriguing anticancer properties. MXenes possess numerous functional groups that enable them to facilitate drug transport, ensuring targeted delivery with increased toxicity towards cancer cells while being cytocompatible.¹¹ MXenes are synthesised by etching the weakly bonded A-group elements from bulk ternary carbides/nitrides of MAX phases. It has a general formula of M_n+1X_nT_x, where M is an early transition metal (such as Sc, Ti, Zr, Hf, Zr, etc.), A is a group (12–16) element of the periodic table, X represents carbon and/or nitrogen, T_x represents surface terminations (such as –F, –Cl, –OH, –O, etc.) and n = 1–3.^12–14 MXene production originated in 2011 using hydrofluoric acid (HF) as its principal etchant.¹⁵ It was in 2015 that it transitioned into a 2D material category. Several alternative methods of synthesis have been developed to address the potential hazards associated with using HF. The methods used include in situ HF etching,¹⁶ molten salt methods,¹⁷ alkali-based procedures,¹⁸ minimally intensive layer delamination (MILD),¹⁹ hydrothermal techniques,²⁰ ball milling approaches²¹ and electrochemical etching.²² However, there exist several limitations associated with the alternative synthesis processes, such as the extended etching times and the limited efficiency in tuning the functional groups of the synthesised Ti₃C₂T_x. These challenges underscore the significance of safer, more effective, and regulated etching strategies in the synthesis of Ti₃C₂T_x MXenes. It is worth mentioning that electrochemical etching has gained attention as a safer, simpler, and more dependable alternative to HF-based techniques for the generation of Ti₃C₂T_x MXenes, offering the potential to produce large quantities of high-quality materials and tune the functional groups. The electrochemical method has emerged as a fluoride free, environmentally sustainable method for the synthesis of MXenes with highly tunable and biocompatible surface terminations (–O, –Cl, and –OH).

The electrochemical etching framework utilizes an electric current that helps the ions to move from the electrolyte into the bulk materials, thus weakening the van der Waals forces that bind the bulk layers together. The etching process effectiveness is greatly influenced by the combination of electrolyte composition, operating voltage, and precursor selection.²³ By altering electrochemical factors like current, voltage, and the electrolyte, it is possible to precisely control the MXene surface terminations. This approach provides a precise method for expelling challenging ‘A-phase’ materials with minimal etching time. It also offers the ability to control functionalization options such as –O, –OH, –Cl, or –F.

Firstly, we synthesised Ti₂CT_x utilizing a milder electrolyte and lower potentials such as HCl at +0.6 V to etch the A layer from the MAX phase.²³ Researchers found that the MXenes underwent complete conversion into carbon derived carbon (CDC) after being etched for 5 days. During the process, they also observed white layer depositions on the platinum counter electrode and that the surface terminations attached to Ti₂C are –O, –C, and –Cl. Another study conducted electrochemical etching of Ti₃C₂T_x MXenes utilizing a binary aqueous electrolyte (NH₄Cl + TMAOH) at a pH greater than 9.²⁴ In the study, etching was carried out at higher potentials of approximately +5 V. It was found that the prepared samples are terminated with –OH as T_x, and the overall etching yield was initially low at around 40%. However, after implementing recycling methods, the yield was increased to 60%. Furthermore, scientists utilized a mixture of LiCl and KCl as etchants through a molten salt procedure to prepare the initial MAX Ti₂AlC phase.²⁵ They then employed a one pot in situ E-etching method at 1.3 V for extended periods of 24 hours, conducted at a high temperature of 500 °C. They found that Ti₂CT_x have surface terminations with –Cl, but after washing with ammonium persulfate, the T_x changed to the –O functional group.²⁵ While electrochemical etching techniques have been employed to synthesize Ti₃C₂, these methods have produced functional groups without precise tuning.

In anticancer applications, the generation of reactive oxygen species (ROS) relies on the idea that increasing ROS levels above a lethal threshold selectively eliminates cancer cells. The –O functional groups attached to Ti₃C₂ MXenes act as carriers for drug delivery, enhancing ROS production towards cancer cells. Additionally, the chlorinated compounds attached to Ti₃C₂ MXenes exhibit a cytotoxic effect, suggesting resilient anticancer activity. A recent study tested the bioavailability of Ti₃C₂T_x on two normal (MRC-5 and HaCaT) and two cancerous (A549 and A375) cell lines.²⁶ It was observed that the prepared Ti₃C₂ MXenes had surface terminations such as –OH, =O, and C=O–OH groups, showing higher cytotoxic effects against cancerous cells compared to normal ones. Additionally, the study noted that MXene suspensions were non-toxic only in HaCaT cells, with cell viability remaining above 70% and the lowest levels of ROS production. In contrast, the highest ROS levels (approximately 89%) were observed in A375 cells.

