Difunctional nano preparation for tumor starvation and photothermal therapy as well as preparation method and application of difunctional nano preparation

Abstract

The invention discloses a difunctional nano preparation for tumor starvation and photothermal therapy as well as a preparation method and application of the difunctional nano preparation. The difunctional nano preparation is prepared from PDA (at) MXene and glucose oxidase loaded on the PDA (at) MXene, wherein the glucose oxidase is loaded on the PDA (at) MXene; the PDA (at) MXene comprises an MXene nanosheet and polydopamine, and the MXene nanosheet is modified by the polydopamine. According to the invention, polydopamine (PDA) is modified on an MXene nanosheet so as to improve the photothermal conversion efficiency, and glucose oxidase (GOX) is loaded on PDA-coated MXene with a large surface area so as to catalyze glucose in situ. The bifunctional nano preparation shows higher photo-thermal response efficiency than that of a single MXene nanosheet, promotes the pharmacokinetic effect of glucose oxidase, and realizes '1 + 1gt, multipurpose 'deep tumor ablation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a bifunctional nanoformulation for tumor starvation and photothermal therapy, its preparation method, and its application. Background Technology

[0002] Photothermal therapy (PTT) uses photothermal agents to convert light energy into heat energy for targeted tumor ablation. PTT offers the advantage of being non-invasive, but typically requires sufficient heat to ablate the tumor. This thermal stimulation usually triggers a self-protective mechanism in cancer cells under heat stress: upregulating the expression of heat shock proteins (HSPs) to promote the proper folding of denatured proteins, thereby promoting cancer cell survival. To date, researchers have proposed several strategies to inhibit HSP expression, including HSP inhibitors and small interfering RNA (siRNA). However, due to tumor heterogeneity, subgroups with different HSP phenotypes exhibit varying degrees of resistance and immune evasion to PTT therapy, leading to the survival of residual tumor lesions.

[0003] To overcome the limitations of photothermal therapy (PTT) in deep tumor ablation and heat stress induction of heterogeneous cells, combining PTT with starvation therapy offers a novel approach. Tumor starvation therapy refers to inhibiting normal tumor metabolism and proliferation by reducing the nutrient supply to the tumor. Glucose oxidase (GOX) is one of the most important metabolic inhibitors, primarily oxidizing glucose to gluconic acid and H2O2, consuming glucose and ATP, increasing intracellular reactive oxygen species (ROS) levels, thereby inhibiting tumor growth. Notably, GOX activity exhibits temperature-dependent properties, with optimal levels observed at 50°C. GOX consumption of ATP leads to a significant downregulation of HSP, thereby weakening heat stress resistance and restoring photosensitivity of tumor cells. Therefore, the combination of PTT and starvation therapy holds promise for achieving ablation of deep and heterogeneous tumors in a safe and mild synergistic manner. However, further research is needed to control the optical stability of the photosensitizer at the optimal temperature and to ensure timely synergy between PTT and cellular starvation metabolism in the small in-situ cellular space.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a bifunctional nano-formulation for tumor starvation and photothermal therapy, its preparation method and application, which aims to achieve photothermal tumor ablation at a safe temperature while consuming glucose to inhibit the expression of heat shock proteins, thereby achieving deep tumor eradication.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a bifunctional nanoformulation for tumor starvation and photothermal therapy is provided, the bifunctional nanoformulation comprising: PDA@MXene, and glucose oxidase loaded on the PDA@MXene; the PDA@MXene comprising: MXene nanosheets, and polydopamine modified on the MXene nanosheets.

[0008] In a preferred embodiment, the mass ratio of PDA@MXene to glucose oxidase in the bifunctional nanoformulation is (800-1200):1.

[0009] In a preferred embodiment, the mass ratio of MXene nanosheets to polydopamine in the PDA@MXene is 1:(2.5-5).

[0010] In a preferred embodiment, the average size of the bifunctional nanoformulation is 50-200 nm.

[0011] In a preferred embodiment, the average size of the MXene nanosheets is 20-100 nm.

[0012] Secondly, a method for preparing a bifunctional nanoformulation as described in the first aspect is provided, comprising the steps of:

[0013] Solution A containing MXene nanosheets and solution B containing dopamine hydrochloride are provided;

[0014] Solution B was added dropwise to solution A, and after stirring, PDA@MXene was obtained.

[0015] The glucose oxidase solution was added dropwise to the dispersion of PDA@MXene, and the mixture was stirred to obtain the bifunctional nano-formulation.

[0016] In a preferred embodiment, the concentration of MXene nanosheets in solution A is 0.1-5 mg / mL; the concentration of dopamine hydrochloride in solution B is 0.5-10 mg / mL; and the volume ratio of solution A to solution B is 1:(0.625-1.25).

[0017] In a preferred embodiment, the concentration of PDA@MXene in the PDA@MXene dispersion is 0.1-5 mg / mL; the concentration of glucose oxidase in the glucose oxidase solution is 50-500 μg / mL; and the volume ratio of the PDA@MXene dispersion to the glucose oxidase solution is 1:(3-8).

[0018] In a preferred embodiment, the stirring conditions are: temperature 10-50℃, rotation speed 200-600 rpm / min, and time 8-24h.

[0019] The preferred technical solution, the method for preparing the MXene nanosheets, includes the following steps:

[0020] Ti3C2 nanosheets were dispersed in N-methylpyrrolidone, sonicated, and then centrifuged for the first time to collect the supernatant.

[0021] Acetone was added to the supernatant, and a second centrifugation was performed to collect the precipitate and obtain the MXene nanosheets.

[0022] Thirdly, the application of a bifunctional nanoformulation as described in the first aspect in the preparation of antitumor drugs is provided.

