Titanium carbide nanosheets with defect structure for photothermal-enhanced sonodynamic therapy

Abstract

Sonodynamic therapy (SDT) has attracted widespread interest in biomedicine, owing to its novel and noninvasive therapeutic method triggered by ultrasound (US). Herein, the Ti₃C₂ MXene nanosheets (Ti₃C₂ NSs) are developed as good sonosensitizers via a two-step method of chemical exfoliation and high-temperature treatment. With the high-temperature treatment, the oxygen defect of Ti₃C₂ MXene nanosheets (H–Ti₃C₂ NSs) is greatly increased. Therefore, the electron (e⁻) and hole (h⁺) generated by US can be separated faster due to the improved degree of oxidation, and then the recombination of e⁻-h⁺ can be prevented with the abundant oxygen defect under US irradiation, which induced the sonodynamic efficiency greatly to improve around 3.7-fold compared with Ti₃C₂ NSs without high-temperature treatment. After PEGylation, the H–Ti₃C₂-PEG NSs show good stability and biocompatibility. In vitro studies exhibit that the inherent property of mild photothermal effect can promote the endocytosis of H–Ti₃C₂-PEG NSs, which can improve the SDT efficacy. In vivo studies further display that the increased blood supply by the mild photothermal effect can significantly relieve hypoxia in the tumor microenvironment, showing photothermal therapy (PTT) enhanced SDT. Most importantly, the H–Ti₃C₂-PEG NSs can be biodegraded and excreted out of the body, showing no significant long-term toxicity. Our work develops the defective H–Ti₃C₂ NSs as high-efficiency and safe sonosensitizers for photothermal-enhanced SDT of cancer, extending the biomedical application of MXene-based nanoplatforms.

Graphical abstract

Highlights

• •Two-step high-temperature methods were used to construct oxygen-defective Ti₃C₂ nanosheets.
• •The oxygen-defective H–Ti₃C₂ NSs displayed an excellent sonodynamic efficacy.
• •The good photothermal efficacy of H–Ti₃C₂-PEG NSs enhanced SDT efficacy.
• •The biodegradable H–Ti₃C₂-PEG NSs showed no significant long-term toxicity.

1. Introduction

Sonodynamic therapy (SDT), which can generate reactive oxygen species (ROS) by sonosensitizers under ultrasound (US) irradiation, has been rapidly developed and recognized as a novel and noninvasive therapeutic method for cancer treatment [[1], [2], [3], [4]]. Compared with other cancer therapy methods, the US with highly focused property, high tissue penetration depth, and minimal damage to the normal tissues, has widely been used for diagnostic and treatment in clinics [[5], [6], [7], [8], [9], [10]]. Therefore, compared with light excited photodynamic therapy (PDT) that is usually applied in superficial tumors, the US affords SDT with the preponderances in the therapy of deep-seated tumors [[11], [12], [13]]. As a major component in SDT, currently, the sonosensitizers are consisted of organic sonosensitizers and inorganic ones, and the efficiency of sonosensitizers plays an important role in ROS generation [1,14]. The organic sonosensitizers are mainly come from organic photosensitizers, such as porphyrins [14,15] (hematoporphyrin, phtotofrin, etc.), Rose Bengal [16], and so on, while the inorganic sonosensitizers mainly include Ti-based nanomaterials [[17], [18], [19]], bimetallic oxide nanoparticles [20], black phosphorus [21], and so on. However, the organic sonosensitizers suffer from the fast obliteration and poor tumor accumulation, which severely restrict the efficacy of SDT [1]. Although these shortcomings can be overcome by the drug delivery system, the intrinsic issues such as photobleaching and phototoxicity still exist in organic sonosensitizer-based SDT. Fortunately, the inorganic sonosensitizers show advantages in high acoustic stability and good sonodynamic effect, however, the biosafety must be considered, and the conversion efficiency of ROS can't be ignored especially [11,22]. Therefore, more efforts of developing inorganic sonosensitizers with high efficiency and good safety should be made at this stage.

From another perspective, tumor microenvironment (TME), with the special characteristics of hypoxia, ischemia, weak acidity, and high concentration of hydrogen peroxide (H₂O₂) [23,24], is the natural barrier for cancer treatment, especially for oxygen (O₂)-dependent cancer treatments such as PDT [25], chemodynamic therapy (CDT) [5,26], radiotherapy (RT) [[27], [28], [29]], and SDT [1,[30], [31], [32]]. For example, the ROS yield would be significantly restricted by severe hypoxia and ischemia, and the condition of tumor hypoxia would be further accentuated with the O₂ consumption during SDT, which causes the vicious cycle and unsatisfied outcome. Therefore, it's meaningful to modulate TME, such as relieving hypoxia and ischemia, to improve the efficacy of SDT. Up to now, several strategies have appeared for tumor-hypoxia relief, like converting high content of H₂O₂ to O₂ in the TME [33], transporting O₂ to the tumor site through the fluorocarbons-based delivery system [34], increasing the blood supply to the tumor site by the mild photothermal effect [22], and so on. Owing to the limited endogenous H₂O₂ in tumor and weak transportation efficiency of O₂ to the tumor sites, the photothermal effect to ameliorate the tumor-hypoxia microenvironment is the simplest and convenient way.