In 2020, researchers further tuned Ti₃C₂ into Ti₃O₂ and assessed the cytotoxicity in vitro against human epithelial breast (MCF-10A), breast cancer (MCF7), human immortalized keratinocytes (HaCaT), and human malignant melanoma (A375) cell lines.²⁷ They synthesized three types of MXene samples: (a) Ti₃C₂, (b) sonicated Ti₃C₂, and (c) thermally oxidized Ti₃C₂, mostly with –O and –OH terminations. The results showed that all three MXene samples were toxic towards the cell lines, with greater toxicity towards melanoma cells. The toxicity of all three increased with the concentration of MXenes. At a concentration of 125 mg L⁻¹, the sonicated and oxidized MXenes were lethal to A375 cells while being relatively less toxic to HaCaT cells. In 2020, another study reported the effect of Chlorin e6 (Ce6) (family of chlorine group) on photodynamic delivery for cancer cell killing and intracellular drug delivery.²⁸ In this study, chlorin e6 was fabricated with gas vesicles (GVs) to study the cytotoxicity of cancerous cells such as a human hypopharyngeal cancer cell line (FaDu-GFP) and the MCF-7 cell line. Ce6 molecules are excited by light from singlets to triplet states improving the energy transfer of O₂ to generate singlet oxygen, which causes oxidative damage leading to cell death. Ce6-GV when compared with free Ce6-based nanoparticles was 200-fold more effective. Furthermore, the cytotoxic effect of chlorine dioxide (an effective disinfectant of chlorine) was studied on small-cell lung cancer (SCLC) cells and human umbilical vein endothelial cells (HUVECs).²⁹ The study reported that apoptotic cell death ranged from 37% to 49% for the DMS114 cell line, while in the HUVEC control it ranged from 19% to 25%.

Ti₃C₂ synthesised through various routes has been explored as a potential anticancer agent.³⁰ However, these studies did not prioritize optimization of ROS promoting functional groups on Ti₃C₂ which are vital for maximising anticancer properties. In some cases, Ti₃C₂ has been combined with photothermal methods to improve its therapeutic efficiency.^31,32 The high surface area of Ti₃C₂ has also been leveraged by using its carrier for drug delivery, including encapsulation of doxorubicin,³¹ which is widely used as a chemotherapeutic agent.

In this work, the Ti₃C₂T_x MXenes are electrochemically etched using chronoamperometry techniques or preliminarily intercalated at −0.6 V followed by etching at a potential of +0.6 V to produce Ti₃C₂O and Ti₃C₂Cl. The synthesis process of the Ti₃C₂(Cl,O) MXenes, as outlined in the ESI,† S1, is illustrated schematically in Fig. 1. Subsequently, the prepared MXenes were subjected to characterization. The produced MXenes were subjected to in vitro analysis as detailed in the ESI,† S2. The prepared MXenes exhibit attributes for in vitro cytotoxicity against cancerous and normal cells. Our research also elucidates oxidative stress phenomena as the potential mechanisms of toxicity. The findings of this study offer crucial insights into the toxicity of Ti₃C₂O and Ti₃C₂Cl in relation to both normal and cancerous cell lines. However, the lack of this information has been a significant barrier to advancing research and development in drug delivery systems for anticancer therapies involving MXenes.

Fig. 1 Schematic diagram of electrochemical synthesis of Ti₃C₂T_x MXenes from Ti₃AlC₂ pristine powder.

2. Materials and methods

2.1 Materials used for the preparation of Ti₃C₂T_x MXenes

Ti₃AlC₂ MAX powder was purchased from Nanochemazone, Aritech (India), ethanol was purchased from analytical CS reagent (India), de-ionized water and Nafion binder were purchased from Sigma Aldrich (India), HCl was purchased from Finer (India), and dimethyl sulfoxide (DMSO) was purchased from SRL (India).

2.2 Materials used for anticancer studies

FaDu, MCF-7, HEK-293, and H9C2 cells were purchased from National Center for Cell Science (Pune, India). RPMI-1640 (AL162A), DMEM (AL219A), trypsin (TCL007), and MTT (TC191) were purchased from Himedia (Himedia Laboratories Pvt Ltd, Mumbai, India). FBS (10270-106) was purchased from Gibco (Gibco, Life technology, E.U, South American). Doxorubicin (D1515) and 2′,7′-dichlorofluorescin diacetate (D6883) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, USA).

