Ti₃AlC₂ (powder, 200-meshes), HF, HCl (36%, w/w, purity >98%), TMAOH (25 wt% in water), AlCl₃ 6H₂O, and Folic acid, sodium dodecyl sulfate (SDS), phosphate buffer saline (PBS), high glucose cell culture medium, and a tetrazolium-based assay were purchased from Sigma-Aldrich. No further treatment was applied to the chemicals unless otherwise stated. An aqueous solution of monobasic potassium phosphate (KH₂PO₄) and dibasic potassium phosphate (K₂HPO₄) was utilized to produce PBS at pH 7. We used ultrapure water throughout all experiments.

Bruker diffractometers (PW1730) were used to carry out X-ray diffraction (XRD) with Cu Kα radiation (λ = 1.5406 Å). In order to characterize the morphology of the samples, transmission electron microscopes (TEM, Philips EM208S 100 KV) and field emission scanning electron microscopes (FESEMs, Hitachi, Japan) were used. To measure absorbance from 200 to 800 nm, Uv/Vis spectrometer (Perkin Elmer Lambda 25) was used. In addition, a JASCO FT-IR-460 spectrometer was utilized to obtain Fourier transform infrared spectroscopy (FT-IR) in the 400 to 4,000 cm⁻¹ range. Using vibrating sample magnetometers (VSM), magnetic nanocomposites were measured at Mahamax, Tehran, Iran).

A sample of 1 g of Ti₃AlC₂ powder was etched in a solution containing 1 g of LiF and 0.3 g of AlCl₃ 6H₂O for 3 days at room temperature with 10 mL of HCl (9 M) (Liu P. et al., 2018). The etching materials were centrifuged several times before being dispersed in 10 mL of TMAOH for 3 days, followed by centrifugation and washing to remove the intercalated Ti₃C₂. In order to prepare a colloidal suspension of Ti₃C₂ nanosheets, a clay-like solid was dispersed in water for hours under bath sonication, then the supernatant was centrifuged at 7,000 rpm, and then a freeze-drier was used to collect the dried samples.

As part of the preparation of Ti₃C₂-Fe₃O₄@SiO₂ nanocomposites, 50.0 mg of Fe₃O₄@SiO₂ nanoparticles synthesized hydrothermally (Darroudi et al., 2020; Ghasemi et al., 2021) was dispersed ultrasonically in 20 mL deionized water, and 100.0 mg of Ti₃C₂ nanosheets were dispersed in 80 mL deionized water and stirred for 30 min (Dai et al., 2017). In both solutions, ultra-sonification was performed for 120 min under an Ar atmosphere. After filtering the suspension to obtain the Ti₃C₂-Fe₃O₄@SiO₂ nanocomposite, it was dried in a vacuum overnight at 60°C. Afterward, 100.0 mg of the prepared Ti₃C₂-Fe₃O₄@SiO₂ and 0.5 g of folic acid were dispersed in 60 mL of deoxygenated water for 1 h with ultrasound. Then, the reaction system was kept in an Ar atmosphere in an oil bath at 60 C for 4 h. Finally, the obtained Ti₃C₂-Fe₃O₄@SiO₂-FA was washed with water and dried with a freeze-dryer.

As part of the preparation of Ti₃C₂@FA nanocomposites, 5.0 mg of folic acid (FA) functionalized polymer was dispersed ultrasonically in 10 mL deionized water, and 100.0 mg of Ti₃C₂ nanosheets were dispersed in 50 mL deoxygenated water and stirred for 30 min, and then mixed through sonicating for extra 90 min under an Ar atmosphere. After filtering the suspension to obtain the Ti₃C₂@FA nanocomposite, it was dried in a vacuum overnight at 60°C. Finally, the obtained Ti₃C₂@FA was washed with water and dried using freeze-dryer.

