Aptamer modified Ti₃C₂ nanosheets application in smart targeted photothermal therapy for cancer

Abstract

Background

Ti₃C₂ is a type of transition metal carbides and nitrides (MXenes) with high light-to-heat conversion efficiency property, which has been widely used in cancer treatment recently. In fact, active targeting delivery of MXenes nanomaterials with targeting molecule could enhance the therapeutic efficacy. However, targeted therapy of MXenes has not been further studied in the past. Aptamers (Apt) with excellent affinity and high specificity properties have been widely used as targeting tools. Predictably, the incorporation of Apt into Ti₃C₂ nanomaterials will offer an unprecedented opportunity in the research fields of cancer targeted therapy.

Results

Transmembrane glycoprotein mucin 1 (MUC1) is overexpressed on the surface of MCF-7 cells, and MUC1 Apt (Apt-M) could target MCF-7 cells with high affinity and specificity. Here, a smart targeting nanotherapeutic system Ti₃C₂/Apt-M was fabricated, which could specifically recognize and enter in MCF-7 cells. Benefitting from the desirable targeted performance of Apt-M, MCF-7 cells completed the ingestion process of Ti₃C₂/Apt-Mf nanosheets within 4 h, and Apt-M facilitated the entry of the Ti₃C₂/Apt-Mf nanosheets into MCF-7 cells. Besides, Ti₃C₂/Apt-M nanosheets exhibited the potential as an outstanding photothermal agent (PTA) because of the photothermal performance inherited from wrapped Ti₃C₂ nanosheets. As demonstrated, upon 808 nm laser irradiation, the Ti₃C₂/Apt-M nanotherapeutic system displayed a satisfactory antitumor effect by targeted photothermal therapy both in vitro and in vivo.

Conclusion

This study provides a new idea for the development of MXenes nanotherapeutic system with high active targeting performance.

Graphical Abstract

Introduction

Targeted delivery of anticancer drugs or nanomaterials to enhance therapeutic efficacy has become a new trend in cancer treatment (Zhu et al. 2022). Active cancer targeting can be achieved by conjugating different tissue-specific groups or ligands to nanomaterials surface, including peptides, antibodies, small molecules, aptamers (Apt) and so on (Arslan et al. 2021; Tan et al. 2021). Particularly, Apt is a class of artificially synthesized short- and single-stranded DNA or RNA sequences, which has a broad range of targets, including small molecules, peptides, proteins, cells and even viruses (Li et al. 2021). Apt have been widely used as promising targeting moieties, which due to their significant advantages, including high selectivity and affinity, high thermal stability, low toxicity and immunogenicity, low molecular weight, easy synthesis and modification (Xie et al. 2021). Such significant advantages make Apt a suitable alternative for peptides, antibodies, small molecules (Liu et al. 2022a; Yuhan et al. 2022). As a result, Apt have to be used as targeting tools for various anticancer drugs or nanomaterials delivery, such as Au nanocage (Yang et al. 2021), carbon spheres (Sargazi et al. 2022), graphene oxide (Du et al. 2020), mesoporous silica (Vandghanooni et al. 2020), MOF (Alijani et al. 2020), and DNA origami (Pan et al. 2020).

Two-dimensional (2D) titanium carbide (Ti₃C₂) nanomaterial is a kind of transition metal carbides and nitrides (MXenes), which was first synthesized by etching Ti₃AlC₂ with HF acid in 2011 (Li et al. 2019; Naguib et al. 2011; VahidMohammadi et al. 2021). To date, nearly 30 types of MXenes have been successfully experimentally synthesized, however, more than 100 types of MXenes have been computationally predicted (Gogotsi and Anasori 2019). Ti₃C₂ nanomaterial was gifted with some exciting and unique properties such as high surface-to-volume ratio, high atomic number, paramagnetic behavior and high light-to-heat conversion efficiency properties, which make it especially suitable nanoplatform for the application of tumor therapy (Huang et al. 2022; Xu et al. 2022; Wu et al. 2022; Han et al. 2018; Tang et al. 2019). With the passively targeted and accumulated in tumor tissues through enhanced permeability and retention (EPR) effects, Ti₃C₂ nanomaterial could be used as cancer treatment and theranostics applications (Lin et al. 2017; Liang et al. 2019; He et al. 2022; Li et al. 2022; Zhang et al. 2022). However, active targeting delivery of Ti₃C₂ nanomaterial could enhance the accumulation of nanomaterials at tumor sites and improve the effect of tumor treatment (Liu et al. 2020, 2017, 2021; Li et al. 2018).