2.3 Material characterization used for Ti₃C₂T_x MXenes

Powdered X-ray diffraction (PXRD) (Rigaku Ultima-IV, Ultima) (X-ray source – copper, scan rate – 1° min⁻¹ to 5° min⁻¹, 2θ range – 5–90°), X-ray photoelectron spectroscopy (XPS) (Thermo Fisher Scientific Pvt. Ltd UK) (source – aluminium K-alpha, kinetic energy – 5 to 1500 eV), FE-SEM (FEI, Apreo Lovac) (source – copper, magnification – 10× to 30 000×, resolution – up to 2 nm), confocal Raman spectrometry (Lab Ram HR Horiba) (range – 300 to 2500 cm⁻¹), high-resolution transmission electron microscopy (HR-TEM) (Jeol Jem 2100 plus, India) (resolution – 1 nm), and a potentiostat/galvanostat (Metro-Ohm, India) (range −10 to +10 V) were used for material characterisation.

2.4 Statistical methods

Statistical analyses were performed using Graph Pad prism 9 software. One-way ANOVA followed by the Bonferroni post hoc test was used to define statistical significance. p < 0.05 was considered statistically significant.

3 Results and discussion

3.1 Synthesis and characterization of Ti₃C₂ (O,Cl)

In order to ascertain the redox potentials of both the working electrode (Ti₃AlC₂) and the electrolyte, initial cyclic voltammetry was carried out with a potential range of −1.5 V to +1.5 V, as shown in Fig. 2(A). The redox potentials of −1.19 V, −1.32, and −0.44 V were observed in the CV curve and also exhibited a linear increase in current from 0 to +1.5 V, followed by a small peak at −0.4 V during the forward scan and a peak at −1.1 V during the backward scan. The counter electrode used was platinum (Pt), while the Ag/AgCl electrode served as the reference.

Fig. 2 (A) Cyclic voltammetry curve for the Ti₃AlC₂ MAX electrode at a potential window of −1.5 V to +1.5 V, (B) a representation of the chronoamperometry curve for 5 s each at potentials of −0.6 V (intercalation) and +0.6 V (etching), (C) chronoamperometry including both intercalation and etching for 6000 s and (D) XRD analysis for the pristine Ti₃AlC₂ Max phase and Ti₃C₂T_x MXene.

1.23 V indicates reduction processes associated with water splitting, and −0.44 V indicates the reduction of titanium species, so the potential values of −0.6 V for intercalation and +0.6 V for etching were selected for the present study. Specifically, −0.6 V was sufficiently negative to facilitate efficient H⁺ ion intercalation while minimizing significant side reactions, such as water reduction or the disruption of Ti–C bond stability. Conversely, the application of +0.6 V was favourable for effectively etching aluminium from the Ti₃AlC₂ MAX phase without compromising the structural integrity of the resulting Ti₃C₂T_x MXenes. The synthesis of Ti₃C₂T_x MXenes involves the application of chronoamperometry, where a potential of −0.6 V was applied for 5 seconds to introduce an ion into the Ti₃AlC₂ compound. This was followed by applying a potential of +0.6 V for 5 seconds to remove the Al ions from the Ti₃AlC₂ MAX compound, as shown in Fig. 2(B). Then, the electrochemical etching process was carried out for a total duration of 6000 s, as depicted in Fig. 2(C), resulting in the production of Ti₃C₂ (O,Cl) MXenes. We subsequently performed delamination on the resultant MXenes to isolate the unetched Ti₃AlC₂ and Ti₃C₂T_x MXenes.

After synthesis, the Ti₃C₂ MXene was washed, sonicated, and centrifuged and the delaminated material was further characterised. XRD analysis of the synthesised Ti₃C₂ MXene is shown in Fig. 2(D); major peaks of (002), (004), (101), (103), (104), (105), (107), (108), and (109) in the diffraction pattern in the 2 theta range from 5° to 75° are consistent with the previously reported Ti₃C₂ MXene.³³ It was observed that the majority of diffraction peaks’ intensity decreased from the pristine sample to Ti₃C₂T_x samples, and also characteristic (i.e. 104) peaks and several broad and low-intensity peaks in the 2 theta range from 5° to 75° were observed; these characteristics indicate the successful reduction of Ti₃AlC₂ to layers of Ti₃C₂ MXene.³⁴ The shift of the (002) peak observed from 10.627° to 10.36° (±0.02°) suggests that the interlayer distance has increased. The characteristic peaks at 39.86° (104) and 42.83° (105) corresponding to Ti₃AlC₂ show reduced intensities, indicating partial etching of Al layers and successful MXene formation via the electrochemical technique. This reduction in intensity indicates that a substantial portion of the Ti₃AlC₂ parent material has undergone successful conversion into Ti₃C₂T_x MXene. The weight percentage of Ti₃C₂T_x formed through electrochemical etching was 73.7%^34,35 which was calculated through formula weight percentage = 1 − 0.2x + 0.013x², where x is the ratio of intensity of the 39.86° peak to the intensity of the 10.36° peak.³⁶ Hence, XRD confirms the successful etching of Ti₃AlC₂ to form Ti₃C₂(O,Cl) MXene, evidenced by the shift in the (002) peak and reduced intensity of the Ti₃AlC₂ (104) peak. The increased interlayer spacing indicates the incorporation of surface terminations and partial removal of aluminium.