Transition metal carbides, nitrides, and carbonitrides (MXenes) are a novel type of two-dimensional (2D) nanomaterials owing to their large surface area, high near-infrared (NIR) absorbance, and substitutable components ability, which have been rapidly used in biomedicine [35]. Amongst MXenes, titanium carbides (Ti₃C₂) is one of the most popular representatives with high absorbance in the NIR Ⅱ region, which has been widely applied in photo-induced cancer therapy [[36], [37], [38]]. Due to easier to be partly oxidized of the surface on Ti₃C₂ NSs, the formation of defect structure, that was TiO₂/Ti₃C₂, could promote the transformation of charge carriers and capture the electron (e⁻) to prevent the recombination of the e⁻ and hole (h⁺), which resulted in the improved photocatalysis owing to the favorable properties [[39], [40], [41]]. Therefore, such unique structure would be better for US-induced cancer therapy. Herein, we developed a new type of MXene-based sonosensitizer by two-step methods of chemical exfoliation and high-temperature treatment for enhanced SDT (Scheme 1). After the high-temperature treatment of Ti₃C₂ NSs (H–Ti₃C₂ NSs), the formed TiO_x/Ti₃C₂ structure could not only promote the separation of US generated e⁻ and h⁺ from the energy-band structure, but also capture electronic to prevent the recombination of e⁻-h⁺ by oxygen vacancy, leading to an excellent sonodynamic effect under US irradiation. Especially, the H–Ti₃C₂ NSs also had relatively high absorbance in NIR ΙΙ window, which could be used for photothermal therapy (PTT). The mild photothermal effect generated by H–Ti₃C₂ NSs could prolong the blood circulation and improve the O₂ supply. Both in vitro and in vivo photothermal-sonodynamic synergistic therapy was achieved by H–Ti₃C₂ NSs with sequential 1064 nm laser and US irradiation. Importantly, the synthesized H–Ti₃C₂-PEG NSs showed excellent biocompatibility, without causing any long-term toxicity obviously. Collectively, our work highlighted that the defective H–Ti₃C₂ NSs via the high-temperature treatment could be a promising sonosensitizer for SDT, which extended the biological application of MXene-based nanoplatforms.

Scheme 1.

Schematic of the preparation of H_H-Ti₃C₂ nanosheets by the chemical exfoliation and high-temperature treatment methods for photothermal enhanced sonodynamic cancer therapy.

2. Results and discussion

2.1. Preparation and characterization of Ti₃C₂ NSs and H–Ti₃C₂ NSs

According to the previous reports, Ti₃C₂ NSs were fabricated by selective etching and chemical exfoliation methods [37,38], and their morphology was mainly single-layer sheet structure revealed by transmission electron microscopy (TEM) image (Fig. 1a). Inspired by the photocatalysis application, the high-temperature method was introduced to treat the above-synthesized Ti₃C₂ NSs. In short, the Ti₃C₂ NSs were dispersed in the mixture of oleylamine (OM) and 1-octadecene (ODE), and then the solution was heated to 320 °C under nitrogen protection. In order to investigate the oxidation degree, the Ti₃C₂ NSs were treated with 1 h (H_L-Ti₃C₂ NSs) and 2 h (H_H–Ti₃C₂ NSs) at such high temperature, respectively. From the TEM images, the microstructure and morphology of Ti₃C₂ NSs did not change too much after high-temperature treatment (Fig. 1b and c). Next, the composition of Ti₃C₂ NSs before and after high-temperature treatment were carefully investigated. X-ray energy dispersive spectrometer (EDS) and element mapping firstly showed that both Ti₃C₂ NSs and H–Ti₃C₂ NSs mainly contained three elements of Ti, C, and O (Fig. S1, S2). Before the high-temperature treatment, the element O existed in the Ti₃C₂ NSs probably due to a certain degree of oxidization in the process of fabricating Ti₃C₂ NSs. With the increased time of high-temperature treatment, the content of O was gradually enhanced, demonstrating the oxidation of Ti₃C₂ NSs. From the X-ray powder diffraction (XRD) spectra, new peaks at 21.7° (TiO₂, JCPDS. 00-049-1433) and 61.1° (TiO, JCPDS. 01-086-2352) appeared (Fig. 1d), further claimed the oxidization of Ti₃C₂ NSs in the process of high-temperature treatment.

Fig. 1.

Preparation and characterization of Ti₃C₂NSs and H–Ti₃C₂NSs prepared by high-temperature method. (a-c) The TEM images of Ti₃C₂ NSs (a), H_L-Ti₃C₂ NSs (b), and H_H-Ti₃C₂ NSs (c). (d) XRD spectra of Ti₃AlC₂, Ti₃C₂ NSs, H_L-Ti₃C₂ NSs, and H_H-Ti₃C₂ NSs. (e-h) XPS spectra of Ti 2p for Ti₃C₂ NSs (e), H_L-Ti₃C₂ NSs (f), and H_H-Ti₃C₂ NSs (g) and corresponding peaks area (h). (i) The NEXAFS spectra of Ti L-edge for Ti₃C₂ NSs and H_H-Ti₃C₂ NSs. (j&k) The Raman spectra of Ti₃C₂ NSs, H_L-Ti₃C₂ NSs, and H_H-Ti₃C₂ NSs (j) and corresponding magnification in the blue area (k). (l) UV–vis–NIR spectra of Ti₃C₂ NSs, H_L-Ti₃C₂ NSs, and H_H-Ti₃C₂ NSs. Insert is the photograph of Ti₃C₂ NSs, H_L-Ti₃C₂ NSs, and H_H-Ti₃C₂ NSs at the same concentration of Ti ions.