[0023] Beneficial Effects: This invention provides a bifunctional nanoformulation for tumor starvation and photothermal therapy. The bifunctional nanoformulation comprises PDA@MXene and glucose oxidase loaded onto the PDA@MXene. The PDA@MXene comprises MXene nanosheets and polydopamine modified onto the MXene nanosheets. This invention provides a method of modifying MXene nanosheets with polydopamine (PDA) to improve photothermal conversion efficiency, while simultaneously loading glucose oxidase (GOX) onto the large surface area of ​​PDA@MXene to catalyze glucose in situ. This bifunctional nanoformulation exhibits higher photothermal response efficiency than MXene nanosheets alone and promotes the pharmacokinetics of glucose oxidase, achieving a "1+1>multiple uses" effect in deep tumor ablation. Attached Figure Description

[0024] Figure 1 This invention relates to the preparation process of bifunctional nano-formulations and their application in deep tumor starvation therapy and biosafe photothermal therapy.

[0025] Figure 2 These are the characterization results of MXene and PDA@MXene in Example 1 of the present invention; wherein, a)-c) are TEM and HR-TEM images of MXene; d)-h) are the hydrodynamic particle size distribution test results of MXene and PDA@MXene (containing 10, 30, and 50 mg of dopamine, respectively); i) is the UV-vis-NIR absorption spectrum of PDA@MXene containing 10, 30, and 50 mg of dopamine; j) is the XRD spectrum of MXene and PDA@MXene; k) is the XPS spectrum of MXene and PDA@MXene; l)-m) are the high-resolution XPS carbon elemental full spectrum of MXene and PDA@MXene.

[0026] Figure 3These are the stability test results of MXene and PDA@MXene in Example 1 of this invention; where a) is the UV-vis-NIR absorption spectrum of MXene stored at room temperature for different times, and the numbers in the figure represent the incubation time (days); b) is the UV-vis-NIR absorption spectrum of PDA@MXene stored at room temperature for different times, and the numbers in the figure represent the incubation time (days); c) is the change of optical density of MXene and PDA@MXene at 808nm over time; d)-e) are the Raman spectra of MXene and PDA@MXene before and after 7 days of storage; f)-g) are the photothermal heating curves of MXene and PDA@MXene after laser irradiation, corresponding to different storage times; h) is a photograph of the dispersion of MXene and PDA@MXene after the photothermal test.

[0027] Figure 4 The results of the photothermal properties investigation of PDA@MXene in this invention are as follows: a) is the UV-vis-NIR absorption spectrum of MXene and PDA@MXene; b) is the concentration-absorbance relationship of MXene and PDA@MXene at 808 nm; c) is the photothermal heating curves (808 nm, 1 W / cm²) of MXene, PDA@MXene, PDA and H₂O at a concentration of 100 μg / mL. 2 d) shows the temperature changes of MXene and PDA@MXene during five laser-on / off cycles; e) shows the heating-cooling curves of MXene and PDA@MXene (808 nm, 1 W / cm²). 2 f) is the relationship between the time obtained by MXene and PDA@MXene from cooling and -lnθ; g) is the UV-vis-NIR absorption spectrum of PDA@MXene and PDA@MXene@GOX; h) is the UV-vis-NIR absorption spectrum of PDA@MXene and PDA@MXene@GOX under 808 nm irradiation (1 W / cm²). 2 The concentration of H2O2 in the GOX-glucose reaction catalyzed by GOX and PDA@MXene@GOX under the following conditions: i) under 808 nm irradiation (1 W / cm²). 2 The O2 concentration in the GOX-glucose reaction catalyzed by PDA@MXene@GOX.

[0028] Figure 5 The results are the photothermal performance test results of PDA@MXene of this invention; wherein, a) are the results of PDA@MXene at 0.25, 0.5, 0.75 and 1 W / cm². 2 Photothermal performance at laser power density; b)-c) are PTT heating curves and thermograms of PDA@MXene at concentrations of 0, 6.5, 12.5, 25, 50 and 100 μg / mL.

[0029] Figure 6 These are the test results of the photothermal enhancement of enzyme activity of PDA@MXene@GOX in this invention; a) the effect of temperature on H2O2 concentration in the GOX-glucose reaction; b) the relative activity of GOX at different temperatures; c) the change of pH over time in the GOX-glucose reaction at different temperatures.

[0030] Figure 7 This is the result of in vitro synergistic tumor starvation and photothermal therapy using PDA@MXene@GOX in Example 1 of the present invention; wherein, a) is the analysis of 4T1 cell uptake of PDA@MXene@GOX, with incubation times of 0 hours, 1 hour, 4 hours, and 6 hours; b) is the cell viability of 4T1 cells after treatment with GOX, PDA@MXene, or PDA@MXene@GOX under laser irradiation or no irradiation (laser irradiation for 5 minutes, 808 nm, 1 W / cm²). 2 c) Assessment of live / dead cells in 4T1 cells stained with Calcein-AM / PI, error bars represent standard deviation (n=3), scale bar: 50 μm; d) Apoptosis analysis of 4T1 cells treated with GOX, PDA@MXene, or PDA@MXene@GOX by flow cytometry using Annexin V-FITC / PI staining; e) Fluorescence images of 4T1 cells treated with GOX, PDA@MXene, or PDA@MXene@GOX by detecting ROS levels using the DCFH-DA kit, scale bar: 50 μm.

[0031] Figure 8 This refers to the tumor retention and photothermal effect of PDA@MXene@GOX in vivo in Example 1 of the present invention; wherein, a)-b) are fluorescence imaging and fluorescence intensity quantification of 4T1 tumor-bearing mice after injection of PDA@MXene@Cy5; c)-d) are thermal imaging and temperature changes of tumor-bearing mice after injection of PBS and PDA@MXene@GOX and treatment under laser irradiation.