The Ti₃C₂T_x MXenes were subjected to further characterization via SEM to validate the alterations in the structure and morphology, as depicted in Fig. 3(A) and (B) for the pristine Ti₃AlC₂ and synthesized Ti₃C₂T_x MXenes. The observed morphology aligns closely with the existing literature, exhibiting a microstructure reminiscent of an accordion. The disparity in surface features between unetched Ti₃AlC₂ and Ti₃C₂T_x was remarkably pronounced. While the former presents a polished surface, the latter experiences delamination and results in a coarser surface when a minimal potential was applied. The morphological transformation serves as a compelling indicator of the efficacy of the electrochemical etching technique.

Fig. 3 SEM images for (A) the pristine Ti₃AlC₂ MAX phase and (B) Ti₃C₂(O,Cl) MXene; (C) Raman analysis for Ti₃AlC₂ and Ti₃C₂(O,Cl); (D) TEM images for Ti₃C₂(O,Cl) MXene at 100 nm; (E) TEM image of Ti₃C₂(O,Cl) at 5 nm and (F) AFM image of Ti₃C₂(O,Cl) with a height of 5.4 nm.

Furthermore, the Raman analysis of the synthesized Ti₃C₂T_x MXenes, illustrated in Fig. 3(C), showed significant peaks at 312 cm⁻¹, 435 cm⁻¹, and 605 cm⁻¹ for the Ti₃AlC₂ MAX sample. Conversely, the Ti₃C₂T_x sample displayed peaks at 323 cm⁻¹, 431 cm⁻¹, 597 cm⁻¹, and 1583 cm⁻¹. The noted transition in the peak from 312 to 323 cm⁻¹ signifies the replacement of the ‘Al’ element in Ti₃AlC₂ MAX with surface terminal groups. The peak at 323 cm⁻¹ corresponds to the vibrational modes of the oxygen atoms.³⁷ The spectral region located at 435 cm⁻¹ is indicative of in-plane (E_g) vibrations of surface groups that are attached to titanium atoms.³⁸ The region around 597 cm⁻¹ can be attributed to the E_g vibrations of carbon atoms in the Ti₃C₂T_x MXene, which possesses terminal hydroxyl (–OH) groups and represents Ti₃C₂(OH).³⁹ Furthermore, the peak at 1548 cm⁻¹ corresponds to the G bands of carbon groups. The Raman examination revealed the presence of Ti–C, Ti–Cl, and Ti–O bonds in the produced Ti₃C₂T_x MXenes. The surface terminations consisted of Cl⁻, O⁻ and OH⁻ groups, resulting in the formation of Ti₃C₂Cl, Ti₃C₂O and Ti₃C₂(OH) species.

The detailed structural alterations in the Ti₃C₂T_x sample are further revealed by low magnitude high resolution transmission electron microscopy (HRTEM). Fig. 3(D) and (E) show the HRTEM image with high crystallinity of the Ti₃C₂(O,Cl) MXenes. Further analysis of Ti₃C₂(O,Cl) shows an interlayer spacing of ∼0.4 nm representing the (004) plane.⁴⁰ Thus, the demonstrated increase in the interlayer gap enhances the interfacial charge transfer and electrolytic ion accessibility to electroactive regions. Additionally, the TEM results align with the XRD findings. The height profile obtained from the atomic force microscopy (AFM) measurement indicates that the thickness of the Ti₃C₂(O,Cl) MXene flakes was 5.4 nm as shown in Fig. 3F. This value was in agreement with the reports that have been made in the past on few-layer flakes.

Further investigation was carried out on the synthesized MXenes utilizing X-ray photoelectron spectroscopy (XPS) analysis, as illustrated in Fig. 4(A to E). The investigation into the formation of surface terminations (T_x) on Ti₃C₂ MXenes encompassed elemental analyses of titanium, carbon, aluminium, chlorine, and oxygen. The XPS spectra for the Ti peak, as depicted in Fig. 4(A), show Ti-2p bonds with spin orbitals of 2p_1/2 and 2p_2/3. In the analysis of the Ti peak, Ti–C bonds appear at 455.03 eV and 461.20 eV (with ΔB.E of ∼6.17), Ti–O bonds at 459.12 eV and 464.96 eV (with ΔB.E of ∼5.7) and Ti–Cl bonds at 460.06 eV. Thus, the Ti peak indicates bonding with C, Cl, and O, forming Ti–C, Ti–Cl, and Ti–O bonds. Furthermore, it was observed that the Ti peak has no bond formation with Al (i.e. no Ti–Al bond), which confirms the successful etching of Al from the sample surface. From the Ti peaks, the ratio of Ti–Cl/Ti–C was found to be 0.57 and the ratio of Ti–O/Ti–C was 2.9 as shown in Table 1, and thus the –O surface terminations are more compared to –Cl terminations as T_x on Ti₃C₂T_x.