In addition, the major elements of Ti, C, and O of Ti₃C₂ NSs before and after high-temperature treatment were analyzed by X-ray photoelectron spectroscopy (XPS). For the Ti 2p region, the peaks at 455, 455.64, 456.52, 458.59, 461.3, 462.29, 463.45, and 464.51 eV corresponded to the Ti–C (2p_3/2), Ti²⁺ (2p_3/2), Ti³⁺ (2p_3/2), TiO₂ (2p_3/2), Ti–C (2p_1/2), Ti²⁺ (2p_1/2), Ti³⁺ (2p_1/2), and TiO₂ (2p_1/2), respectively (Fig. 1e–g, Fig. S3). From the spectra, we found that the peak of Ti–O band gradually increased while the Ti–C bond decreased with the high-temperature treatment. By calculating the integral area, the peak area of Ti–O band increased from ~39.6% to ~51.3% (H_L-Ti₃C₂ NSs), and further to ~67.7% (H_H–Ti₃C₂ NSs), while those of the Ti–C bond decreased from ~19.2% to ~18.1%, then to ~7.9%, respectively, indicating that the Ti₃C₂ NSs were oxidized to form TiO_x by the high-temperature treatment (Fig. 1h). In addition, the C 1s region possessed two main peaks at 281.34 and 284.92 eV, which corresponded to the Ti–C and C–C bonds, respectively (Fig. S4). The intensity of Ti–C bond also decreased gradually with the prolonged time of high-temperature treatment, indicating that the Ti₃C₂ NSs were partly transformed into TiO_x. Besides, the changes of O1s spectra revealed that the peak intensity of TiO_x increased after high-temperature treatment compared with Ti₃C₂ NSs without treatment, further indicating that the formation of TiO_x on the surface of Ti₃C₂ NSs (Fig. S5). In order to further prove the change of composition, the Ti L-edge near-edge X-ray absorption fine structure (NEXAFS) was used to identify the Ti 3d electronic state changes on the surface of Ti₃C₂ NSs before and after high-temperature treatment. There were slight changes within 455–457 eV (Fig. 1i, Fig. S6), which was caused by the crystal-field splitting of the Ti 3d oribitals, and the formed TiO_x nanostructures were sensitive to the oxidation state [42].

Raman spectrum was a significant method to analyze the surface message and the change process of nanomaterials. According to the Raman spectra, there were three characteristic peaks around 260, 400, and 600 cm⁻¹ for Ti₃C₂ NSs; meanwhile, a new peak around 155 cm⁻¹ appeared in the samples after the high- temperature treatment, and the intensity increased with the long-time treatment (Fig. 1j and k), which demonstrated that the TiO_x was generated and accumulated on the surface of Ti₃C₂ NSs [39,40,43]. From the UV–vis–NIR absorbance spectra, all the samples before/after high-temperature treatment showed good NIR II absorbance, which could be used for NIR II photo-induced cancer therapy. By the high-temperature treatment, the absorbance spectra and the color of samples slightly changed (Fig. 1l), indicating such treatment did not affect the unique photo properties of Ti₃C₂ NSs. Collectively, it could be directly or indirectly identified that the TiO_x were generated and accumulatively deposited on the surface of Ti₃C₂ NSs by the high-temperature treatment.

In order to improve the dispersity, stability, and promote biomedical applications, the amphiphilic polymer of DSPE-PEG ₍₂₀₀₀₎ was used to modify H–Ti₃C₂ NSs (H–Ti₃C₂-PEG NSs) by noncovalent interaction. After the surface coating, the dynamic light scattering (DLS) of the H–Ti₃C₂-PEGNSs showed an average size of ~164 nm (Fig. S7), and the final sample showed high stability in the different physiological solutions including H₂O, PBS, 0.9% NaCl, and RPMI in 4 °C (Fig. S8). Through this high-temperature treatment in the organic phase, the PEGylated H–Ti₃C₂ NSs showed excellent stability and biocompatibility, which would be used in the field of biomedicine.

2.2. Sonodynamic and photothermal performance of H–Ti₃C₂-PEG NSs

After high-temperature treatment for Ti₃C₂ NSs, the TiO_x was generated on the surface of Ti₃C₂ NSs due to the changes of composition and structure, making the H–Ti₃C₂-PEG NSs to be promising effective sonosensitizers (Fig. 2a). To investigate the sonodynamic efficiency of H–Ti₃C₂-PEG NSs, the 1,3-diphenylisobenzofuran (DPBF) was used as the molecular probe to detect ROS under US irradiation. In the presence of ROS, the characteristic peak at 418 nm decreased. The Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs were irradiated by US at the same condition, respectively. Compared with Ti₃C₂ NSs and commercial TiO₂, both H_L-Ti₃C₂-PEG NSs and H_H-Ti₃C₂-PEG NSs could obviously decrease the absorbance of DPBF (Fig. 2b c, Fig. S9), and the attenuation ratios were calculated to be ~18% for H₂O, ~23% for Ti₃C₂ NSs, ~45% for the commercial TiO₂, ~64% for H_L-Ti₃C₂-PEG NSs, and ~85% for H_H-Ti₃C₂-PEG NSs) after US irradiation for 6 min (Fig. 2d), indicating that the H–Ti₃C₂-PEG NSs had much stronger efficiency of ROS generation than that of Ti₃C₂ NSs and the commercial TiO₂. The longer time of high-temperature treatment, the higher the sonodynamic effect of the H–Ti₃C₂-PEG NSs. The H_H-Ti₃C₂-PEG NSs incubated with DPBF without US irradiation showed no ROS generation (Fig. S10). In addition, we also used the probe of diphenylamine (DPA) to verify the ROS (¹O₂) generation, it was clear that the H_H-Ti₃C₂-PEG NSs under US could generate enough ¹O₂ (Fig. S11). Afterwards, the tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) probes were also used to detect other types of ROS, like hydroxyl radical (∙OH), however, no obvious signal could be detected (Fig. S12), indicated no ∙OH generation under US irradiation. Moreover, electron spin resonance (ESR) measurements were further conducted to further identify the ROS type generated in this process. Obviously, the typical characteristic peak of 1:1:1 occurred, indicating the ¹O₂ generation by US irradiated Ti₃C₂ NSs [44]. In addition, the H–Ti₃C₂-PEG NSs showed a higher ability in producing ¹O₂ under US irradiation, revealing that the H–Ti₃C₂-PEG NSs indeed could act as better sonosensitizers than Ti₃C₂ NSs (Fig. 2e). Importantly, the inorganic sonosensitizer had the advantages of good ultrasonic stability and continuous ROS generation. To evaluate ROS generation stability, the H_H-Ti₃C₂-PEG NSs were treated with US for five cycles, and the stability performance was reflected by the decline of DPBF probe, which showed no significant change (Fig. 2f, Fig. S13). Moreover, after US irradiation for different times, the UV–vis–NIR absorbance spectra and the DLS sizes of H_H-Ti₃C₂-PEG NSs had no obvious change. Therefore, the H_H-Ti₃C₂-PEG NSs possessed excellent stability under US irradiation (Fig. S14).