In order to examine Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin’s anticancer properties, it was examined on the cervix tumor model. The C57BL/6 mice (25–30 g) were purchased from the Laboratory Animal Center of Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. The Ethical Committee approved animal experimentation protocols of the Experimental Animal Center at MUMS, Mashhad, Iran. Standard food and water were provided during the experiments, and mice were housed in a laboratory environment. The TC1 cells (murine cervix cancer cell lines) were obtained from Pastour Institute (Tehran, Iran) and cultured in RPMI-1640 medium with 10% heat-inactivated FBS and 1% streptomycin at 5% CO₂ at 37°C. 2.5 × 10⁶ TC1 cells per 100 mL were injected in the left flank region of the mouse subcutaneously injected into mice, and approximately 2 weeks later, the tumor size reached 80–100 mm³ (Ghaemi et al., 2012). The participants were divided into four groups randomly according to the following instructions (n = 6 in each group): I. Control group (Untreated group), II. Cisplatin as a standard regimen for the treatment of cervical cancer (administered twice at a 3-day interval using 5 mg/kg, intraperitoneally; IP), III. Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin (administered twice at a 3-day interval using 5 mg/kg, IP), and IV. Ti₃C₂@Cisplatin (administered twice at a 3-day interval using 5 mg/kg, IP), followed by applying an external circular magnet (10 mm by 10 mm, 0.4 T surface field strength). The tumor size of the animals was measured once every other day. In order to determine the tumor volume (V), the formula V = AB²/2 was used, where A is the primary axis length and B is the minor axis length (Ghaemi et al., 2012). The cervical tumors of each mouse were removed on day 18 to undergo further investigation by hematoxylin and eosin staining (H&E).

The results of all experiments are expressed as the mean and standard error. One-way ANOVA using LSD post hoc test was used to analyze the significance of the results using SPSS software (SPSS Inc., Armonk, NY, USA). Statistical significance was determined by p .05.

Through sonication treatment, multi-layer Ti₃C₂ was converted into ultrathin Mxene nanosheets (Figure 1A). After this step, magnetic nanoscale Mxene sheets exhibited a large planar structure and good dispersity, which are ideal for biomedical applications (Soleymaniha et al., 2019). An HCl/LiF etchant was used initially to remove the Al layer from Ti₃AlC₂. In this manner, the Ti₃AlC₂ can be exfoliated using HCl/LiF etching, but the resulting multi-layer Ti₃C₂ nanosheets remain close together, exhibiting large particle sizes, which is incompatible with the demands of biomedicine (Naguib et al., 2011). Due to this, ultrasonication was used to complete the separation process and simultaneously reduce particle size. To increase controllability, magnetic nanoparticles were intercalated into Mxene layers to form heterostructures, thereby ensuring the confinement of Ti₃C₂ nanosheets. The Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier can store the charged functional groups of the anticancer drug (Cisplatin) owing to adequate hydroxyl groups on the surface (Figure 1A). For chemotherapy purposes, Cisplatin can be targeted released from nanocarriers under inner or external stimulation. As a result of their nanoscale size, Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier would circulate easily within blood vessels and could passively penetrate tumor cells (Li X. et al., 2017). The Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier can be reached cancerous cells using an external magnetic field and would not leave the cancer cells with blood circulation (Figure 1B).

The morphology and structure of constructed nanocomposites are characterized using TEM and FESEM images. As illustrated in Figure 2A, the Mxene Ti₃C₂ nanosheet, after etching the Al layer and following intercalation by TMAOH, the FESEM image depicted that closely pack layer structure of MAX phase exfoliating into nano size thickness sheets. Also, the EDAX analysis of the etched sample confirmed the surface modification by Al ion. TEM image also revealed that the titanium carbide nanosheets have an average lateral size of approximately 35 nm (Figure 3A). Furthermore, the formation of Mxene nanosheets is confirmed by XRD analysis, in which no peak at 38̊ was exhibited through intercalation, while the characteristic peaks from stacked Mxene nanosheets appeared. As shown in Figure 2C, the Brightfield FESEM image of Ti₃C₂-Fe₃O₄@SiO₂-FA composite nanosheets revealed a small Fe₃O₄@SiO₂ magnetic core-shell with an average size of approximately 31.3 nm on the surface of Ti₃C₂ Mxene nanosheets with a thickness of ∼31 nm. It is clear from the TEM image (Figure 3A) that the core shells of Fe₃O₄@SiO₂ species are in core-shell morphology. As shown in Figure 2D, the TEM and SEM images of functionalized Ti₃C₂ Mxene by FA had a surface of sheet-like morphology with a thickness of around ∼29.3 nm (Figure 2B). According to FESEM images of Ti₃C₂@FA nanosheet surface become roughened and covered by some spheres composed of Ti₃C₂@FA nanocomposite. Furthermore, the vanishing or decreasing peaks in the 30–40-degree range confirm the 2D structure of functionalized Ti₃C₂ nanosheets (Figure 4D).