[0032] Figure 9 This describes the in vivo antitumor effect of PDA@MXene@GOX in Example 1 of this invention; wherein, a) is the change in body weight of mice during PBS+laser, PDA@MXene with / without laser, and PDA@MXene@GOX with / without laser treatment; b) is the change in relative tumor volume of mice under different treatment conditions; c) is the tumor weight of mice at the end of the treatment period; d) is a photograph of tumor resection after different treatments; e) is a photograph of mice with different tumor loads before and after different treatments; f) is the glucose concentration in the tumor after different treatments (n=3); g) is a tumor section from each group of mice at the end of the test, stained with H&E, TUNEL and HSP 70, respectively.

[0033] Figure 10 This refers to the hematological tests and H&E staining results of major mouse organs (heart, liver, spleen, lung, and kidney) in Example 1 of this invention; wherein, a) is the whole blood analysis of mice after treatment with PBS or PDA@MXene@GOX, RBC: red blood cells, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin content, MCHC: mean corpuscular hemoglobin concentration, WBC: white blood cells, PLT: platelets, MPV: mean platelet volume, and the data are expressed as mean ± standard deviation (n=4); b) is the hemolysis rate of PDA@MXene@GOX; c) is the H&E staining of sections of major mouse organs (including liver, heart, kidney, spleen, and lung) after treatment, scale bar: 100μm. Detailed Implementation

[0034] This invention provides a bifunctional nanoformulation for tumor starvation and photothermal therapy, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0035] Due to tumor heterogeneity, different tumor cell subpopulations express heat shock protein phenotypes with varying photosensitivity, leading to uncoordinated resistance to photothermal therapy (PTT) among tumor cell populations. PTT combined with tumor starvation therapy can target specific metabolic pathways, enhancing the photosensitivity of cancer cells and eliminating deep tumors that are not easily stimulated by light. However, this therapy requires ensuring precise synergistic effects within the effective time window and cellular space, while also ensuring the local safety of the photothermal effect.

[0036] Based on this, embodiments of the present invention provide a bifunctional nanoformulation for tumor starvation and photothermal therapy, the bifunctional nanoformulation comprising: PDA@MXene, and glucose oxidase loaded on the PDA@MXene; the PDA@MXene comprises: MXene nanosheets, and polydopamine modified on the MXene nanosheets.

[0037] Specifically, MXenes are a novel class of two-dimensional (2D) materials composed of transition metal carbides and nitrides, which have attracted considerable attention in recent years in fields such as energy storage, catalysis, sensing, and biomedicine. MXenes exhibit excellent properties, including metallic conductivity, surface activity, and high photothermal conversion efficiency. However, due to the hydrolysis and oxidation of oxygen-containing functional groups on MXenes, they are prone to degradation or aggregation in physiological environments.

[0038] Polymers such as poly(acrylic acid), polyethylene glycol, polyvinylpyrrolidone, and hyaluronic acid have been used to stabilize MXene. However, some molecules can only couple with MXene in toxic organic solvents, while others form surface passivations, thereby reducing the light absorption and thermal conductivity of MXene. Polydopamine (PDA), a melanin-inspired biomaterial, is considered a good candidate for surface modification due to its abundant surface chemical groups, which promote strong adhesion and inherently high photothermal conversion capabilities. Therefore, this embodiment uses PDA-modified MXene to obtain a bifunctional nanoformulation with two-dimensional topological stability and high loading rate, while also possessing efficient ROS metabolism and photothermal conversion capabilities.

[0039] The principle of the bifunctional nanoformulation of this invention for starvation therapy and biosafe photothermal therapy of deep / heterogeneous tumors is as follows: Figure 1 As shown. Specifically, small-sized MXene nanosheets (2D MXene) were prepared via an aqueous exfoliation method. After being modified with PDA molecules in a simple, green aqueous environment, 2D MXene formed leaf-shaped PDA@MXene. Figure 1 (Referring to PDA@MXene). PDA@MXene exhibits excellent 2D surface expansion and improved adhesion properties, enabling it to be used to load GOX molecules and form bifunctional nanoformulations PDA@MXene@GOX.

[0040] During application, under low-intensity 808nm laser irradiation, PDA@MXene first initiates physical ablation of the tumor, causing a slight increase in local temperature at around 50°C. This significantly increases GOX enzyme activity, consumes intratumoral glucose, and reduces metabolic levels within the tumor, thereby downregulating HSP expression to alleviate thermal stress. The bifunctional nanoformulation of this invention effectively combines the inherent photothermal advantages of PDA@MXene with the pharmacokinetic properties of GOX biocatalytic molecules, achieving safe and deep tumor ablation.

[0041] In one embodiment, the mass ratio of PDA@MXene to glucose oxidase in the bifunctional nanoformulation is (800-1200):1.

[0042] In one embodiment, the mass ratio of MXene nanosheets to polydopamine in the PDA@MXene is 1:(2.5-5).

[0043] In one embodiment, the average size of the bifunctional nanoformulation is 50-200 nm.

[0044] In one embodiment, the average size of the MXene nanosheets is 20-100 nm.

[0045] This invention provides a method for preparing the bifunctional nano-formulation as described above, comprising the following steps:

[0046] Solution A containing MXene nanosheets and solution B containing dopamine hydrochloride are provided;

[0047] Solution B was added dropwise to solution A, and after stirring, PDA@MXene was obtained.

[0048] The glucose oxidase solution was added dropwise to the dispersion of PDA@MXene, and the mixture was stirred to obtain the bifunctional nano-formulation.

[0049] In one embodiment, the concentration of MXene nanosheets in solution A is 0.1-5 mg / mL; the concentration of dopamine hydrochloride in solution B is 0.5-10 mg / mL; and the volume ratio of solution A to solution B is 1:(0.625-1.25).