Fig. 2.

Sonodynamic and photothermal performance of Ti₃C₂NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs. (a) The schematic illustration of sonodynamic and photothermal properties of H–Ti₃C₂-PEG NSs. (b&c) ROS generation by the Ti₃C₂ NSs (b) and H_H-Ti₃C₂-PEG NSs (c) with DPBF under US irradiation. (d) Comparison of the sonodynamic performance of H₂O, commercial TiO₂, Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs detected by DPBF probe (n = 3 independent samples). (e) The ESR spectra showing ROS (¹O₂) generation for H₂O, Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs under the same US irradiation. (f) The ROS generation stability of the H_H-Ti₃C₂-PEG NSs with DPBF under US irradiation in five cycles. (g) The mechanism of TiO₂ NPs and H–Ti₃C₂-PEG NSs under US irradiation. (h) The ESR spectra demonstrating the oxygen vacancy signal of Ti₃C₂ NSs, H_L-Ti₃C₂ NSs, and H_H-Ti₃C₂ NSs at g = 2.03. (i) The photothermal heating curves of H₂O, Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs (50 ppm, 1W·cm⁻²). (j) The photothermal profile of H_H-Ti₃C₂-PEG NSs after 1064 nm laser exposure to obtain steady temperature and turn off the laser to cool down. (k) The heat stability of H_H-Ti₃C₂-PEG NSs in five cycles laser On/Off. Error bars = standard deviation (n = 3). Data are presented as mean values ± SD. A representative image of three biological replicates from each group is shown.

Next, the mechanism of enhanced SDT efficiency by the H–Ti₃C₂ NSs was systematically investigated. As an important semiconductor photocatalyst, the formed TiO_x on the surface of Ti₃C₂ NSs with different oxidation degrees could promote the separation of e⁻-h⁺, which could improve the ROS generation efficiency. Meanwhile, Ti₃C₂ NSs treated by the high-temperature might generate a large amount of oxygen vacancy (V_o) that could capture the e⁻ to prevent the recombination of e⁻-h⁺ pairs, thus ulteriorly increasing the ROS generation effect. Therefore, to prove the above sonodynamic mechanism of H–Ti₃C₂-PEG NSs, the room-temperature ESR was measured, which could determine the V_o of the sample [45,46]. The Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs revealed the V_o signal located at g = 2.03, and the signal intensity was gradually enhanced with the prolonged high-temperature treatment time (Fig. 2h), indicating that the V_o structure could improve the ability to capture e⁻. Furthermore, the photoluminescence (PL) spectra of Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs were future measured to determine the oxygen vacancy structure. The PL spectra showed that the emission intensities of H_L-Ti₃C₂-PEG NSs and H_H-Ti₃C₂-PEG NSs were much lower than that of Ti₃C₂ NSs (Fig. S15), probably due to that most of the V_o in H_H-Ti₃C₂-PEG NSs could capture the photo-excited electrons, then the decreased excite energy reduced the emission intensity. According to the above results, the probable mechanism was confirmed as fellows (Fig. 2g). Owing to the TiO_x formation on the surface of Ti₃C₂ NSs, the electron transfer could be accelerated from the valence band (VB) to the conduction band (CB) when receiving the US irradiation. More importantly, the V_o of Ti₃C₂ NSs treated by high-temperature could capture e⁻ and prevent the recombination of e⁻-h⁺ pairs. Therefore, the O₂ molecules captured the e⁻ and generated large amounts of ¹O₂, leading to the high generation efficiency of ROS (for example, ¹O₂) with H–Ti₃C₂ NSs rather than Ti₃C₂ NSs under US irradiation. In brief, the formation of TiO_x and oxygen vacancy of Ti₃C₂ NSs contributed to the improved sonodynamic effect.