Along with characterized nanocarrier, it exhibited a multi-layer structure of constructed Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin nanosheets that Fe₃O₄@SiO₂ component in core-shell morphology is firmly attached to the surface of Ti₃C₂ nanosheets in the average size of 27.2 nm on the sheets with thickness 30.5 nm (Figure 2E). In Figure 2F, FESEM images of Ti₃C₂@FA and the multi-layer Ti₃C₂ are shown, where a typical accordion-like structure of the multi-layer Ti₃C₂ can be observed with a thickness of approximately 38.5 nm.

The FESEM of MAX phase exhibited in Figure 2, confirmed the accordion-like structure of Ti₃AlC₂ structure, in which, through the ultrasonic process, the weak covalent bonds between the multi-layer Ti₃C₂ nanosheets are destroyed, confirmed through TEM image (Figure 3B). The EDAX analysis also confirmed the presence of Ti, Al, and C elements in the structure of the MAX phase (Figure 5A). Overall, the TEM image of Ti₃C₂ nanosheets revealed further details about their morphology and structure (Figure 3), which are approximately 29–41 nm in transverse dimension. Further, TEM results suggest that Ti₃C₂ nanosheets could be used as drug delivery nanocarriers due to their small thickness and size, which enhances blood circulation (Wang et al., 2013). An XRD pattern for Ti₃C₂ nanosheets, shown in Figure 4, demonstrates the etching of Al layers from Ti₃AlC₂ by using LiF/HCl etchant successfully removed Al layers from Ti₃AlC₂ through the most intense XRD peak (104) of Ti₃AlC₂ (2θ ≈ 39°). As a consequence of the introduction of lithium and hydroxyl ions into the Ti₃C₂ layers, the main peak (002) of Ti₃AlC₂ moved from 10.5° to 9.2°, since the distance between adjacent Ti₃C₂ layers in Ti₃C₂-Fe₃O₄@SiO₂-FA, Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin, and Ti₃C₂@FA is augmented by the presence of these ions (Wang et al., 2021). The aqueous dispersion of Ti₃C₂ nanosheets appears dark-colored, with the incident light scattered by colloidal nanosheets. The formation of Ti₃C₂ is also confirmed by X-ray diffraction (XRD).

As indicated by previous studies (Ma et al., 2021), Ti₃C₂ MXene exhibited negatively charged oxygen-containing groups, which could absorb the positively charged Fe₃O₄@SiO₂ (Zhang et al., 2022). Analysis of this reaction procedure was conducted using Fourier transform infrared (FTIR). Following iron oxide and silane core shells, Fe₃O₄@SiO₂, Ti₃C₂-Fe₃O₄@SiO₂-FA, and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin FIT-IR profiles are illustrated in Figure 6. FT-IR spectroscopy was used to verify the chemical modifications. According to the spectra analysis of Ti₃C₂-Fe₃O₄@SiO₂-FA, the FTIR spectrum showed C–H stretch at 2,820–2,950 cm⁻¹, and C–O stretch at 1,335–1,192 cm⁻¹. In the range between two peaks of 1,620 and 1,440 cm⁻¹, strong bond vibrations are observed for NH and NH₂. The NH₃ ⁺ stretch is a wide peak at 3,230–2,600 cm⁻¹. In addition to C=O stretch at 1,694 cm⁻¹ and C–O stretch at 1,340–1,180 cm⁻¹, C=C stretch in aromatic compounds was also found at 1,600–1,450 cm⁻¹ in the FA molecule (Figure 6). The FTIR spectra of Ti₃C₂-Fe₃O₄@SiO₂-FA exhibited a strong band at 1,626 cm⁻¹ attributing to the N-H vibration of FA. Furthermore, the FTIR spectrum of Ti₃C₂-Magnetic nanosheets showed a new peak at 575 cm⁻¹, correlated with Fe-O stretching vibration of Fe₃O₄, demonstrating Fe₃O₄ deposition on Ti₃C₂ nanosheets in comparison with the FTIR spectrum of Ti₃C₂ nanosheets. Ti₃C₂-Magnetic NPs exhibit a significant reduction in the FTIR band associated with hydroxyl group at 3,411 cm⁻¹ and carbonyl group at 1,690 cm⁻¹ (Figure 6). The Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin showed weak amine bands, suggesting that the NH₂ was conjugated with the OH groups of the nanocomposites and Folic acid towards the Cisplatin. Moreover, the bending vibration at 1,650 cm⁻¹ indicates that Cisplatin has been functionalized, as well as the reaction between the Folic acid and Pt (II) complex.