Fig. 4 XPS analysis of Ti₃C₂(O,Cl) MXene for (A) Ti peak fitting, (B) Al peak fitting, (C) Cl peak fitting, (D) O peak fitting and (E) C peak fitting and (F) FTIR analysis for Ti₃C₂(O,Cl) MXene.

Table 1 Toxicity of Ti₃C₂T_x MXenes on different cell lines

Ref.	Cancer cell	Normal cells	ROS (MFI/%)	Cell viability (%)	Effect of –O and –Cl	Dose in mg L^−1 (incubation time)	Effects
26		HaCaT	29	70	—	50–500 (24 h)	No toxicity
		MRC-5	—	—
	A549
	A375		89	>70			Highest ROS
27	MCF7			>70	–O: high anticancer activity	62.5–500 (24 h)	Toxicity increases with the Ti3C2 concentration
	A375			>70
		HaCaT		>70	—
		MCF-10A		>70	—
28	FaDu-GFP	—	500 MFI	∼ <5	–Cl: some anticancer activity	24 h with light exposure	DCF fluorescence recorded using a plate reader.
	MCF-7	—	400 MFI	∼ <10			Mean fluorescence is intensity not reported.
29		HUVEC	—	>75	–Cl: moderate anticancer activity	1–5 (48 h)	Cl has potential for anticancer activity and is less toxic for normal cells. Apoptosis by FACS
	SCLC	—	—	<54		0.1–10 (48 h)
Present work		H9C2		>65	Synergistic effect of Cl and O gives a high anticancer effect	6.25–500 (24 h)	Less toxic than Doxo for normal cell lines
		HEK-293	>16	>65
	FaDu		>91	<30
	MCF7	—	—	>70			More toxic to FaDu and MCF-7

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To further investigate the absence of Al in the prepared sample, the Al-2p peak was fitted as shown in Fig. 5(B). It was observed that Al oxides emerged at 75 eV, forming an Al–O bond, which confirms the removal of Al from the sample surface, resulting in the formation of Ti₃C₂(O,Cl). In addition to the Ti peak and Al peak, we have also investigated the Cl peaks, O peaks, and C peaks, as shown in Fig. 4(C), (D) and (E), respectively, to acquire a greater comprehension regarding the formation of surface termination groups. Likewise, from the peak of O-2s illustrated in Fig. 4(C), it is evident that –O appears as Ti–O, Ti–OH and Al–O at 530.74 eV, 531.86 eV, and 532.89 eV, respectively. From the O peak, it was observed that the ratio of Ti–O/Ti–OH was 0.657, indicating that Ti–OH predominates over the Ti–O bond as represented in Table 2. From the Cl-1s peak illustrated in Fig. 4(D), Cl-2p establishes bonds with Ti as Ti–Cl at 198.73 eV and 200.32 eV (with ΔB.E of approximately 1.6). The XPS analysis indicates that Al has been removed from the surface of the sample, resulting in the formation of surface terminations such as –Cl, –OH, and –O. Consequently, a higher level of –O and –Cl surface terminations as T_x bonded to Ti₃C₂T_x significantly enhances the properties that contribute to the efficacy of anti-cancer activity.

Fig. 5 Cytotoxic effect of MXene on FaDu, MCF-7, H9C2, and HEK-293 cells (A, B, C, and D, respectively) for a time duration of 24 h. Data are shown as mean ± SD (n = 3) of three independent experiments. *** p < 0.001, ** p < 0.01, and * p < 0.05 vs. respective control.

Table 2 XPS analysis for the Ti peak and the O peak

Ti peak
Compound	2p3/2 (eV)	2p1/2 (eV)	FWHM	ΔB.E (eV)	Ratio
Ti–C	455.03	461.20	2.09 and 2.2	6.17
Ti–O	459.59	464.86	2.3 and 2.2	5.7

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O peak
Compound	2s (eV)	Ratio
Ti–O	531.01
Ti–OH	532.84

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Furthermore, Fourier transform infrared spectroscopy (FTIR), as shown in Fig. 4(F), was used to analyse the functional groups on the surface of Ti₃C₂T_x. The peaks observed at 3471 cm⁻¹, 2860–2932 cm⁻¹, 1650 cm⁻¹, 1495 cm⁻¹, 1222–1387 cm⁻¹, 1093 cm⁻¹, 863 cm⁻¹ and 658 cm⁻¹ correspond to the stretching vibrations of O–H, C–H, Ti–OH, Ti–O, C=O, C–H, C–Cl, and Ti–O, respectively, which were in accordance with the findings of prior investigations from XPS and Raman analyses.