[0050] In one embodiment, the concentration of PDA@MXene in the PDA@MXene dispersion is 0.1-5 mg / mL; the concentration of glucose oxidase in the glucose oxidase solution is 50-500 μg / mL; and the volume ratio of the PDA@MXene dispersion to the glucose oxidase solution is 1:(3-8).

[0051] In one embodiment, the stirring conditions are: temperature 10-50℃, rotation speed 200-600 rpm / min, and time 8-24h.

[0052] In one embodiment, the method for preparing the MXene nanosheets includes the following steps:

[0053] Ti3C2 nanosheets were dispersed in N-methylpyrrolidone, sonicated, and then centrifuged for the first time to collect the supernatant.

[0054] Acetone was added to the supernatant, and a second centrifugation was performed to collect the precipitate and obtain the MXene nanosheets.

[0055] Specifically, within the MXene family, carbides, especially Ti3C2, exhibit good biocompatibility both in vitro and in vivo. The MXene nanosheets prepared using commercially available Ti3C2 nanosheets in this embodiment also demonstrate good biocompatibility.

[0056] In a more specific embodiment, the ultrasonic treatment conditions in the preparation method of the MXene nanosheets are: power 300-800W and 19-25kHz, time 3-6h.

[0057] In a more specific embodiment, in the method for preparing the MXene nanosheets, the conditions for the first centrifugation are: rotation speed 6000-10000 rpm, time 3-10 min.

[0058] In a more specific embodiment, in the method for preparing the MXene nanosheets, the conditions for the second centrifugation are: rotation speed 12000-18000 rpm, time 10-20 min.

[0059] This invention also provides an application of the bifunctional nanoformulation described above in the preparation of antitumor drugs.

[0060] The present invention will be further described below through specific embodiments.

[0061] Example 1

[0062] 1. Materials and Methods

[0063] 1.1 Materials

[0064] Large-size Ti3C2 nanosheets (colloidal aqueous solution) were purchased from Suzhou Beike Nanotechnology Co., Ltd. (China). Dopamine hydrochloride and α-D-glucose (ACS grade) were purchased from Aladdin Reagent Inc. (Shanghai, China). Glucose oxidase was obtained from Sigma-Aldrich (USA). Hydrogen peroxide (H2O2) assay kit, BCA protein assay kit, glucose assay kit, Hoechst 33342 staining kit, Mito-tracker red, and PI-Annex-V-FITC apoptosis assay kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Live / dead cell staining kit and 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Thermo-Scientific Co., Ltd. (USA). Cell Counting Kit-8 (CCK-8) was purchased from GlpbioCo., Ltd. (Shanghai, China). Cyanine 5 (Cy5) dye was purchased from Wuhan Duofluor Inc.

[0065] 1.2 Characterization

[0066] TEM images were acquired using a field emission transmission electron microscope (JEOL JEM-2100F) operating at an accelerating voltage of 200 kV. SEM images were acquired using a scanning electron microscope (Tescan VEGA3). AFM images were acquired using a scanning probe microscope (Bruker MultiMode 8). Hydrodynamic diameters and zeta potentials were measured using dynamic light scattering (Zetasizer-90, Malvern Instrument, UK). UV-Vis spectra were acquired using a UV-Vis spectrophotometer (Biochrom WPA visible spectrophotometer). Infrared thermal images were captured using a thermal imager (FLIRC3-X) under laser illumination. X-ray photoelectron spectroscopy (XPS) was measured using an X-ray photoelectron spectrometer (ThermoFisher Scientific Nexsa), with the binding energy corrected for anisotropic carbon (284.8 eV) during measurement. Raman spectra were acquired using an integrated confocal microscopy Raman spectrometer equipped with a 532 nm laser (Renishaw micro Raman spectroscopy system) at 1% laser power with a cumulative time of 10 s. The material structure and phase composition were analyzed using X-ray diffraction (RigakuSmartLab 9kW-Advance). The chemical functional groups of the material were determined using FTIR spectroscopy (Nicolet 5700).

[0067] 1.3 Preparation of MXene nanosheets

[0068] MXene nanosheets were prepared using a water sonication method in an ice bath. Large-sized Ti3C2 nanosheets were centrifuged at 15,000 rpm for 10 minutes to obtain a precipitate, which was then redispersed in N-methylpyrrolidone (NMP) at a concentration of 0.5 mg / mL. The dispersion was sonicated in an ultrasonic homogenizer tip at 600 W and 19–25 kHz for 4 hours under N2 protection, with an on / off interval of 4 seconds. The resulting dispersion was then centrifuged at 8,000 rpm for 5 minutes to remove large, aggregated nanosheets, and the supernatant was carefully collected. To obtain smaller-sized Ti3C2 nanosheets, a large amount of acetone was added to the supernatant, followed by centrifugation at 15,000 rpm for 15 minutes. The precipitate containing the smaller-sized Ti3C2 nanosheets was collected and washed three times with water. Finally, the smaller-sized Ti3C2 nanosheets, known as MXene nanosheets (abbreviated as MXene), were collected and stored at 4°C for future use, with excess oxygen removed by bubbling with N2.