Transmembrane glycoprotein mucin 1 (MUC1) was a heterodimeric protein overexpressed on the surface of tumor cells in most malignant tumors including breast, ovarian and lung cancers (Correa et al. 2022). Moreover, MUC1 aptamer (Apt-M) has been developed to target MUC1-expressing cancer cells such as MCF-7 with high affinity and specificity, which could enhance anticancer nanomaterials delivery in cancer therapy (Khan et al. 1239).

Herein, a smart targeting nanotherapeutic system Ti₃C₂/Apt-M (Scheme 1) was developed for cancer-targeted photothermal therapy (PTT). In this work, the Apt-M was covalently bound to the surface of nanotherapeutic system, which enable MCF-7 cells ingest more Ti₃C₂/Apt-M nanosheets. As expected, the temperature of Ti₃C₂/Apt-M nanotherapeutic system raised rapidly under laser irradiation, which achieve the purpose of PTT. Furthermore, the Ti₃C₂/Apt-M nanotherapeutic system inhibited tumor growth using targeted PTT has been systematically demonstrated in vitro and in vivo. This study provides a new idea for the development of MXenes nanotherapeutic system with smart targeting performance.

Scheme 1

Schematic illustration of Ti₃C₂/Apt-M nanotherapeutic system for smart targeted photothermal therapy for cancer

Results and discussion

Synthesis and characterization

Ti₃C₂ nanosheets were prepared from bulk Ti₃AlC₂ by chemical exfoliation method (Lin et al. 2017). After HF etching to remove the Al layer, the prepared Ti₃C₂ bulk showed an accordion-like structure (Additional file 1: Fig. S1). After further intercalation with TPAOH, the prepared Ti₃C₂ nanosheets has an ultrathin 2D sheet-like morphology with excellent hydrophilicity and dispersibility (Fig. 1a). The theoretical thickness of Ti₃C₂ nanosheet is 0.474 nm (Lin et al. 2017). AFM profile showed the average lateral size of Ti₃C₂ nanosheet was about 100 nm and the thickness was lower than 1.74 nm, which proved that the prepared Ti₃C₂ nanosheets had fewer layers (Additional file 1: Fig. S2). The EDS (Additional file 1: Fig. S3) and XPS (Additional file 1: Fig. S4) results showed that the Al element was completely removed in the prepared Ti₃C₂ nanosheets (Additional file 1: Figs. S3, S4). XRD further revealed that only the (002) peak at 6.0° was still evident in Ti₃C₂ nanosheets (Fig. 1b). Taken together, these results proved that Ti₃C₂ nanosheets was successfully prepared.

Fig. 1

Synthesis and characterization. (a) TEM image of Ti₃C₂ nanosheets (inset shows a digital photo of Ti₃C₂ nanosheets dispersed in water exhibiting Tyndall effect). (b) XRD patterns of Ti₃AlC₂ ceramic bulks and Ti₃C₂ nanosheets. (c) FT–IR spectra, (d) Dynamic light scattering analysis and (e) zeta potential analysis of Ti₃C₂, Ti₃C₂-PEG and Ti₃C₂/Apt-M nanosheets. (f) Hemolysis assay of Ti₃C₂, Ti₃C₂-PEG and Ti₃C₂/Apt-M nanosheets at different concentrations

After the surface modification of COOH–PEG–COOH, the stretching vibration of C=O in carboxyl group at ~ 1729 cm⁻¹ was found in FT–IR spectra of Ti₃C₂-PEG (Fig. 1c) (Yathindranath et al. 2013). The –NH₂ modified Apt-M was immobilized on the surface of Ti₃C₂-PEG nanosheets by EDC-NHS reaction. FT–IR spectra of Ti₃C₂/Apt-M showed the characteristic absorption peak of amide bond at ~ 1648 cm⁻¹ (amide peak I, stretching vibration of C=O), ~ 1575 cm⁻¹ (amide peak II, bending vibration of N–H) and ~ 1344 cm⁻¹ (amide peak III, stretching vibration of C–N) (Du et al. 2020). In addition, the hydrodynamic diameter of Ti₃C₂/Apt-M (139 nm) increased slightly compared with those of Ti₃C₂-PEG (119 nm) and Ti₃C₂ (101 nm) (Fig. 1d). Moreover, the zeta potential of Ti₃C₂, Ti₃C₂-PEG and Ti₃C₂/Apt-M was measured as −22.10, −27.43 and −37.30 mV, respectively (Fig. 1e). These results indicated the successful PEG modification and Apt-M functionalization.