The optical properties of the Ti₃C₂-PEG NSs were studied afterwards. Similar to the Ti₃C₂ NSs, both H_L-Ti₃C₂-PEG NSs and H_H-Ti₃C₂-PEG NSs had good absorbance in NIR I and NIR II regions (Fig. S16). Because the laser of NIR ΙΙ (1000–1700 nm) has a higher tissue penetration depth than NIR Ι (700–1000 nm), the 1064 nm laser was selected here as the excitation source for potential PTT. The photothermal performance of Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs showed similar results at the concentration of 50 ppm (Ti ions) under the NIR II laser irradiation (Fig. 2i). For the comprehensive assessment, the photothermal property of Ti₃C₂ NSs, H_L-Ti₃C₂-PEG NSs, and H_H-Ti₃C₂-PEG NSs at the various concentration (0, 25, 50, 100, and 200 ppm) and various power densities (0.5, 0.75, 1, 1.25, and 1.5 W ▪ cm⁻²) were carefully investigated (Fig. S17), and the photothermal curves showed the concentration-dependent and power-dependent behavior of Ti₃C₂ NSs before and after high-temperature treatment. Furthermore, the photothermal conversion efficiency (η) of Ti₃C₂ NSs and H_H-Ti₃C₂ NSs was calculated to be ~50.8% and ~49.6%, respectively, much higher than the previously reported Ti₃C₂-based PTT agents (Fig. 2j, Fig. S18). To evaluate the photothermal stabilities of H_H-Ti₃C₂-PEG NSs, the laser on/off through five cycles and the photothermal absorbance were not changed obviously, which showed excellent photothermal stability (Fig. 2k, Fig. S19).

2.3. In vitro Sonodynamic and photothermal performance of H–Ti₃C₂-PEG NSs

Based on the above results, the H_H-Ti₃C₂-PEG NSs possessed great photothermal and sonodynamic efficiency. Next, the in vitro PTT and SDT properties of H_H-Ti₃C₂ NSs were evaluated (Fig. 3a). Firstly, the cytotoxicity of the H_H-Ti₃C₂-PEG NSs was assessed by the standard methyl thiazolyl tetrazolium (MTT) assay, which showed that H_H-Ti₃C₂-PEG NSs had no obvious cytotoxicity even the concentration was as high as 100 μg mL⁻¹ at the different incubation time (6, 12, and 24 h) (Fig. 3b). Next, the standard MTT assay was utilized to evaluate the therapeutic efficiency by the H_H-Ti₃C₂-PEG NSs (Fig. 3c). Using 1064 nm laser irradiation for 10 min (<1 W ▪ cm⁻¹), the temperature of the cell culture medium maintained at ~ 42 °C, and the cell viability was as high as ~79.5%. When the cells were irradiated by US only (40 KHz, 3W ▪ cm⁻¹, 1 min per cycle, 5 cycles), the cell viability was decreased to ~ 53.4%. Interestingly, the cell viability was as low as ~13.4% with the combined 1064 nm laser irradiation and then US treatment. It might be the reason that the mild photothermal effect promoted the endocytosis and enhanced efficiency of SDT. To verify this hypothesis, the Cy5.5-labeled H_H-Ti₃C₂-PEG NSs were incubated with 4T1 cells for different times. It could be found that the fluorescence intensity of Cy5.5 increased with time, and the group of H_H-Ti₃C₂-PEG NSs plus laser irradiation showed a higher fluorescence intensity than that in the H_H- Ti₃C₂-PEG NSs group (Fig. 3d and e). confirmed the hypothesis that the mild photothermal effect could improve the efficiency of SDT by promoting the endocytosis of H_H- Ti₃C₂-PEG NSs.

Fig. 3.

In vitro mild PTT-enhanced SDT of H_H-Ti₃C₂-PEG NSs. (a) Schematic illustrating of H_H-Ti₃C₂-PEG NSs for the mild PTT-enhanced SDT. (b) Relative cell viability of 4T1 cells with the H_H-Ti₃C₂-PEG NSs with various concentrations for 6, 12, and 24 h (n = 6 biologically independent samples). (c) Relative cell viability of 4T1 cells after different treatments (G 1-G 7, detailed at the end of the caption) (***P < 0.001, **P < 0.01, or *P < 0.05). (d) Confocal images of 4T1 cells incubated with Cy5.5-conjugated H_H-Ti₃C₂-PEG NSs for different times with/without 1064 nm laser irradiation. (e) Quantitative analysis of the fluorescence intensity in (d) (n = 6 cells examined over independent micrographs). (f) Flow-cytometry apoptosis assay of 4T1 cells after different treatments stained with Annexin-FITC and PI. (g) Confocal images of 4T1 cells after incubation H_H-Ti₃C₂-PEG NSs followed by staining with Calcein AM (green, live cells) and propidium iodide (red, dead cells) after different treatments (G 1-G 7). (h) Confocal images of 4T1 cells stained with DAPI (blue, nuclei) and DCFH-DA (green, intracell ROS) after various treatments (G 1-G 7). G 1: Control, G 2: H_H-Ti₃C₂-PEG, G 3: Laser, G 4: US, G 5: H_H-Ti₃C₂-PEG + Laser, G 6: H_H-Ti₃C₂-PEG + US, G 7: H_H-Ti₃C₂-PEG + Laser + US. H_H-Ti₃C₂–PEG NSs: 50 ppm, NIR laser: 1064 nm, < 1W ▪ cm⁻², 10 min, T < 42 °C; US irradiation: 40 kHz, 3W ▪ cm⁻², 1 min per cycle, 5 cycles. Error bars = standard deviation (n = 6). Data are presented as mean values ± SD. A representative image of three biological replicates from each group is shown.