3.2 Results of the in vitro cytotoxicity assay

The biological activity of the Ti₃C₂(O,Cl) MXene on two normal (H9C2 and HEK-293) and two cancerous (FaDu and MCF-7) cell lines were determined. The results concerning cell viability, obtained using an MTT assay for 24 h and 48 h, are shown in Fig. 5(A)–(D) and 6(A) and (B). The viability of the cells decreased significantly with increasing concentration of Ti₃C₂(O,Cl) MXene for time points of 24 and 48 h (p < 0.001). The cytotoxicity of the tested Ti₃C₂(O,Cl) MXene material was strongly dependent on the cell line. The highest cytotoxicity was observed in the FaDu cell line followed by MCF-7 as shown in Fig. 5(A) and (B) for 24 h. However, in H9C2, Doxo 20 μM showed more cytotoxicity compared to Ti₃C₂(O,Cl) MXene, Fig. 5(C). Notably, Ti₃C₂(O,Cl) MXene exhibited significantly lower cytotoxic effects on H9C2 and HEK-293 cells compared to FaDu and MCF-7 cell lines over a 24-h time period, as illustrated in Fig. 5(C) and (D) for 24 h. Moreover, the observed cytotoxic effects were higher against cancerous cells in comparison to the normal ones.

Fig. 6 Cytotoxic effect of Ti₃C₂(O,Cl) MXene on FaDu (A) and HEK-293 (B) cells for a time duration of 48 h. Data are shown as mean ± SD (n = 3) of three independent experiments. *** p < 0.001, ** p < 0.01, and * p < 0.05 vs. respective control.

Furthermore, the presence of dual oxygen (O) and chlorine (Cl) functional groups on Ti₃C₂(O,Cl) MXene appears to enhance preferential cytotoxicity toward cancer cells due to differential redox thresholds, likely due to these groups’ capacity to generate localized oxidative stress or disrupt specific cancer cell processes more effectively.⁴¹ Cancer cells, with higher metabolic rates and altered redox balances, may be more susceptible to the reactive oxygen species (ROS) or cellular disruptions triggered by these functional groups,⁴² explaining the observed preference in cytotoxicity towards cancer cells over the normal ones. Moreover, selectivity was not ligand-receptor mediated but arose from cancer cells’ intrinsic susceptibility to oxidative stress. Similarly, a previously reported study also compared the effects of Ti₃C₂T_x nanosheets on two non-malignant cell lines (MRC-5 and HaCaT) and two cancer lines (A549 and A375), observing that the nanomaterial was markedly more toxic to the tumor cells than to healthy cells.²⁶ Moreover, a recent study evaluated the short-term cytotoxicity of several Ti₃C₂T_x MXene variants and their carbide precursors (TiC, Ti₂AlC, and Ti₃AlC₂) and found significantly greater cell death in HeLa cancer cells than in normal human fibroblasts. These selective effects have been linked to elevated reactive oxygen species generation and stronger MXene-membrane interactions in malignant cells.⁴³ A recent study focused on antibacterial applications highlights that Cl-terminated Ti₃C₂ MXenes generate higher ROS compared to other functional groups (e.g., –OH and –F). Combined with oxygen groups, this supports the plausibility of O/Cl synergy in cancer-selective toxicity.⁴⁴ Additionally, surface functionalization of O and –Cl of MXenes enhances their interactions with cancer cell membranes, promoting selective uptake and ROS-mediated cell death.⁴⁵