The Ti₃C₂/Apt-M nanosheets showed excellent stability and dispersibility in various physiological media (Additional file 1: Fig. S5). Furthermore, the Ti₃C₂/Apt-M nanosheets didn’t cause significant hemolysis (< 5%) even at a high concentration of 200 µg mL⁻¹ (Fig. 1f, Additional file 1: Fig. S6) (Chang et al. 2022). Interestingly, the UV–vis absorption of Ti₃C₂/Apt-M at 808 nm did not change significantly compared to Ti₃C₂ (Additional file 1: Fig. S7).

Photothermal performance

To assess the photothermal performance of Ti₃C₂/Apt-M nanosheets, the extinction coefficient (α) was evaluated to be 16.9 Lg⁻¹ cm⁻¹ according to Lambert–Beer law (Fig. 2a), which was superior to other Ti₃C₂ MXenes composite nanosheets, such as Ti₃C₂-IONP@PEG-GOD nanoparticle (8.57 Lg⁻¹ cm⁻¹) (Liang et al. 2019) and Ti₃C₂-PEG-OVA-Mn²⁺ nanoparticle (6.40 Lg⁻¹ cm⁻¹) (Liu et al. 2022b). Furthermore, the photothermal conversion efficiency (η) of Ti₃C₂/Apt-M nanosheet was calculated to be as high as 31.76% (Fig. 2b, c), which was significantly higher than that of Ti₃C₂-IONP@PEG-GOD nanoparticle (27.27%) (Liang et al. 2019) and Ti₃C₂-PEG-OVA-Mn²⁺ nanoparticle (28.17%) (Liu et al. 2022b). The temperature of Ti₃C₂/Apt-M solutions raised with the increase of Ti₃C₂/Apt-M concentration and laser power density, suggesting the photothermal properties of the Ti₃C₂/Apt-M nanosheets were concentration dependent (Fig. 2d, e) and power dependent (Additional file 1: Fig. S10). It was noteworthy that the temperature of the Ti₃C₂/Apt-M nanosheets (100 µg mL⁻¹) could reach 67.6 °C under 808 nm irradiation in 10 min, which was enough for photothermal tumor ablation (Lu et al. 2022). Moreover, Ti₃C₂/Apt-M nanosheets exhibited satisfactory photothermal stability in five laser on/off cycles (Fig. 2f).

Fig. 2

The photothermal performance of Ti₃C₂/Apt-M nanosheets. a Mass extinction coefficient of Ti₃C₂/Apt-M nanosheets at 808 nm (inset shows UV–vis spectra of Ti₃C₂/Apt-M nanosheets at different concentrations). b Photothermal performance of Ti₃C₂/Apt-M nanosheets dispersed in aqueous solution under 808 nm laser irradiation. c Calculation of time constant and photothermal-conversion efficiency of Ti₃C₂/Apt-M nanosheets at 808 nm laser irradiation. d Infrared thermal images and e temperature changes of Ti₃C₂/Apt-M nanosheet aqueous solutions with different concentrations (20, 40, 60, 80 and 100 µg mL⁻¹) under 808 nm laser irradiation. f Temperature curve of Ti₃C₂/Apt-M nanosheet aqueous solution under five 808 nm laser on/off cycles

Intracellular endocytosis and targeting performance analysis

The affinity and specificity of Apt-M with cancer cells were verified by flow cytometry. A significant increase of fluorescence intensity from Apt-Mf in MCF-7 cells was observed, which was significantly stronger than that of the MCF-7 cells incubated with Apt-Cf (Fig. 3a). Meanwhile, no obvious change was observed in HepG2 cells when incubated with Apt-Mf or Apt-Cf, implying that Apt-M had specificity to MCF-7 cells (Fig. 3b).

Fig. 3

Active targeting performance analysis of Ti₃C₂/Apt-M nanosheets. Flow cytometry analysis of a MCF-7 and b HepG2 cells after incubation with Apt-Cf or Apt-Mf. Flow cytometric analysis of (c) MCF-7 and (d) HepG2 cells incubated with Ti₃C₂/Apt-Mf nanosheets for different time (0, 1, 2, 3, 4, 5 and 6 h). Flow cytometric analysis of e MCF-7 and f HepG2 cells incubated with Ti₃C₂/Apt-Mf, Ti₃C₂/Apt-Mf with free Apt-C, Ti₃C₂/Apt-Mf with free Apt-M for 4 h. g CLSM images of MCF-7 and HepG2 cells incubated with Ti₃C₂/Apt-Mf for 2, 4 and 6 h. Scale bar: 50 μm. h CLSM images of MCF-7 and HepG2 cells incubated with Ti₃C₂/Apt-Mf, Ti₃C₂/Apt-Mf with free Apt-M, Ti₃C₂/Apt-Mf with free Apt-C for 4 h. Scale bar: 50 μm