Next, the live/dead co-staining assay was conducted and further confirmed that the mild PTT could enhance the SDT therapeutic effect (Fig. 3g). It also found that the mild photothermal effect could improve the cell membrane permeability and increase the cellular uptake of the H_H-Ti₃C₂-PEG NSs. To further demonstrate the treatment, the study of apoptosis assay based on the typical Annexin V-FITC and PI was conducted (Fig. 3f). The cell viabilities in the groups of H_H-Ti₃C₂-PEG + Laser and H_H-Ti₃C₂-PEG + US were ~85.8% and ~74.9%, respectively, however, the majority of cells were killed by the H_H-Ti₃C₂-PEG + Laser + US, with only ~4% of cells survived. To investigate the mechanism of cell apoptosis by the H_H-Ti₃C₂-PEG NSs, the ROS staining assay was tested by using 2,7 - dichlorofluorescein diacetate (DCFH-DA, green color). The strongest green signal was observed in the group of H_H-Ti₃C₂-PEG NSs + Laser + US, indicating that the enhanced uptake of H_H-Ti₃C₂-PEG NSs by the mild photothermal effect was helpful for a large amount of ROS generation under US irradiation, as compared with that of H_H-Ti₃C₂-PEG NSs + US group (Fig. 3h, Fig. S20). These results demonstrated that the mild photothermal could promote endocytosis and the H_H-Ti₃C₂-PEG NSs could generate ROS under US irradiation to kill the cancer cells.

For evaluating the synergistic effect of H_H-Ti₃C₂-PEG NSs in vivo, the blood circulation was firstly studied to monitor the biocompatibility and tumor accumulation potential. After the intravenous (i.v.) injection of H_H-Ti₃C₂-PEG NSs into the Balb/c mice bearing 4T1 tumor, the pharmacokinetics indicated that the H_H-Ti₃C₂-PEG NSs had good biocompatibility and stability (Fig. 4a). It would be more accurate to know the concentration of H_H-Ti₃C₂-PEG NSs in the tumor site, which could help to design the optimal schedule to initiating treatment and reducing the damage to the surrounding normal tissue. Then photoacoustic (PA) imaging was used to investigate the biodistribution owing to the great absorbance in NIR windows. In vitro PA imaging showed that the higher concentration of the H_H-Ti₃C₂-PEG NSs, the stronger the PA signal (Fig. 4b, Fig. S21). In vivo PA imaging demonstrated that PA signal located at the tumor site became stronger with the prolonged circulating time due to the enhanced permeability and retention (EPR) effect, with the highest signal appeared in the tumor site at 12 h post injection, and it could retain in the tumor site for a longer time, providing the long-term window for cancer treatment (Fig. 4c and d). Afterwards, the biodistribution of H_H-Ti₃C₂-PEG NSs in the tumor was quantitatively analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) to determine Ti ions. It could be found that the tumor uptake of the H_H-Ti₃C₂-PEG NSs was ~5.8% ID g⁻¹ after i. v. Injection for 12 h, evidencing an efficient tumor accumulation of the H_H-Ti₃C₂-PEG NSs (Fig. 4e).

Fig. 4.

In vivo tumor accumulation and mild PTT-defeated tumor hypoxia via H_H-Ti₃C₂-PEG NSs. (a) Blood circulation of H_H-Ti₃C₂-PEG NSs post i. v. injection. (b) PA imaging of H_H-Ti₃C₂-PEG NSs. (c) In vivo PA imaging of 4T1 tumor-bearing mice. (d) Time-dependent tumor PA signals at 800 nm based on PA imaging data in (c). (e) Biodistribution of H_H-Ti₃C₂-PEG NSs in mice after 12 h by i. v. injection. (f) IR thermal images of 4T1 tumors under the 1064 nm laser irradiation. (g) Tumor slices staining of different treatments. H_H-Ti₃C₂–PEG NSs: 20 mg ▪ kg⁻¹; NIR laser: 1064 nm, < 1W ▪ cm⁻², 15 min (n = 3 biologically independent mice). Data are presented as mean values ± SD.

2.4. In vivo Sonodynamic and photothermal performance of H–Ti₃C₂-PEG NSs

Encouraged by the excellent properties indicated by the in vitro studies, the in vivo sonodynamic and photothermal performances of H–Ti₃C₂-PEG NSs were investigated. First of all, the in vivo photothermal property of the H_H-Ti₃C₂-PEG NSs for the NIR ΙΙ laser-induced hyperthermia was studied (Fig. 4f). 4T1 tumor-bearing mice were exposed to the 1064 nm laser irradiation after i. v. Injection for 12 h (1 W ▪ cm⁻², 5 min), then the tumor temperature was quickly increased to ~50.3 °C, that can achieve the condition of mild photothermal. (Fig. S22). However, the temperature of the control group was less increased, mainly due to the strong NIR Ⅱ absorbance and the efficient tumor accumulation of the H_H-Ti₃C₂-PEG NSs. The complex TME and abnormal blood vessels lead to severe hypoxia in the solid tumor, which greatly reduces the therapeutic effect of ROS. Based on the previous reports, the mild photothermal effect could promote blood circulation and increase the number of red blood cells, thus increasing the oxygen-carrying capacity of hemoglobin, which could reverse the hypoxic microenvironment and increase the effect of ROS-based therapies [22,47]. PA imaging of oxyhemoglobin saturation was detected in disparate points after irradiation with different times (5 min, 15 min, 25 min, T < 42 °C). It was obvious that the blood oxygen was time-dependent enhanced and reached the maximum at 10 min (Fig. S23). Later, an immune-fluorescence hypoxia staining assay was conducted (Fig. 4g). The hypoxia signals of the H_H-Ti₃C₂-PEG NSs + Laser group were significantly decreased compared with other control groups, which suggested that the mild photothermal effect could relieve the tumor hypoxia, so that the sonodynamic effect could be enhanced.