[0069] 1.4 Preparation of PDA@MXene and PDA@MXene@GOX

[0070] PDA@MXene is a self-polymerization of dopamine synthesized on MXene nanosheets. The MXene nanosheets obtained above were redispersed in a solvent mixture consisting of 5 mL water, 2.5 mL ethanol, and 0.25 mL ammonium hydroxide (25 wt%–28 wt%) to obtain solution A, with a final concentration of MXene nanosheets of 0.5 mg / mL. The solution was then sonicated for 15 minutes. Simultaneously, dopamine hydrochloride was dissolved in water to obtain solution B, with a final concentration of dopamine hydrochloride of 2 mg / mL. Solution B (4.84 mL–9.68 mL) was then added dropwise to solution A (7.75 mL) while stirring at 400 rpm / min. After stirring for 12 hours, the PDA@MXene dispersion was collected and purified by ultrafiltration using a 30 kDa MWCO tube for several cycles, with pH monitored to ensure neutrality. GOX (200 μg / mL, 200 μL) was added dropwise to a PDA@MXene solution (1 mg / mL, 1 mL) under ultrasonic bath conditions, and stirred overnight at room temperature to prepare PDA@MXene@GOX. Unbound GOX was removed by centrifugation, and the resulting PDA@MXene@GOX was stored at 4°C until further use. The GOX loading efficiency was determined by the Coomassie Brilliant Blue method.

[0071] 1.5 PTT conversion efficiency determination

[0072] To evaluate the photothermal conversion performance of PDA@MXene, the PDA@MXene dispersion was diluted with water to a specific concentration, and 100 μL of the diluted PDA@MXene dispersion was transferred to a centrifuge tube. Subsequently, at a power density of 1 W / cm³... 2 Temperature was monitored at predetermined time intervals using a thermal imager under an 808 nm laser. For comparison, MXene nanosheet dispersions with the same concentration as the PDA@MXene dispersion were also prepared and placed under the same experimental conditions to study their photothermal conversion capabilities.

[0073] 1.6 Catalytic Activity Study of PDA@MXene@GOX

[0074] First, the effect of temperature on the catalytic activity of GOX was investigated. A fixed amount of PDA@MXene@GOX (equivalent to 10 μg GOX) was dispersed in PBS, and glucose was added to achieve a final concentration of 0.05 M, yielding the reaction mixture. The reaction system containing the reaction mixture was placed in different water bath temperatures (25, 35, 46, 55, and 65 °C), and the pH value of the reaction mixture was recorded every minute. After 15 minutes, the reaction was terminated, and the hydrogen peroxide (H2O2) concentration was measured using an H2O2 kit to assess the catalytic activity of GOX. Furthermore, the oxygen concentration of the reaction mixture was monitored using a dissolved oxygen meter (Shanghai Remy Co., Ltd.).

[0075] 1.7 Cell Experiments

[0076] The mouse breast cancer cell line 4T1 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin and 100 μg / mL streptomycin, and at 37°C in a 5% (v / v) CO2 atmosphere.

[0077] 1.7.1 Cytotoxicity assay and in vitro antitumor activity assay

[0078] To evaluate the cytotoxicity and in vitro antitumor activity of PDA@MXene@GOX, 4T1 cells were seeded at a density of 5000 cells per well in 96-well plates and incubated for 12 hours to ensure tight cell adhesion. Cytotoxicity assay: PDA@MXene dispersions were diluted to different concentrations with culture medium and added to the wells; after 24 hours of incubation, cell viability was assessed using the CCK-8 assay; each concentration was repeated three times, with cells incubated only with culture medium serving as a control group with 100% viability. In vitro antitumor activity assay: PDA@MXene@GOX was diluted to different concentrations with culture medium and added to the wells as in the cytotoxicity assay; after 4 hours of incubation, the laser treatment group was treated with 1 W / cm². 2 Cells were irradiated with an 808nm laser at 37℃ for 5 minutes; subsequently, they were incubated at 37℃ for 24 hours, and cell viability was analyzed using the CCK-8 assay. Each concentration was repeated three times, with cells incubated only with culture medium serving as a control group with 100% viability. Viability was calculated using the following formula: Cell viability = Experimental group OD value / Control group OD value * 100%.

[0079] 1.7.2 Detection of endocytosis efficiency

[0080] Cellular uptake and localization of PDA@MXene@GOX were investigated using confocal laser scanning microscopy (CLSM, Leica SP8). To this end, the fluorescent dye Cy5 was physically adsorbed onto PDA@MXene@GOX, and unbound Cy5 was removed using a 30 kDa MWCO tube through several ultrafiltration cycles. 4T1 cells were seeded at a density of 50,000 cells per well in confocal culture dishes and incubated for 12 hours. Subsequently, a PDA@MXene@GOX-Cy5 dispersion (equivalent to 1 μg Cy5) was added to the culture dishes. After 4 hours of incubation, cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342 for confocal imaging.

[0081] 1.7.3 Assessment of intracellular H2O2 levels

[0082] 4T1 cells were seeded at a density of 50,000 cells per well in 24-well plates and cultured overnight. Then, fresh culture medium containing either PDA@MXene (20 μg / mL), GOX (21.5 ng / mL), or PDA@MXene@GOX (containing 20 μg / mL PDA@MXene and 21.5 ng / mL GOX) was added to the cells. After 4 hours of incubation, the laser-treated group of cells was treated at 1 W / cm². 2 The cells were exposed to an 808 nm laser for 5 minutes. They were then washed three times with DPBS and fresh culture medium containing DCFH-DA (10 μM) was added. After incubation for 20 minutes, the cells were observed using an inverted fluorescence microscope (488 nm, FITC channel).

[0083] 1.7.4 Cell liveness / death staining

[0084] 4T1 cells were loaded at 2 × 10⁴ cells per well. 5 Cells were seeded at a density of 1000 cells / well in 6-well plates and cultured overnight. Then, fresh culture medium containing the drug was added to each well; the drug was either PDA@MXene (20 μg / mL), GOX (21.5 ng / mL), or PDA@MXene@GOX (containing 20 μg / mL PDA@MXene and 21.5 ng / mL GOX). For the laser-treated group, an 808 nm laser was used at 1 W / cm². 2 Cells were irradiated at a rate of 5 min for 12 hours. After incubation, the cells were washed three times with fresh PBS and stained with calcein-AM and PI. Finally, the cells were observed using a confocal laser scanning microscope (CLSM).