To reveal the smart targeting performance of Ti₃C₂/Apt-M nanosheets, flow cytometry was used to verify the fluorescence intensity of MCF-7 or HepG2 cells after incubation with Ti₃C₂/Apt-Mf for different time. The fluorescence intensity of MCF-7 cells was significantly higher than that of HepG2 cells at the same time (Fig. 3c, d). Moreover, the fluorescence intensity of MCF-7 cells was not significantly increased after 4 h. The results indicated that MCF-7 cells completed the ingestion process of Ti₃C₂/Apt-Mf nanosheets within 4 h, and Apt-M facilitated the entry of the Ti₃C₂/Apt-Mf nanosheets into MCF-7 cells. For competitive study, MCF-7 cells were pre-incubated with free Apt-M or Apt-C. As expected, the fluorescence intensity of MCF-7 cells was significantly decreased after pre-incubated with free Apt-M (Fig. 3e). Meanwhile, negligible changes in fluorescence intensity were observed in HepG2 cells after pre-incubation with free Apt-C or Apt-M, indicating the ingestion of Ti₃C₂/Apt-M nanosheets into cells was mediated by Apt-M (Fig. 3f). Furthermore, CLSM was conducted to investigate the ingestion behavior and targeting efficacy of Ti₃C₂/Apt-M nanosheets, and the results indicated that the ingestion of Ti₃C₂/Apt-M nanosheets into cells was mediated by Apt-M, which in accordance with the flow cytometry results (Fig. 3g, h).

In vitro targeted photothermal therapy against tumor cell growth

The in vitro anticancer performances of Ti₃C₂/Apt-M nanosheets were evaluated by MTT assay. Negligible toxicity on the MCF-7 and HepG2 was observed after 24 or 48 h treatment with Ti₃C₂-PEG, Ti₃C₂/Apt-C or Ti₃C₂/Apt-M nanosheets, even at a high concentration of 400 µg mL⁻¹ (Additional file 1: Fig. S11). Furthermore, the targeted photothermal therapy against cell growth was assessed under 808 nm laser irradiation. The results showed that only 43.1% of MCF-7 cells survived in Ti₃C₂-PEG + Laser group (Fig. 4a). With the aid of the excellent targeting ability of Apt-M, the viability of MCF-7 cell treated with Ti₃C₂/Apt-M + laser was only 22.7% under the smart-targeted PTT. It was notable that the HepG2 cell viabilities of Ti₃C₂/Apt-M + Laser group were not significantly different from that of Ti₃C₂-PEG + Laser group. Furthermore, compared with Ti₃C₂-PEG and Ti₃C₂/Apt-C, Ti₃C₂/Apt-M nanosheets showed excellent targeted therapeutic effects on MCF-7 at different concentrations (Additional file 1: Fig. S12). The MTT results further confirmed the targeted therapeutic effect of Ti₃C₂/Apt-M nanosheets were mediated by Apt-M, which making them more ingested by MCF-7 cells and displayed a concentration-dependent anticancer performance.

Fig. 4

In vitro targeted photothermal therapy against tumor growth. a Cell viability of MCF-7 and HepG2 cells after 4 h incubation with different nanomaterials and treatments. b CLSM images of MCF-7 cells stained with Calcein-AM (green fluorescence) and PI (red fluorescence) staining. Scale bar: 100 μm. c Flow cytometry apoptosis analysis of Annexin V-FITC and PI strained MCF-7 cells. d Quantification analysis of the percentages of apoptotic cells after various treatments in flow cytometry

Additionally, to reveal the desirable targeted PTT effect by Ti₃C₂/Apt-M, cells after treatments were stained with Calcein-AM and PI, respectively, and observed by CLSM. Ti₃C₂/Apt-M could effectively ablate the MCF-7 cells under laser irradiation as evidenced by the number of dead cells with red fluorescence (PI), which was in consistent with the MTT results (Fig. 4b). The targeted PTT against MCF-7 cell was further investigated by flow cytometry analysis. The percentage of apoptotic cells (including early and late apoptotic cells) induced by Ti₃C₂/Apt-M + Laser reached 52.17%, which exhibited the highest therapeutic efficacy among all treatments (Fig. 4c–d).