Then, we studied the anti-tumor effect of the H_H-Ti₃C₂-PEG NSs via the mild PTT- enhanced SDT of the 4T1 tumor-bearing mice (Fig. 5a). All the mice were randomly divided into seven group: (1) control; (2) H_H-Ti₃C₂-PEG NSs (i.v. Injection, 20 mg ▪ kg⁻¹); (3) Laser (1064 nm, < 1W ▪ cm⁻², 15min, T < 42 °C); (4) US (40 kHz, 3W ▪ cm⁻², 1 min per cycle, 15 cycles); (5) H_H-Ti₃C₂-PEG NSs + Laser; (6) H_H-Ti₃C₂-PEG NSs + US; (7) H_H-Ti₃C₂-PEG NSs + Laser + US. The H_H-Ti₃C₂-PEG NSs were administrated intravenously. According to the above PA imaging results, the 1064 nm laser and US irradiation were subsequently applied to the tumors after 12 h post injection. After treatments, the tumor growth of different groups was monitored (Fig. 5b). Compared with the control group, the tumors grew rapidly in the groups of H_H-Ti₃C₂-PEG NSs, Laser, US, and H_H-Ti₃C₂-PEG NSs + Laser, but slowly in the H_H-Ti₃C₂-PEG NSs + US group, which showed a certain inhibitory effect, indicating that the H_H-Ti₃C₂-PEG NSs had a good sonodynamic effect for tumor inhibition. From the tumor inhibitory rate statistics, the groups of H_H-Ti₃C₂-PEG NSs, Laser, US, and H_H-Ti₃C₂-PEG NSs + Laser only had the extremely low inhibitory effect, while the H_H-Ti₃C₂-PEG NSs + US group possessed ~54.9% inhibitory rate, and yet the tumors could not achieve ablation thoroughly. When treated with H_H-Ti₃C₂-PEG NSs + Laser + US, the tumors were completely suppressed (Fig. 5c), probably due to that the mild photothermal effect alleviated the hypoxic microenvironment, which further enhanced SDT efficiency. Importantly, the tumors in H_H-Ti₃C₂-PEG NSs + Laser + US group did not recur, which significantly increased the overall survival. However, the size of tumors in the other control groups reached the death criteria gradually, revealing that the mild photothermal-enhanced SDT had an obvious synergistically therapeutic outcome with the H_H-Ti₃C₂-PEG NSs (Fig. 5d).

Fig. 5.

In vivo mild PTT-enhanced SDT of H_H-Ti₃C₂-PEG NSs. (a) Schematic illustration to show the combination of PTT and SDT of H_H-Ti₃C₂-PEG NSs. (b) Average tumor growth curves on mice with different treatments (G 1-G 7, detailed at the end of the caption), (***P < 0.001, **P < 0.01, or *P < 0.05). (c) Tumor inhibition rates of different treatments (G 1-G 7). (d) Survival curves of mice after various treatments. (e) Micrograph of DCFH-DA stained tumor slices with different treatments. (f) H&E stained tumor slices G 1: Control, G 2: H_H-Ti₃C₂-PEG, G 3: Laser, G 4: US, G 5: H_H-Ti₃C₂-PEG + Laser, G 6: H_H-Ti₃C₂-PEG + US, G 7: H_H-Ti₃C₂-PEG + Laser + US. H_H-Ti₃C₂–PEG NSs: 20 mg ▪ kg⁻¹; NIR laser: 1064 nm, < 1 W ▪ cm⁻², 15 min, T < 42 °C; US irradiation: 40 kHz, 3W ▪ cm⁻², 1 min per cycle, 15 cycles. (n = 5 biologically independent mice). Data are presented as mean values ± SD.

To further explore the mechanism of synergistic therapy mediated by H_H-Ti₃C₂-PEG NSs, the ROS levels in the tumors after the various treatments were evaluated via DCFH-DA staining (Fig. 5e). It was clear that the groups of control, H_H-Ti₃C₂-PEG NSs, Laser, US, and H_H-Ti₃C₂-PEG NSs + Laser only induced weak green fluorescence in tumor slices, while the groups of H_H-Ti₃C₂-PEG NSs + US and H_H-Ti₃C₂-PEG NSs + Laser + US showed enhanced green fluorescence in the tumor slices. The strongest green fluorescence intensity appeared in H_H-Ti₃C₂-PEG NSs + Laser + US group, and the intensity was 2.8 -fold higher than that of the control group (Fig. S24). Therefore, the mild PTT could alleviate the hypoxic tumor environment and promote the generation of ROS under US irradiation. Thereafter, the hematoxylin and eosin (H&E) staining of tumor slides was conducted after the various treatments at 24 h (Fig. 5f). In the groups of H_H-Ti₃C₂-PEG NSs + US and H_H-Ti₃C₂-PEG NSs + Laser + US, nearly all the tumor cells were damaged severely while the other groups had little impact on tumor cells. These results confirmed that the mild PTT could enhance the efficacy of SDT to achieve a good synergistic effect by the H_H-Ti₃C₂-PEG NSs.