In order to examine the presence of synthetic magnetic core shells and nanosheets, SEM and EDAX were used. As shown in Figure 5, magnetic nanoparticles with an average width of 27–40 nm have a microstructure.

The distribution maps of Ti, Fe, Si, C, and O elements on Ti₃C₂-Fe₃O₄@SiO₂-FA composite nanosheets confirmed the coexistence of Ti and Fe elements and the uniform distribution of Fe₃O₄@SiO₂ on the surface. An EDAX analysis of Ti₃C₂-Fe₃O₄@SiO₂-FA composite nanosheets (Figure 6B) shows that Ti, Fe, and Si elements are present, indicating that Fe₃O₄@SiO₂ was successfully functionalized on the surface of Ti₃C₂ Mxene nanosheets. Furthermore, through loading Cisplatin on the nanocarrier, the existence of Pt would be exhibited in the EDAX analysis, confirming the construction of the Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin and Ti₃C₂@FA-Cisplatin (Figures 6E, F). Insets in Figure 5 provide a list of results of the elemental analysis of Fe₃O₄@SiO₂-uncoated, Ti₃C₂-Fe₃O₄@SiO₂-FA-coated, and Ti₃C₂@FA-coated nanoparticles, in which 6.76%, 10.79%, and 15.62% of nanoparticles’ weights can be found to be respectively. Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin contains 2.89% Pt, while Ti₃C₂@FA-Cisplatin contains 1.92% Pt. The elemental analysis indicates that since Fe₃O₄@SiO₂ and adsorbed-Mxene on Fe₃O₄@SiO₂, the remaining weight is comprised of Fe and Ti (from Fe₃O₄ and Ti₃C₂). Thus, the weight% of Fe and Ti is estimated to be 26.63% and 16.13%. The number ratio of compounds is determined to be C (8): Fe (26): Ti (16), using the mentioned weight% ratio. Therefore, 3.03:1 and 2.05:1 have been determined for the Fe₃O₄:Ti₃C₂ number and weight ratios. FT-IR analysis illustrates the existence of Ti₃C₂ MXene nanosheets as indicated by the elemental analysis results. Regarding the elemental analysis of Ti₃C₂@FA nanosheets, it should be noted that the weights% of C and Ti atoms are, respectively, determined to be 84.38% and 15.62% (Figure 6D). The C: Ti ratio is determined to be 5.41:1. In this case, C is attributed to both Ti₃C₂ and Folic acid coated on the nanosheets. Therefore, we conclude that the Fe₃O₄@SiO₂:Ti₃C₂ weight% ratio is 26.53:16.13, with a 25.5% weight ratio of Fe in the total weight of the nanocarrier.

Due to the presence of magnetic Fe₃O₄ nanoparticles in Ti₃C₂-Magnetic composite nanosheets, the Mxene can modulate their magnetic properties by applying an external magnetic field (Figure 7), which suggests that magnetic fields can be used for further nanomedicine applications. Figure 7 shows the unique magnetic property of Ti₃C₂-Magnetic NPs composite nanosheets, which exhibit a saturation magnetization of 23.5 emu g⁻¹.

Considering the magnetic properties of Fe₃O₄ core-shell nanomaterials (Gao and Wang, 2014; Oravczová et al., 2021), a magnetically controlled nanocarrier based on Ti₃C₂-Fe₃O₄@SiO₂-FA was investigated. It is shown in Figure 7B that Fe₃O₄@SiO₂, Ti₃C₂-Fe₃O₄@SiO₂-FA, and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin nanocarriers show field-dependent magnetization curves. It was determined that Fe₃O₄@SiO₂ has the highest saturation magnetization (60.0 emu/g) (Figure 7A), whereas Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin nanocarrier (6.5 emu/g) exhibits significantly lower saturation magnetization than Ti₃C₂-Fe₃O₄@SiO₂-FA (23.5 emu/g), owing to the presence of Ti₃C₂ nanosheets, the remanence of Ti₃C₂ nanocarriers is lower than the remanence of Fe₃O₄@SiO₂ nanosheets (Liu et al., 2008). In conclusion, the Ti₃C₂-Fe₃O₄@SiO₂-FA and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin nanocarriers show hysteresis loops, suggesting they could be magnetically controlled drug carriers. Furthermore, previous studies have indicated that saturation magnetizations of 16.3 emu/g would be sufficient for magnetic control (Deng et al., 2014).