In vivo-targeted photothermal therapy against tumor growth

Motivated by the desirable in vitro-targeted PTT performance, the MCF-7 cell xenograft mice models were established and used for the targeted PTT in vivo. As expected, the surface temperature of tumor sites in Ti₃C₂/Apt-M + Laser treatment mice increased rapidly to 61.4 °C, which was significantly higher than other laser irradiated groups, suggesting that the Ti₃C₂/Apt-M nanosheets could smartly target MCF-7 cells and increase the accumulation in tumor areas (Additional file 1: Fig. S13). The body weight fluctuations of all mice were negligible during the treatment period (Additional file 1: Fig. S14), indicating the satisfactory biosafety of the Ti₃C₂/Apt-M nanosheets in vivo treatment. More importantly, the tumor volume of mice treated with Ti₃C₂/Apt-M + Laser was suppressed effectively, while the tumors in other treatment groups continuously grew at similar rates (Fig. 5a–c). Moreover, the tumor weight in Ti₃C₂/Apt-M + Laser treatment group was significantly lower than other treatment groups (Fig. 5d). The results strongly suggested that Apt-M mediated targeted PTT display excellent therapeutic effect for suppressing the tumor growth, although the photothermal conversion efficiency of Ti₃C₂/Apt-M nanosheets is not the most prominent among MXenes and other inorganic photothermal agents (Additional file 1: Table S2).

Fig. 5

In vivo targeted photothermal therapy against tumor growth. a Digital photos of excised tumors from sacrificed mice at the end of therapy. b Time-dependent tumor volume curves of tumor-bearing mice after different treatments in 17 d. c Representative digital photographs of tumor-bearing mice after different treatments. d Tumor weight of excised tumors from sacrificed mice at the end of therapy. e H&E staining, TUNEL staining and Ki-67 staining of excised tumor tissue after different treatments. Scale bar: 100 μm

Furthermore, the tumors were harvested and stained by hematoxylin–eosin (H&E) and immunohistochemistry (IHC). For H&E staining, Ti₃C₂/Apt-M + Laser treatment group showed obvious tumor tissue shrinkage and damage compared to the other groups (Fig. 5e). TDT-mediated dUTP nick-end labelling (TUNEL) IHC staining was taken to further reveal the apoptosis of tumor cells, and the high expression of this marker was shown in the tumor from mice treated with Ti₃C₂/Apt-M + Laser. Similarly, Ki-67 IHC staining results verified the least cell proliferation with the treatment of Ti₃C₂/Apt-M + Laser in tumor tissues compared with other groups, while further demonstrating the better targeted therapeutic effect on inhibiting the tumor growth. Moreover, to investigate the biosafety of the proposed targeted PTT, the major organs (heart, liver, spleen, lung, and kidney) were gathered and sectioned for H&E staining and any toxic effects or inflammation could not be observed in these organs (Fig. S15). Overall, Ti₃C₂/Apt-M was a highly promising targeted photothermal agent (PTA) for in vivo tumor targeted therapy with satisfactory biosafety.

Conclusions

In summary, this work successfully designed a distinctive Ti₃C₂/Apt-M nanosheets for smart-targeted photothermal therapy of cancer in vitro and in vivo. Especially, the Ti₃C₂ nanosheets covalently linked Apt-M were endowed with smart targeting properties, which could specifically recognize and enter in MCF-7 cells. Moreover, Ti₃C₂/Apt-M nanosheets exhibited the potential as an outstanding PTA because of the photothermal performance inherited from wrapped Ti₃C₂ nanosheets. As demonstrated, upon 808 nm laser irradiation, the Ti₃C₂/Apt-M nanosheets displayed a satisfactory antitumor effect by targeted photothermal therapy both in vitro and in vivo. This work expanded the application of MXenes for targeted therapy. More commendable, different types of Apt could be used for different types of tumors, so as to achieve precision medicine and personalized medicine.