[0085] 1.7.5 Apoptosis Analysis

[0086] The 4T1 cell line was loaded at 2 × 10⁻⁶ cells per well. 5Cells were cultured at a density of 1000 cells / well in 6-well plates and allowed to grow overnight. Then, fresh culture medium containing the drug was added to each well; the drug was either PDA@MXene (20 μg / mL), GOX (21.5 ng / mL), or PDA@MXene@GOX (containing 20 μg / mL PDA@MXene and 21.5 ng / mL GOX). After 4 hours of incubation, the illumination group was controlled at 1 W / cm². 2 Cells were irradiated with an 808 nm laser at a power density for 5 min. After 24 hours of incubation, cells were stained with the Annexin V-FITC / PI apoptosis detection kit and observed using a confocal laser scanning microscope (CLSM). Cells were then digested and quantitatively analyzed using flow cytometry (BD Biosciences). Furthermore, cells were stained with Mito-tracker red and Annexin-V to further analyze mitochondrial activity and apoptotic cell death. These stained cells were observed and analyzed using CLSM.

[0087] 1.8 Animals

[0088] Eight-week-old female BALB / c mice were purchased from Shenzhen Top Biotech Co., Ltd. This research and animal protocol have been approved by the Animal Ethics Subcommittee of the Hong Kong Polytechnic University. 1×10 6 4T1 cells were inoculated into the right axilla of each mouse to establish a 4T1 tumor xenograft model.

[0089] 2. Results and Discussion

[0090] 2.1 Preparation of PDA@MXene@GOX

[0091] The preparation process of PDA@MXene@GOX and its application in deep tumor starvation therapy and biosafe photothermal therapy, such as Figure 1 As shown. The MXene nanosheets prepared in this embodiment exhibit a two-dimensional thin-layer morphology with micron-sized structures, as shown. Figure 2 As shown in a)-c). From Figure 2 As shown in b), after ultrasonic treatment, large Ti3C2 nanosheets were effectively broken into small, blade-shaped, and uniform MXene nanosheets. Figure 2 As shown in c), the good crystallinity of MXene nanosheets was maintained, indicating that the mechanical force provided by ultrasound can break larger nanosheets without significantly affecting their crystal structure. Figure 2As shown in d), the average hydrodynamic size (DLS) of PDA@MXene is 45.66 nm. Zeta potential measurements revealed that PDA@MXene exhibits a stronger negative charge (-27.3 mV) compared to MXene. Then, dopamine molecules were functionalized on the MXene nanosheet surface through oxidation and polymerization. To optimize the modification efficiency of PDA on MXene, 10, 30, and 50 mg of dopamine were incorporated into the reaction mixture, respectively. Figure 2 As shown in e)-g), the thickness of PDA@MXene gradually increases with increasing dopamine levels. Figure 2 As shown in e) and h), 10 mg of dopamine can provide PDA@MXene with an average size of approximately 100 nm, which is beneficial for endocytosis and tumor penetration. Therefore, 10 mg of dopamine was chosen for the preparation of PDA@MXene. The ultraviolet-visible-near-infrared (UV-vis-NIR) absorption spectrum is as follows: Figure 2 As shown in i), by Figure 2 As shown in i), the optical absorption capacity of PDA@MXene is enhanced compared to MXene, indicating that PDA molecules have excellent intrinsic optical absorption capacity.

[0092] XRD pattern as follows Figure 2 As shown in j), by Figure 2 As shown in the figure (j), both MXene and PDA@MXene exhibited the typical sharp diffraction peak (002) of MXene at approximately 2θ = 6.18 degrees. The XPS spectra are shown below. Figure 2 As shown in k), by Figure 2 As shown in Figure k), MXene exhibits strong peaks at 458.8 eV and 464.8 eV, corresponding to Ti₂P₃ / ₂ and Ti₂P₁ / ₂, respectively. Furthermore, MXene shows a peak at 565.3 eV, indicating Ti₂S, and a strong peak at 688.3 eV, indicating F₁s. The high-resolution XPS spectrum is shown below. Figure 2 As shown in l) and m), by Figure 2 As shown in Figure 1), the C1s spectrum of MXene can be decomposed into four peaks at 279.3, 282.8, 284.5, and 286.3 eV, corresponding to C-Ti, CC, CO, and -C=O, respectively, indicating a change in the surface composition of the material. Figure 2 As shown in m), no C-Ti bonds were detected in the C1s spectrum of PDA@MXene, indicating that MXene does not exist on the surface of PDA@MXene. These results collectively demonstrate the successful preparation of PDA@MXene nanosheets.

[0093] 2.2 Stability of MXene and PDA@MXene in physiological environments

[0094] The dispersibility and stability of PDA@MXene in the physiological environment are key prerequisites for its photothermal therapy applications. Figure 3 As shown in a)-b), MXene exhibits a 30% decrease in light absorption by day 3, while PDA@MXene still shows stable UV-Vis-NIR absorption even by day 6. The Raman spectra of MXene and PDA@MXene are as follows: Figure 3 As shown in d) and e). By Figure 3 From d), we can see that MXene is at 158cm -1 A significant peak is observed at this point, attributed to the in-plane vibrations of Ti and C atoms; after seven days of room temperature incubation, the characteristic Raman peak decreases significantly, suggesting oxidation and structural damage to Ti3C2. Conversely, the characteristic Raman peak is significantly lowered by… Figure 3 As shown in e), PDA@MXene maintained clear characteristic Raman peaks, indicating that its structural stability was not significantly affected. These results demonstrate that PDA modification significantly improves the degradation resistance of MXene, endowing it with superior stability.