It has been demonstrated that Ti₃C₂ nanosheets (Karlsson et al., 2015) and Fe₃O₄ (Darroudi et al., 2020) can be further developed into drug delivery nanocarriers for cancer therapy due to the abundance of hydroxyl functional groups and their large surface areas. For evaluating the drug loading/release ability of Ti₃C₂-Fe₃O₄@SiO₂-FA and Ti₃C₂@FA nanocarriers, Cisplatin, a chemotherapy drug used to combat cancer, was used as a model drug. After vigorous stirring and sonication under an Ar atmosphere, Cisplatin was loaded on the surface of Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier. Due to the strong electrostatic interactions between the negatively charged surface of the Ti₃C₂-Fe₃O₄@SiO₂-FA and Ti₃C₂@FA nanocarriers and the positively charged Cisplatin, we achieved a high drug loading content (Liu et al., 2019). This can be seen in the shaded part of the UV–Vis spectrums (Figure 8A), where the acquired Ti₃C₂-Fe₃O₄@SiO₂-FA and Ti₃C₂@FA exhibit the characteristic absorption peak of Cisplatin compared to the Ti₃C₂-Fe₃O₄@SiO₂-FA after loading with Cisplatin. In this case, the combination between both Ti₃C₂-Fe₃O₄@SiO₂-FA and Ti₃C₂@FA nanocarriers and Cisplatin would be a successful result of the interaction between the hydroxyl groups of Ti₃C₂-Fe₃O₄@SiO₂-FA and Ti₃C₂@FA nanocarriers and Cisplatin (Zhao et al., 2019). Furthermore, since the constructed titanium carbide nanocarriers are negatively charged, it may offer better cell accessibility and hydrophilic properties (Chen et al., 2016a).

A UV-vis spectroscopy study was conducted to assess the capacity of the Ti₃C₂-Fe₃O₄@SiO₂-FA and Ti₃C₂@FA nanocarriers to load different weight ratios of Cisplatin (0.5, 1, 1.5, 2, 3). By using a standard calibration curve for Cisplatin under 293 nm irradiation (Figure 8C), the concentration of Cisplatin was calculated and incorporated into the drug loading equation. As shown in Figures 8A–C greater ratio of Ti₃C₂-Fe₃O₄@SiO₂-FA results in an increase in the drug loading capacity. In accordance with the equation for drug loading, the drug loading efficiency at a mass ratio of three of the Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier was calculated to be 244.01% based on the drug loading formula, while the Ti₃C₂@FA-Cisplatin loading efficiency was 134.01%. These are considerably higher than the average drug loading efficiency for single Ti₃C₂ nanosheets (89%) and most drug delivery nanocarriers (10%–50%) (Liu et al., 2020). It is assumed that the increase in drug loading capacity can be attributed to two factors: 1) In the case of the Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier, there are more electrostatic sites available for loading Cisplatin 2) The Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier provides sufficient surface area for Cisplatin to be loaded.

This study examined the pH-dependent drug release of Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin and Ti₃C₂@FA-Cisplatin under several pH values (7.4 and 4.5, respectively). Figure 8D exhibited that at pH 7.4 and 4.5 during a 6-h period, 31.59%, and 89.01%, of Cisplatin are released from Ti₃C₂-Fe₃O₄@SiO₂-FA -Cisplatin, respectively, suggests that it is more readily released in an acidic microenvironment. In comparison, Ti₃C₂@FA-Cisplatin exhibited a pH-responsive release profile; during 6 h, Cisplatin release was measured to be 44.31% and 23.04%, corresponding to pH 4.5 and 7.4, respectively. Due to the altered interaction between Cisplatin and Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarriers and the greater solubility of Cisplatin at lower pH values, this result can be attributed to the altered interaction between drug and Ti₃C₂-titanium carbide nanocarriers. Notably, at pH 7.4, a large number of hydroxyl groups on the surface of Ti₃C₂-Fe₃O₄@SiO₂-FA become deprotonated and negatively charged, in contrast to Cisplatin’s positive charge (Gong et al., 2016). The solubility and hydrophilic properties of Cisplatin increase with an increase in protonation of the amino group under a pH value of 4.5 (Du et al., 2010). Meanwhile, the hydroxyl groups on the surface of the nanocarrier Ti₃C₂-Fe₃O₄@SiO₂-FA are protonated in this process, would result in a repulsive interaction between Cisplatin and Ti₃C₂-Fe₃O₄@SiO₂-FA nanocarrier (Mu et al., 2019). Since Cisplatin releases from Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin in an acidic environment, the pH-triggered drug release action has a significant impact.

This study aimed to determine whether Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin and Ti₃C₂@Cisplatin inhibited the growth of cervical carcinomas in mice using a tumor model. Figure 9A in each group of mice, Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin, Ti₃C₂@Cisplatin, and Cisplatin alone were treated, and tumor weight and size were measured. The results of in vivo experiments indicated a decrease in tumor size and weight in mice after treatment with Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin (Figures 9B, C). This cervical cancer model shows that Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin exhibits enhanced anticancer activity compared with Cisplatin alone Ti₃C₂@Cisplatin nanosheets, which significantly increases Cisplatin’s antitumor activity. According to the in vivo release profile of nanocomposite, Cisplatin alone and Ti₃C₂@Cisplatin nanosheets follow a one-compartment model in vivo. Since these magnetic nanocomposites consist of multiple compartments, they would be released sequentially, followed by the continuous release resulting from burst releases.

A significant challenge facing the treatment of cervical cancer is how to prevent the accumulation of cancer-fighting drugs in healthy tissue while improving the local accumulation of these drugs at the tumor site (Koning et al., 2010). A new therapeutic approach for treating localized cancer may be possible with nanoparticles with magnetic properties (Kumar and Mohammad, 2011). Recent research reports that a polymeric nanocapsule containing 5-Fu could treat colon cancer similarly (Li S. et al., 2008). A study by Shakeri-Zadeh et al. found that 5-Fu had an increased tendency to cause colon tumors when loaded into magnetic nanoparticles (Shakeri-Zadeh et al., 2014). In this regard, our in vivo experiments have shown that when Cisplatin is loaded into Mxene-magnetic nanosheets, it would have a sustained release in a pH-responsive manner, prolonged half-life, and significantly increased tumor uptake, while there was no predicted efficiency for Mxene Ti₃C₂@Cisplatin.

An increased area of tissue necrosis was observed in the cervix tumor after Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin treatment (Figure 10A). It was revealed that Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin increased percentages of necrosis in cervical tissue compared to Cisplatin alone and Ti₃C₂@Cisplatin as the standard chemotherapeutic regimen in cervical cancer (Figure 10B; p = .05). Using H&E staining, arrows indicate the necrosis area.

To examine the anti-proliferative potential of Cisplatin, Ti₃C₂@FA@Cisplatin, and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin, cells were exposed to the rising concentrations (0–1,500 ppm) for 24 h and 72 h. A reduction of nearly 90% in cell viability was observed at the highest concentration of free Cisplatin. In contrast, although both Ti₃C₂@FA@Cisplatin and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin had radical negative effects on cell viability, neither was as effective as Cisplatin. This finding also reflects in the determined IC50 values and could be attributed to variations in the drug loading/release profiles between Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin and Ti₃C₂@FA@Cisplatin. IC50 values were found to be for Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin (24 h): 855 μg/mL, (72 h): 60 μg/mL. Additionally, IC50 in terms of the amount Ti₃C₂@FA@Cisplatin (24 h): 130 μg/mL, (72 h): 30 μg/mL (Figure 11).

In a 3-D cell structure (Figure 12), Ti₃C₂@FA@Cisplatin and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin were assessed for their specific anti-cancer abilities. The spheroid size of the treated group differed significantly from the control group after 3 days. Spheroid area did not change significantly after 3 days in the control group, while it significantly decreased in Cisplatin, Ti₃C₂@FA@Cisplatin, and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin treated groups. As a result of the loss of cell membrane integrity and the loss of viability of the core cells of Ti₃C₂@FA@Cisplatin and Ti₃C₂-Fe₃O₄@SiO₂-FA-Cisplatin spheroids, the peripheral cells have already disappeared. A three-dimensional model of cervical cancer cells showed that treatments could prevent proliferation and progression, and consequently, the cancerous cells are more likely to die (Figure 12). Spheroid growth is not only significantly retarded in the presence of nanoforms, but also a corona of dead and fragmented cells is visible after approximately 3 days of treatment.