Experimental

Materials

Ti₃AlC₂ (> 98%, 200-meshes) ceramics powder was purchased from Shanghai Mclean Biochemical Technology Co., Ltd. Hydrofluoric acid (HF, AR, 40.0%), tetrapropylammonium hydroxide (TPAOH, AR, 40.0%) and Dimethyl Sulfoxide (DMSO) were obtained from Shanghai Adamas reagent Co., Ltd. The COOH-PEG-COOH (MW 4000) was purchased from Shanghai Yare Biotechnology Co., Ltd. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide Hydrochloride (EDC•HCl) and N-Hydroxysuccinimide (NHS) were acquired from Shanghai Aladdin reagent Co., Ltd. The phosphate buffered saline (PBS), trypsin, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and penicillin/streptomycin were obtained from Shanghai Sigma–Aldrich Trading Co., Ltd. Fetal Bovine Serum (FBS) were obtained from Biological Industries reagent Co., Ltd. The Roswell Park Memorial Institute-1640 (RPMI-1640) medium was purchased from Biosharp reagent Co. Hoechst 33342 reagent, Calcein AM/PI cytotoxicity assay kit and Annexin V-FITC/PI apoptosis assay kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd. MUC1 Apt (Apt-M), control Apt (Apt-C), 5-Carboxyfluorescein (FAM) labeled Apt-M (Apt-Mf) and FAM-labeled control Apt (Apt-Cf) sequences (Additional file 1: Table S1) were synthesized and HPLC purified by Shanghai Sangon Biotech Co., Ltd.

Characterizations

Scanning Electron Microscope (SEM) images were measured by TESCAN MAIA3 microscope. Transmission Electron Microscope (TEM) images were obtained by JEOL JEM-2100F transmission electron microscope at 200 kV accelerating voltage, and the elements composition of Ti₃C₂ nanosheets was analyzed by X-MaxN 80 T IE250 energy dispersive spectroscopy (EDS). Atomic Force Microscope (AFM) measurement was carried out on Bruker Dimension Icon system at typical rate of 3 min per image. X-ray diffraction (XRD) patterns were recorded on a Rigaku SmartLab SE diffractometer, with the angles from 5° to 65° and the rate of 2° min⁻¹. X-ray photoelectron spectroscopy (XPS) spectra was detected by a Thermo Scientific K-Alpha XPS energy spectrometer. Fourier transform infrared (FT–IR) spectra were recorded by a Thermo Scientific NICOLET iS20 Spectrometer. Hydrodynamic size and zeta potentials of nanomaterials were measured using Malvern Zetasizer Nano ZS90 nano particle potential meters. PerkinElmer Lambda35 spectrometer was used to record UV–vis absorption spectra. The laser irradiation was conducted by a high-power 808 nm multimode pump (Cnilaser DPS–808–Q) laser of Changchun new industry photoelectric technology Co., Ltd. The temperature and thermal images were recorded on an FOTRIC 323pro infrared thermal imager. The absorbance value of 96-well plate was measured by Tecan Infinite M200PRO microplate reader. Beckman Coulter CytoFLEX flow cytometer was used for the flow cytometry experiments. The confocal laser scanning microscopy (CLSM) images were recorded using an Olympus FV1200V inverted CLSM.

Preparation of Ti₃C₂ nanosheets (MXenes)

Ti₃C₂ nanosheets were prepared from Ti₃AlC₂ by chemical exfoliation method (Lin et al. 2017). In brief, Ti₃AlC₂ ceramics (1.0 g) were slowly suspended in 20 mL of a 40% HF aqueous solution (Please take personal protection when using this hazards reagent), stirred at room temperature for 3 d. Then, the precipitate was washed with deionized water and dispersed in 20 mL TPAOH aqueous solution (25 wt.%), stirred for 3 d at room temperature. Afterwards, the sediment was collected by centrifugation (3500 rpm, 10 min) and washed with deionized water for 6–8 times to make the pH of the solution greater than 6.0. The Ti₃C₂ was prepared after centrifugation at 3500 rpm for 60 min.

Preparation of Ti₃C₂-PEG and Ti₃C₂/Apt nanosheets

To attach COOH-PEG-COOH on the surface of Ti₃C₂ nanosheets, the 2.5 mL COOH–PEG–COOH (10.0 mg mL⁻¹) slowly dissolved in 2.5 mL Ti₃C₂ aqueous suspension (1.0 mg mL⁻¹) and stirred for 6 h at room temperature. Then, the Ti₃C₂-PEG nanosheets were obtained by centrifugation and washed three times with deionized water to remove excess COOH-PEG-COOH. Afterwards, 500 μL EDC•HCl (500 mM) and 500 μL NHS (100 mM) were slowly added to 1.0 mL Ti₃C₂-PEG nanosheets suspension (2.0 mg mL⁻¹) and stirred for 30 min at room temperature. The activated Ti₃C₂-PEG nanosheets were obtained by centrifugation and washed with deionized water for 3 times. Then, Apt was dispersed in the activated Ti₃C₂-PEG nanosheets and stirred for 6 h at room temperature. The Apt modified Ti₃C₂/Apt nanosheets were obtained by centrifugation and washed 3 times with deionized water to remove excess Apt.

Hemolysis assay

Erythrocytes were collected from mouse blood by centrifugation (3000 rpm, 3 min). Then, 4% erythrocytes (v/v, 1.0 mL) were incubated with PBS, H₂O, and different concentrations of nanosheets (1.0 mL, 50–200 µg mL⁻¹) at 37 °C for 8 h, respectively. After that, the supernatant was obtained by centrifugation (3000 rpm, 3 min) and its absorbance value was measured at 540 nm. The percentage of hemolysis can be measured from Eq. (1):

$$\mathrm{Hemolysis }\left(\mathrm{\%}\right)=\frac{{A}_{1}}{{A}_{0}} \times 100\mathrm{\%}$$

(1)

where A₀ represents the absorbance of the supernatant after the erythrocytes were incubated with H₂O, and A₁ denotes the absorbance after incubated with other nanosheets solutions.

Photothermal performance of Ti₃C₂/Apt-M nanosheets

To evaluate the photothermal performance of Ti₃C₂/Apt-M nanosheets, these nanosheets with various concentrations (20, 40, 60, 80 and 100 μg mL⁻¹) were irradiated with the 808 nm laser at a power density of 1.5 W cm⁻² for 10 min. Furthermore, the nanosheets irradiated with various power density (0.5, 1.0, 1.5, 2.0 and 2.5 W cm⁻²) were also carried out. The temperature and thermal images were recorded by infrared thermal imager. The photothermal conversion efficiency (η) of Ti₃C₂/Apt-M was calculated according to Roper’s report (Roper et al. 2007), as shown in Eq. (2):

$$\upeta =\frac{hS\left({T}_{max,NP}-{T}_{surr}\right)-{Q}_{dis}}{I\left(1-{10}^{-{A}_{808}}\right)}$$

(2)

where h is the heat transfer coefficient, S is the surface area of the container, T_max is the maximum temperature of the solution, T_surr is the surrounding temperature, I is the laser power density, and A₈₀₈ is the absorption value of the material at 808 nm. Q_dis is the heat generated after water and container absorbs light. To calculate hS, Eq. (3, 4) was introduced:

$${Q}_{dis}=hS({T}_{max,{H}_{2}O}-{T}_{surr})$$

(3)

$${\tau }_{s}=\frac{{m}_{D}{C}_{D}}{hS}$$

(4)

m_D is the mass of water, C_D is the heat capacity of water (4.2 J·g⁻¹·°C⁻¹), τ_s is the sample system time constant, which was calculated by formula (5 , 6):

$$t=-{\tau }_{s}ln\theta$$

(5)

$$\theta =\frac{{T}_{surr}-T}{{T}_{surr}{-T}_{max}}$$

(6)

According to the obtained data and Eq. (2), the photothermal conversion efficiency of the Ti₃C₂, Ti₃C₂-PEG, Ti₃C₂/Apt-C and Ti₃C₂/Apt-M nanosheets was determined to be 36.72%, 33.25%, 32.19% and 31.76%, respectively.

The photothermal stability of Ti₃C₂/Apt-M was evaluated by five repeated laser on/off irradiations.

Affinity analysis between Apt and cells

MCF-7 and HepG2 cells were donated by Hunan University. MCF-7 and HepG2 cell line were cultured in RPMI-1640 with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO₂. Then, the cells were collected and washed with PBS 2–3 times. Subsequently, the cells were blown to form a cell suspension and incubated with Apt-Mf or Apt-Cf for 2 h, respectively. Finally, the fluorescence intensity of the cells was evaluated by flow cytometry after the cells were washed and resuspended in 500 μL PBS.

Intracellular endocytosis analysis

For the intracellular endocytosis analysis, cells were seeded in 6-well plates (1 × 10⁵ cells/well) for 24 h, and co-incubated with Ti₃C₂/Apt-Mf nanosheets (75 μg mL⁻¹) for 0, 1, 2, 3, 4, 5 and 6 h, respectively. The flow cytometry analysis was conducted after cells were gently washed three times with PBS.

In addition, cells were incubated in a CLSM culture dish at a density of 2 × 10⁵ cells/dish for 24 h. After that, 75 μg mL⁻¹ of Ti₃C₂/Apt-Mf nanosheets were added into the dish and co-incubated with the cells for 2, 4 and 6 h. The cells were washed with PBS gently. Afterwards, the cells were stained with Hoechst 33342 and observed by CLSM.

Targeting performance analysis

The targeting performance was analyzed by flow cytometry and CLSM. The cells were seeded in CLSM culture dish (2 × 10⁵ cells/dish) or 6-well plates (2 × 10⁵ cells/well) for 24 h. After that, the original medium was replaced with fresh medium containing Ti₃C₂/Apt-Mf (75 μg mL⁻¹), Ti₃C₂/Apt-Mf (75 μg mL⁻¹) + 2 OD Apt-M, Ti₃C₂/Apt-Mf (75 μg mL⁻¹) + 2 OD Apt-C, respectively. Then, the cells were analyzed by flow cytometry or CLSM after cultured for 4 h.

In vitro cytotoxicity assay

The cytotoxicity of Ti₃C₂-PEG, Ti₃C₂/Apt-C and Ti₃C₂/Apt-M nanosheets was evaluated by the standard MTT method. The cells were seeded into 96-well plates at 5 × 10³ cells/well for 24 h. Then, different concentrations (0, 25, 50, 100, 200 and 400 µg mL⁻¹) of samples were added and incubated with cells for another 24 or 48 h. Afterwards, the culture medium was removed and cells were washed three times with PBS (200 μL). After that, the cell viabilities were detected by the MTT assay. The absorbance at 570 nm was measured on a microplate reader.

In vitro-targeted antitumor effect

Cells were seeded into 96-well plates at 5 × 10³ cells/well for 24 h. Then, cells were incubated with PBS, Ti₃C₂-PEG (75 μg mL⁻¹), Ti₃C₂/Apt-C (75 μg mL⁻¹) and Ti₃C₂/Apt-M (75 μg mL⁻¹) for 4 h. Afterwards, cells were washed with PBS three times and then irradiated with 808 nm laser at a power density of 1.5 W/cm² for 10 min. Then, the cell viability was detected by MTT method after the cells were cultured for another 12 h.

Live/dead cells staining was used to visualize the targeted antitumor effect in vitro. After incubated with different groups of nanomaterial and irradiated with 808 nm laser, the cells were washed with PBS several times and stained by Calcein-AM and PI solution according to the manufacturer's protocol. Then, the CLSM was used to record the fluorescence images of living cells (green fluorescence) and dead cells (red fluorescence).

Furthermore, apoptosis analysis was performed to visualize the targeted antitumor effect. After incubated with different groups of nanomaterial and irradiated with 808 nm laser, cells were collected and stained by Annexin V-FITC and PI solution according to manufacturer’s protocol. Then, flow cytometry was used to evaluate the cell signals after the cells were washed with PBS three times to remove redundant dyes.

In vivo targeted antitumor effect

Animal experiments were performed under the permission of experimental animal ethics committee of Shanxi Datong University. BALB/c nude mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The xenograft tumor model was established by injecting 5 × 10⁶ cells of MCF-7 into the groin region of mice. The tumor volume was calculated and recorded according to the formula: tumor volume = (tumor length) × (tumor width)²/2. When the tumor volume reached approximately 100 mm³, all mice were randomly divided into four groups with five mice for each group, including: (1) Control group (PBS), (2) Ti₃C₂-PEG + Laser group (dose of Ti₃C₂-PEG = 15 mg kg⁻¹), (3) Ti₃C₂/Apt-C + Laser group (dose of Ti₃C₂/Apt-C = 15 mg kg⁻¹), (4) Ti₃C₂/Apt-M + Laser group (dose of Ti₃C₂/Apt-M = 15 mg kg⁻¹). All mice were injected intravenously with PBS, Ti₃C₂-PEG, Ti₃C₂/Apt-C or Ti₃C₂/Apt-M separately. After 4 h, the tumors of mice in groups (2), (3) and (4) were irradiated with 808 nm laser (1.5 W cm⁻²) for 10 min. Afterwards, the body weight and tumor volume of mice were measured for continuous 17 d. Finally, all the dissected tumors were weighted up and collected for hematoxylin–eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Antigen Ki-67 immunofluorescence staining. Additionally, the major organs including heart, lung, liver, spleen and kidney were obtained as well to make H&E-stained sections for observing whether these nanosheets could cause tissue damage.

Statistical analysis

Analysis of the data was conducted using SPSS version 21.0. The significance of the differences between the experiments and control groups was analyzed by one-way analysis of variance (ANOVA). Statistical significance was set at *P < 0.05, **P < 0.01 and ***P < 0.001.