There could be a metabolic variation among the cells that leads to this outcome. The observed differences in cytotoxicity across cell lines likely result from variations in cellular metabolism, membrane properties, and oxidative stress responses.^41,46 Cancer cells like FaDu and MCF-7 often exhibit higher metabolic activity and increased ROS production,⁴⁷ making them more susceptible to oxidative stress induced by Ti₃C₂(O,Cl) MXene. These cells may also have altered membrane characteristics that enhance the uptake of nanoparticles, leading to increased cytotoxicity. Normal cell lines such as H9C2 and HEK293 are known to possess stronger antioxidant defense systems, including higher expression of enzymes like superoxide dismutase (SOD), catalase, and glutathione peroxidase, which help mitigate ROS-mediated cytotoxicity.^48,49 In contrast, many cancer cells exhibit elevated basal ROS levels due to mitochondrial dysfunction and oncogenic signaling, making them more susceptible to additional oxidative stress-induced damage.⁴¹ Eukaryotic cells tightly regulate ROS production and scavenging to maintain redox homeostasis. Cancer cells, however, frequently exhibit dysregulated ROS metabolism, which supports both survival and progression under oxidative stress.^50,51 MXenes, due to their surface chemistry, can exacerbate oxidative conditions selectively in tumor cells.^52,53 The increased cytotoxicity observed in FaDu cells after 48 hours may thus reflect a time-dependent accumulation of ROS that overwhelms the already stressed antioxidant systems in cancer cells. It is now recognized that ROS mediate a complex network of interactions within the tumor microenvironment, influencing stromal cells, angiogenesis, and immune responses. The therapeutic modulation of ROS, though promising, remains context-dependent and is influenced by factors such as ROS type, level, localization, and tumor stage.^50,51 Our findings suggest that dual-functionalized Ti₃C₂(O,Cl) MXenes may tip this redox balance selectively in favor of cancer cell apoptosis. As in MTT testing, the Ti₃C₂(O,Cl) MXene tested material displayed inverse proportionality between the number of living cells and concentration, as well as higher selectivity against cancerous cells. The results obtained suggest that these effects may be a consequence of differences between the morphology and the permeability of normal and cancerous cells.

3.3 Mechanisms of toxicity

Carbon-based nanomaterials have been shown to exhibit cytotoxicity due to reactive oxygen species (ROS) that play a key role in cell metabolism and survival.⁵⁴ DCF-DA assays were conducted to determine whether the decreased cell viability after Ti₃C₂(O,Cl) MXene treatment is caused by oxidative stress phenomena in cancerous FaDu and normal HEK-293 cells for 24 h. As shown in Fig. 7 and 8, after 24 h of incubating MXene with 100 and 250 μg mL⁻¹ and Doxo 1 μM in FaDu cells, a significant increase in ROS production (a parameter proportional to the level of intracellular reactive oxygen species) was observed as compared to control untreated cells (52.61, 91.44, and 94.36%, respectively, to 100, 250 μg mL⁻¹ Ti₃C₂(O,Cl) MXene and Doxo 1 μM) (p < 0.001; Fig. 7).

Fig. 7 Ti₃C₂(O,Cl) MXene and Doxo induced reactive oxygen species (ROS) production in FaDu cells for 24 h. Data are shown as mean ± SD (n = 3) of three independent experiments. *** p < 0.001 vs. control.

Fig. 8 MXene and Doxo induced reactive oxygen species (ROS) production in HEK-293 cells for 24 h. Data are shown as mean ± SD (n = 3) of three independent experiments. *** p < 0.001 and * p < 0.05 vs. control.

Similarly, to check the selective ROS production effect of MXene in normal HEK-293 cells, we have treated Ti₃C₂(O,Cl) MXene 100 μg mL⁻¹ and Doxo 1 μM in HEK-293 cells for 24 h. Interestingly, less ROS production was observed in HEK-293 cells compared to FaDu cells for 24 h, as depicted in Fig. 8. This result suggests that Ti₃C₂(O,Cl) MXene has more selectivity towards cancer cells as compared to the normal cell line. The results obtained allow us to propose ROS generation as the possible mechanism of the cytotoxicity of Ti₃C₂(O,Cl) MXene.

However, our results demonstrated a dose-dependent and significantly higher cytotoxicity of Ti₃C₂(O,Cl) MXene in cancer cell lines FaDu and MCF-7 compared to normal cell lines H9C2 and HEK-293. This enhanced cytotoxicity in cancerous cells is likely due to differences in intracellular internalization mechanisms and ROS production between malignant and non-malignant cells. Cancer cells are known to exhibit altered membrane characteristics that facilitate greater uptake of nanoparticles, leading to a higher intracellular concentration of Ti₃C₂(O,Cl) MXene, which amplifies ROS production and induces oxidative stress. Additionally, cancer cells often experience elevated baseline oxidative stress due to their metabolic demands and rapid proliferation, rendering them more vulnerable to further ROS generation upon MXene exposure. The heightened ROS levels overwhelm cancer cells’ already compromised antioxidant defences, resulting in cell damage and death. In contrast, normal cells such as H9C2 and HEK-293 typically maintain stronger antioxidant defences and lower baseline ROS levels, enabling them to better neutralize the ROS induced by Ti₃C₂(O,Cl) MXene. This ROS-mediated mechanism aligns with previous studies highlighting the selective cytotoxicity of MXenes toward cancer cells, supporting their potential as targeted anticancer agents.

3.4. Comparative analysis with previous MXene reports

A direct comparison with earlier MXene reports highlights the benefits of our electrochemical dual functionalization. We observe a (002) XRD peak shift from 10.627° to 10.360° (Δ2θ = 0.27°), slightly larger than the Δ2θ = 0.22° shift reported for Cl terminated Ti₃C₂Cl_x obtained via molten salt etching.⁵⁵ High resolution XPS reveals a Ti–Cl/Ti–C area ratio of 0.57, markedly higher than the ∼0.35 ratio in HF etched Ti₃C₂T_x samples.⁵⁶ Biologically, our IC₅₀ of ≈48 μg mL⁻¹ on FaDu cells (SI = 2.7) compares favorably to ≈50 μg mL⁻¹ (SI ≈ 1.8) reported for Ti₃C₂ MXenes against A549 carcinoma.⁵⁷ These results confirm that the incorporation of both –O and –Cl terminations via chronoamperometric etching yields superior interlayer expansion, surface chemical functionality, and selective anticancer efficacy relative to conventional MXene synthesis methods.

3.5 Comparative advantages of dual-functionalized Ti₃C₂(O,Cl) MXenes in targeted cancer therapy

Dual functionalized Ti₃C₂(O,Cl) MXenes show strong cytotoxicity against squamous carcinoma and adenocarcinoma cells (IC₅₀ ≈ 48 μg mL⁻¹) with a selectivity index (SI) of ≈2.7, which is better than that of iron oxide nanoparticles (IC₅₀ ≈ 305 μg mL⁻¹; SI ≈ 2.2)⁵⁸ and comparable to that of hyaluronic acid-capped AuNPs (IC₅₀ ≈ 34.8 μg mL⁻¹; SI ≈ 26.9)⁵⁹ as represented in Table 3. MXenes with distinct –O/–Cl terminations catalyze localized ROS formation, taking advantage of cancer cells’ higher basal oxidative stress in the absence of external stimuli, while their nearly unity photothermal conversion allows synergistic thermal ablation. In contrast, AuNPs use mainly plasmon-induced heating and receptor-mediated uptake for selectivity, and iron oxide systems rely on Fenton chemistry in acidic tumor microenvironments and magnetic targeting.⁵⁸ Additionally, the high conductivity and modifiable surface chemistry of MXenes enable loading of drugs, imaging, and multimodal therapy in a unified platform. Critically, they eschew sophisticated instrumentation demands like NIR lasers or magnetic fields, which simplifies their clinical translation and minimizes cost.

Table 3 Comparative metrics for Ti₃C₂(O,Cl) MXene versus AuNPs and IONPs in anticancer activity

Nanomaterial	IC50 (cancer)	IC50 (normal)	SI (normal/cancer)	Mechanism	Ref.
Ti3C2(O,Cl) MXene	48 μg mL^−1 (FaDu)	130 μg mL^−1 (HEK-293)	2.7	Dual O/Cl terminations generate localized ROS, exploiting cancer cells’ heightened redox sensitivity	Present study
HA-capped AuNPs	34.8 μg mL^−1 (MDA-MB-231)	935.97 μg mL^−1 (L929)	26.9	Photothermal conversion plus HA-mediated CD44-targeted uptake	59
Fe3O4@Glu-Safranal IONPs	305 μg mL^−1 (HepG2)	680 μg mL^−1 (HEK-293)	2.23	Fenton reaction-driven ROS generation in the acidic tumor microenvironment	58

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6. Conclusion

The current investigation demonstrates the successful etching of scalable synthesized Ti₃C₂(O,Cl) MXenes via electrochemical methods. XRD reveals that 73.7% of Ti₃C₂(O,Cl) was successfully etched from the Ti₃AlC₂ MAX phase, and the etched Ti₃C₂(O,Cl) MXenes exhibit a consistent structure, as agreed by TEM and SEM analyses. Raman and XPS analyses, along with FTIR, reveal that the surface terminations on Ti₃C₂ MXenes include –O, –Cl, and –OH. These surface terminations suggest a strengthened cytotoxic effect on cancer cells. This investigation elucidated multiple facets of the in vitro cytotoxicity of Ti₃C₂(O,Cl) MXene in relation to both cancerous and normal cells, confirming a notable toxicity level of approximately 91% against cancer cells. Moreover, the phenomena associated with oxidative stress are recognized as possible mechanisms of toxicity in our investigation. MXenes have demonstrated toxicity towards anticancer cells; the present study offers the latest insights into these compounds. Concerns regarding toxicity have impeded further research and development in the realms of nanomedicine and targeted cancer therapies.

Data availability

Data generated at BITS Pilani Hyderabad Campus are available upon request. Experiments were conducted at BITS Pilani Hyderabad Campus. Experimental data supporting the findings of this study are available from the corresponding author, Prof Afkham Mir (mirafkham@hyderabad.bits-pilani.ac.in), on request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the SPARKLE grant from BITS Pilani, Hyderabad Campus.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00302d

‡ The authors contributed equally to this work.

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