Lastly, biosafety is the most important issue to be considered for the wide biomedicine application of nanomaterials, especially for inorganic nanomaterials [48,49]. Firstly, the H&E staining showed that the main organs (heart, liver, spleen, lung, kidney, and brain) of mice had no obvious morphological changes in different time (Fig. 6a). Then, we moved to study the degradation behavior of the synthesized H_H-Ti₃C₂-PEG NSs. The H_H-Ti₃C₂-PEG NSs were dispersed in various solutions of H₂O, PBS, and RPMI (10% FBS) at 37 °C for different incubation times (Fig. 6b–d). According to the UV–vis–NIR spectra, the absorbance of H_H-Ti₃C₂-PEG NSs around 808 nm decreased after 21 days, and the photograph of H_H-Ti₃C₂-PEG NSs showed the fading color, which evidenced the biodegradation of H_H-Ti₃C₂-PEG NSs. However, a relatively smaller change in the 1640 cell culture medium, probably due to the reason that the cell culture medium contained 10% FBS could partially protect the degradation of Ti₃C₂ nanosheets. Furthermore, TEM and XRD were used to explore the morphology and component after the biodegradation of H_H-Ti₃C₂-PEG NSs. TEM image showed the mixture of nanosheets and nanodots, and the XRD patterns should the amorphous structure in the process the degradation (Fig. S25). Next, we discovered the in vivo distribution profile and the metabolic pathway of H_H-Ti₃C₂-PEG NSs (Fig. 6e and f). At 24 h after i. v. injection, the biodistribution showed the relatively high accumulation of H_H-Ti₃C₂-PEG NSs in liver (~36.5% ± 1.8% ID g⁻¹), spleen (~38% ± 3.0% ID g⁻¹), and lung (~14% ± 4.5% ID g⁻¹). However, the content dropped to 12% ± 0.6% ID g⁻¹ in liver, 13.7% ± 1.8% ID g⁻¹ in spleen, and 2.2% ± 0.2% ID g⁻¹ in lung at the 30^th day later, respectively, showing that most of the H_H-Ti₃C₂-PEG NSs had been metabolized out of the body. In order to investigate the metabolism pathway of the H_H-Ti₃C₂-PEG NSs, the feces and urine of mice were collected to detect the content of Ti through ICP-OES. It could be found that H_H-Ti₃C₂-PEG NSs were mainly excreted through the hepatic metabolism due to the high concentration of Ti ions in the feces. Moreover, there was no significant change in the body weight of mice during the treatment, indicating that the H_H-Ti₃C₂-PEG NSs had no obvious toxicity at the injection dose (20 mg ▪ kg⁻¹) (Fig. 6g). Subsequently, the blood was collected after injection of H_H-Ti₃C₂-PEG NSs for blood routine and blood biochemical tests. All these hematological indexes had no significant difference among all the groups (Fig. S26). In brief, the H_H-Ti₃C₂-PEG NSs could be used as safe sonosensitizers for enhanced SDT without causing any long-term toxicity.

Fig. 6.

Biosafety evaluation of H_H-Ti₃C₂-PEG NSs. (a) H&E staining of mice major organs before and post i. v. injection with H_H-Ti₃C₂-PEG NSs at 1, 14, and 30 days. (b-d) UV–vis–NIR spectra of H_H-Ti₃C₂-PEG NSs in H₂O (b), PBS (c), and RPMI (d) for different days. Insert are the corresponding photograph. (e) Biodistribution of H_H-Ti₃C₂-PEG NSs. (f) The detected Ti mass in urine and feces. (g) The body weight variation of mice after various treatments. (n = 5 biologically independent mice). Data are presented as mean values ± SD.

3. Conclusion

In summary, the oxygen-defective Ti₃C₂ NSs (H–Ti₃C₂ NSs) were successfully established by two-step methods of chemical exfoliation and high-temperature treatment. After high-temperature treatment, the H–Ti₃C₂ NSs displayed an excellent sonodynamic efficacy, and the efficiency was enhanced by the long-time high-temperature treatment. Importantly, the oxygen defect structure of H–Ti₃C₂ NSs treated by the high-temperature also could prevent the recombination of e⁻-h⁺ pairs, which further enhanced the sonodynamic effect under US irradiation. Meanwhile, the H–Ti₃C₂ NSs also had relatively high absorbance in the NIR ΙΙ window, which could produce heat under laser irradiation. After PEGylation, the H–Ti₃C₂-PEG NSs showed great stability and biocompatibility. The good photothermal efficacy of H–Ti₃C₂-PEG NSs promoted the endocytosis of the H–Ti₃C₂-PEG NSs, and further enhanced the efficacy of SDT. In vivo studies showed that the mild photothermal effect could accelerate blood circulation and alleviate the hypoxia tumor microenvironment to realize PTT enhanced SDT. Importantly, the biodegradable H–Ti₃C₂ NSs were excreted out of the body without inducing any long-term toxicity. In brief, our work developed the H–Ti₃C₂ NSs as a high-efficiency and safe sonosensitizer for photothermal-enhanced SDT, which highlighted the extension of the biomedical application of MXene-based nanoplatforms.

CRediT authorship contribution statement

Guangqiang Li: Writing – review & editing, Writing – original draft, designed the experiments, synthesized the materials, performed the sonodynamic and photothermal experiments, performed the cells experiments, wrote the paper, reviewed and edited the paper. Xiaoyan Zhong: Writing – review & editing, Writing – original draft, performed animal experiments, wrote the paper, reviewed and edited the paper. Xianwen Wang: Writing – review & editing, synthesized the materials, performed the sonodynamic and photothermal experiments, reviewed and edited the paper. Fei Gong: Writing – review & editing. Huali Lei: Writing – review & editing. Yangkai Zhou: Writing – review & editing. Chengfei Li: Writing – review & editing. Zhidong Xiao: Writing – review & editing, Writing – original draft. Guoxi Ren: Writing – review & editing, Formal analysis. Liang Zhang: Writing – review & editing, Formal analysis. Zhiqiang Dong: Writing – review & editing, Formal analysis. Zhuang Liu: Writing – review & editing, Formal analysis. Liang Cheng: Writing – review & editing, Writing – original draft.

Declaration of Competing interest

The authors declare no competing financial interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: