Fabrication of Polydopamine-Coated High-Entropy MXene Nanosheets for Targeted Photothermal Anticancer Therapy

Abstract

Transition metal carbides, nitrides, and carbonitrides (MXenes) have emerged as a promising class of 2D materials that can be used for various applications. Recently, a new form of high-entropy MXenes has been reported, which contains an increased number of elemental species that can increase the configurational entropy and reduce the Gibbs free energy. The unique structure and composition lead to a range of intriguing and tunable characteristics. Herein, the fabrication of high-entropy MXene TiVNbMoC₃T_x (T = surface terminations) with a layer of polydopamine is reported, followed by immobilization of a phthalocyanine-based fluorophore for imaging and the peptide sequence QRHKPREGGGSC for targeting the epidermal growth factor receptor (EGFR) overexpressed in cancer cells. The resulting nanocomposite exhibits high biocompatibility and superior photothermal property. Upon laser irradiation at 808 nm, the light-to-heat conversion efficiency is up to 56.1%, which is significantly higher than that of conventional 2D materials. In vitro studies show that these nanosheets could be internalized selectively into EGFR-positive cancer cells and effectively eliminate these cells mainly through photothermal-induced apoptosis. Using 4T1 tumor-bearing mice as an animal model, the nanosheets could accumulate at the tumor and effectively eradicate the tumor upon laser irradiation without causing noticeable adverse effects to the mice.

Keywords: cancer therapy; epidermal growth factor receptor; high‐entropy MXenes; photothermal therapy; polydopamine.

Figure 1

Synthetic route and antitumor mechanism of HE‐M@PDA‐Pc‐QRH.

Figure 2

A) A photograph showing the appearance of HE‐M and HE‐M@PDA‐Pc‐QRH dispersed in deionized water. B) Normalized hydrodynamic diameters and C) zeta potentials (n = 3) of HE‐M, HE‐M@PDA, HE‐M@PDA‐Pc, and HE‐M@PDA‐Pc‐QRH dispersed in deionized water. TEM images of D) HE‐M and E) HE‐M@PDA‐Pc‐QRH. F) EDS and G) AFM images of HE‐M@PDA‐Pc‐QRH. H) UV–Vis spectra of the various nanocomposites and free Pc in deionized water with 0.5% (v/v) Tween 20 (100 µg mL⁻¹ or 2.5 µm of Pc). I) Fluorescence spectra of HE‐M@PDA, HE‐M@PDA‐Pc‐QRH, and free Pc in deionized water with 0.5% (v/v) Tween 20 (100 µg mL⁻¹ or 2.5 µm of Pc) (λ _ex = 610 nm). J) Concentration‐dependent and K) laser power‐dependent photothermal effects of HE‐M@PDA‐Pc‐QRH in deionized water upon irradiation with an 808 nm laser. L) Temperature cycling curve for HE‐M@PDA‐Pc‐QRH in deionized water (100 µg mL⁻¹). Each cycle involves laser irradiation (808 nm, 1.5 W cm⁻²) for 6 min followed by natural cooling for 8 min.

Figure 3

A) Confocal images of HEK‐293, HepG2, HT29, and 4T1 cells after incubation with HE‐M@PDA‐Pc or HE‐M@PDA‐Pc‐QRH (10 µg mL⁻¹) for 8 h. Scale bar = 25 µm. B) Corresponding quantified intracellular fluorescence intensities (n = 50 cells). Data are reported as the mean ± standard deviation (SD) for three independent experiments. n.s., not significant; ****p < 0.0001. C) Cell viabilities of 4T1 cells after incubation with various concentrations of HE‐M or HE‐M@PDA‐Pc‐QRH for 8 h. Cell viabilities of HEK‐293, HepG2, HT29, and 4T1 cells after incubation with various concentrations of HE‐M@PDA‐Pc‐QRH for 8 h in the D) absence and E) presence of laser irradiation at 808 nm (1 W cm⁻²) for 10 min. Data are reported as the mean ± SD for four independent experiments. F) Fluorescence imaging of 4T1 cells after the above treatment and staining with calcein‐AM (green, live cells) and PI (red, dead cells). Scale bar = 100 µm. G) Flow cytometric analysis of 4T1 cells after incubation with HE‐M@PDA‐Pc‐QRH (100 µg mL⁻¹) for 8 h, followed by laser irradiation at 808 nm (1 W cm⁻²) for 1, 3, 5, and 10 min, using a Dead Cell Apoptosis kit with Annexin V‐FITC and PI.

Figure 4

A) In vivo fluorescence images of 4T1 tumor‐bearing mice after intravenous injection with saline (200 µL) with or without the presence of HE‐M@PDA‐Pc or HE‐M@PDA‐Pc‐QRH (200 µg) recorded after different periods of time. The apparently extremely high fluorescence intensity at the bottom part is just an artifact. B) Change in the corresponding quantified fluorescence intensity per unit area of the tumor with time. Data are reported as the mean ± SD (n = 4). *p < 0.05. C) Representative fluorescence images of some major organs and the tumor harvested at 6 h post‐injection. D) Corresponding quantified fluorescence intensities per unit area. Data are reported as the mean ± SD (n = 3). **p < 0.01. E) Representative in vivo infrared thermal images of one of the mice from each of the above three groups recorded upon laser irradiation at 808 nm at the tumor at 6 h post‐injection for different periods of time. F) Change in the temperature at the tumor with irradiation time for the three mice.

Figure 5

A) Timeline of the in vivo study of the PTT efficacy of HE‐M@PDA‐Pc‐QRH against 4T1 tumor‐bearing nude mice. B) Tumor growth curves for the mice after receiving different treatments: G1: intravenous injection with saline (200 µL); G2: intravenous injection with HE‐M@PDA‐Pc in saline (200 µg, 200 µL); G3: intravenous injection with HE‐M@PDA‐Pc‐QRH in saline (200 µg, 200 µL); G4: intravenous injection with HE‐M@PDA‐Pc in saline (200 µg, 200 µL), followed by laser treatment (808 nm, 1 W cm⁻², 10 min) at 6 h post‐injection and on day 8; G5: intravenous injection with HE‐M@PDA‐Pc‐QRH in saline (200 µg, 200 µL), followed by laser treatment (808 nm, 1 W cm⁻², 10 min) at 6 h post‐injection and on day 8. C) Changes in the body weights of the mice for G1 to G5; D) Weights and E) sizes of the tumors harvested from the different groups of the mice on day 15 (n = 5). B–D) Data are reported as the mean ± SD (n = 5). n.s., not significant; **p < 0.01; ***p < 0.001; and ****p < 0.0001. F) H&E‐stained images of different organ and tumor slides from the mice scarified on day 15 after different treatments. G) Representative immunohistochemical images of the tumor sections prepared at the end of the above treatments and stained for TUNEL assay and detection of CD31, Ki67, and PCNA. Scale bar = 100 µm.

Figure 6

A) Timeline of the in vivo study of the systemic toxicity of HE‐M@PDA‐Pc and HE‐M@PDA‐Pc‐QRH against C57BL/6 mice. B) Measurements of the hematologic parameters and blood and urine biochemical indexes for the renal and liver functions of the mice being intravenously injected with saline (200 µL) with or without the presence of HE‐M@PDA‐Pc or HE‐M@PDA‐Pc‐QRH (200 µg), followed by housing for 7 or 28 d. Data are reported as the mean ± SD (n = 10). C) H&E‐stained images of different organ slides from the mice scarified on day 8 or 29 after the above treatments.