[0095] Subsequently, the photothermal stability of PDA@MXene under 808nm laser irradiation was systematically evaluated. Figure 3 As shown in f) and g), PDA@MXene maintains a stronger photothermal conversion capability compared to MXene. After being placed at room temperature for 3 and 7 days, PDA@MXene can still rapidly reach approximately 65℃ (ΔT approximately 40℃) under 808nm laser irradiation, indicating that PDA modification improves the photothermal stability of MXene. Furthermore, from... Figure 3 As shown in Figure h), MXene exhibited significant aggregation after photothermal heating, while PDA@MXene maintained good dispersion after heat treatment. These results collectively indicate that PDA@MXene demonstrates enhanced photothermal stability compared to MXene.

[0096] 2.3 Investigation of the photothermal properties of PDA@MXene

[0097] The UV-Vis-NIR absorption spectra of MXene and PDA@MXene are as follows: Figure 4 As shown in a), by Figure 4 As shown in a), at the same Ti3C2 concentration, PDA@MXene exhibits stronger light absorption, especially in the near-infrared region. The concentration-absorbance relationship between MXene and PDA@MXene at 808 nm is as follows: Figure 4 As shown in b), by Figure 4As shown in b), MXene and PDA@MXene exhibit a strong linear correlation between absorbance and concentration, and their extinction coefficients at 808 nm are calculated to be 25 Lg. -1 cm -1 and 33.1Lg -1 cm -1 .

[0098] Next, the photothermal heating capabilities of MXene, PDA, and PDA@MXene were quantitatively analyzed. Figure 4 From c), we can see that PDA@MXene at 1W / cm 2 The highest temperature rise (ΔT = 41.1 °C) was achieved within 6 minutes under laser irradiation, exceeding that of PDA (30.9 °C) and MXene (38 °C). Then, the temperature rise of PDA@MXene at 0.25, 0.5, 0.75, and 1 W / cm² was analyzed. 2 The photothermal performance at laser power density is as follows: Figure 5 As shown in a) of the diagram. From Figure 5 From a) we know that PDA@MXene at 1W / cm 2 The highest temperature reached was about 65°C under laser irradiation. Figure 5 Figures b) and c) show the PTT heating curves and thermograms of PDA@MXene at concentrations of 0, 6.5, 12.5, 25, 50, and 100 μg / mL. The results indicate that the temperature increase of PDA@MXene is positively correlated with its concentration; even at a low concentration (25 μg / mL), the temperature can still rise to 50 °C.

[0099] Depend on Figure 4 As shown in d), the temperature change of PDA@MXene did not decrease significantly during the five temperature increases, demonstrating its excellent photothermal stability. Finally, this embodiment compared the photothermal conversion efficiency (PCE) of PDA@MXene and MXene, and recorded the time-dependent temperature change curves during a single heating / cooling cycle. The PCE was calculated using a previously reported method at an 808nm laser and a power density of 1W / cm². 2 The PCE results are as follows Figure 4 As shown in e)-f). The photothermal conversion efficiency (PCE) of MXene and PDA@MXene at 808 nm is 46% and 51.6%, respectively. Table 1 lists the photothermal conversion efficiency (PCE) of typical current photothermal therapy (PTT) agents, including materials such as Ti3C2, gold nanorods, black phosphorus, and graphene. The comparison shows that PDA@MXene exhibits the highest PCE, indicating that PDA@MXene is a promising candidate material for photothermal therapy applications.

[0100] Table 1 Comparison of photothermal conversion efficiency of different two-dimensional materials

[0101]

[0102] The references listed in Table 1 are as follows:

[0103] [1]X.Yu,

[0104] [2] G.Cai, Z.Yu, P.Tong, D.Tang, Ti3C2 MXene quantum dot-encapsulated liposomes for photothermal immunoassays using a portable near-infraredimaging camera on a smartphone, Nanoscale, 11 (2019) 15659-15667.

[0105] [3]H.Lin,S.Gao,C.Dai,Y.Chen,J.Shi,A Two-DimensionalBiodegradableNiobium Carbide(MXene) for Photothermal Tumor Eradication in NIR-I andNIR-II Biowindows,J Am Chem Soc,139(2017)16235-16247.

[0106] [4] H. Lin, X. Wang, L. Yu, Y. Chen, J. Shi, Two-Dimensional UltrathinMXeneCeramic Nanosheets for Photothermal Conversion, Nano Lett, 17 (2017) 384-391.

[0108] [5]J.Zeng,D.Goldfeld,Y.Xia,A Plasmon-Assisted optofluidic(PAOF)

[0109] system for measuring the photothermal conversion efficiencies of goldnanostructures and controlling an electrical switch, Angewandte Chemie International Edition, 52 (2013) 4169-4173.

[0110] [6] Y. Zeng, Z. Guo, Synthesis and stabilization of black phosphorus and phosphorene: Recent progress and perspectives, Iscience, 24 (2021).

[0111] [7] L.Cheng, C.Wang, L.Feng, K.Yang, Z.Liu, Functional nanomaterials for phototherapies of cancer, Chemical reviews, 114 (2014) 10869-10939.

[0112] 2.4 Photothermal enhancement of enzyme activity by PDA@MXene@GOX

[0113] To investigate the effect of temperature on GOX activity, this example quantifies the hydrogen peroxide concentration in the GOX-glucose catalytic reaction at different temperatures to evaluate GOX activity. Figure 6 As shown in a)-b), within the temperature range of 25-55℃, the activity of GOX increases with increasing temperature. However, at 65℃, the activity of GOX decreases slightly, possibly due to enzyme inactivation. The change in pH over time in the GOX-glucose catalytic reaction is consistent with the H2O2 detection results, and GOX reaches its peak catalytic activity at approximately 55℃, which is highly consistent with the temperature required for photothermal therapy applications.

[0114] To verify whether the photothermal effect of PDA@MXene can effectively enhance the catalytic activity of GOX, this embodiment loads an appropriate amount of GOX onto the PDA@MXene surface via physical adsorption. The loading efficiency of GOX on PDA@MXene was determined to be 32.9% using the Coomassie Brilliant Blue protein assay. Figure 4 As shown in g), compared with PDA@MXene, the UV-vis absorption of PDA@MXene@GOX slightly increased before 500 nm after GOX loading. Subsequently, the enzyme activities of GOX and PDA@MXene@GOX were compared with and without 808 nm laser irradiation, and evaluated by measuring the generation of key reactants H2O2 and O2 in the GOX-glucose reaction. The results are shown in g). Figure 4 As shown in h) and i). By Figure 4 As shown in h), the amount of H2O2 generated by PDA@MXene@GOX under laser irradiation increased significantly, and its enzyme activity was almost four times that of free GOX, indicating that the enzyme activity was significantly enhanced under laser irradiation. Figure 4 As shown in i), compared with the PDA@MXene@GOX group treated alone, the group treated with both PDA@MXene@GOX and laser exhibited a more significant decrease in oxygen concentration and higher enzyme activity. These results collectively indicate that the photothermal effect of PDA@MXene can significantly enhance the catalytic activity of GOX, providing an effective strategy for developing photothermal / starvation synergistic therapy for tumors.

[0115] 2.5 In Vitro Synergistic Tumor Starvation and Photothermal Therapy

[0116] This embodiment investigates the efficacy of PDA@MXene@GOX in in vitro tumor starvation and photothermal therapy. First, confocal fluorescence imaging was used to assess the cellular uptake level of PDA@MXene@GOX in 4T1 tumor cells. Cyanocyanine 5 (Cy5) fluorescent dye was attached to the surface of GOX@PDA@MXene to generate a fluorescent signal representing PDA@MXene@GOX. PDA@MXene@GOX-Cy5 was co-cultured with 4T1 cells for 1 h, 4 h, and 6 h, and images were then captured under a fluorescence microscope. Figure 7 As shown in (a), 4T1 cells exhibited time-varying cellular uptake behavior. The highest fluorescence intensity, reaching saturation, was observed after 4 hours of co-culturing with PDA@MXene@GOX-Cy5. Furthermore, the red fluorescence signal of Cy5 indicates that the primary site of action for the endocytosed PDA@MXene@GOX-Cy5 nanoparticles is located in the cytoplasm.

[0117] This embodiment used the CCK-8 assay and fluorescence live / dead staining to quantitatively assess the effect of PDA@MXene@GOX on the apoptosis level of 4T1 cells. The results are as follows: Figure 7 As shown in b). Figure 7 As shown in b), under dark conditions, PDA@MXene did not exhibit significant cytotoxicity in the concentration range of 0-20 μg / mL, indicating that the PDA@MXene material has good biosafety.

[0118] However, cell viability decreased by 50% after treatment with 20 μg / mL PDA@MXene and light irradiation. Starting from 5 ng / mL, GOX induced significant cytotoxicity, with 21.5 ng / mL GOX causing a 40% decrease in cell viability, indicating that GOX-mediated glucose depletion triggered cell starvation and apoptosis. 5 μg / mL PDA@MXene@GOX (GOX concentration of 21.5 ng / mL) resulted in lower cell viability compared to the same concentration of GOX, suggesting that PDA@MXene@GOX may have a higher cellular uptake rate.

[0119] In the PDA@MXene@GOX + laser group treated with both bifunctional nanoparticles and laser, cell viability decreased to 10%, significantly lower than in the PDA@MXene@GOX group treated with only bifunctional nanoparticles or the GOX group treated with only GOX. This demonstrates the synergistic effect of starvation and photothermal therapy. Figure 7 As shown in c), both GOX and PDA@MXene@GOX induced a small amount of cell death. When the laser was turned on, PDA@MXene@GOX induced significant cell death within the laser-irradiated area. The cell death rate in the PDA@MXene@GOX + laser group was significantly higher than that in the PDA@MXene + laser group and the GOX + laser group, indicating that the photothermal effects of PDA@MXene and GOX may synergistically promote cell death. Subsequently, the apoptosis rate of 4T1 cells was analyzed by flow cytometry. Figure 7 As shown in d), the apoptosis rates of cells in the GOX or PDA@MXene@GOX groups after 24 hours of treatment were 22.8% and 19.4%, respectively. The highest apoptosis rate, approximately 44.1%, was observed in the PDA@MXene@GOX + laser group.

[0120] To further explore the mechanism behind the synergistic anti-tumor effect of PDA@MXene@GOX, this embodiment used the DCFH-DA probe to detect changes in intracellular ROS levels to determine whether PDA@MXene@GOX induces ROS metabolism through GOX. Figure 7As shown in Figure e), compared with the control group, the cellular ROS level in the PDA@MXene+laser group did not change significantly. However, the ROS level in the PDA@MXene@GOX+laser group was significantly higher than that in the GOX+laser group. This may be because PDA@MXene increases the temperature under irradiation, effectively enhancing the activity of GOX, thereby increasing the production of H2O2. This embodiment also evaluated the effect of PDA@MXene@GOX on cellular mitochondrial activity using mitochondrial fluorescence staining and mitochondrial membrane potential staining. The results showed that PDA@MXene@GOX+laser treatment could significantly induce mitochondrial apoptosis and reduce mitochondrial membrane potential. These results indicate that PDA@MXene@GOX enhances the catalytic activity of GOX through the increased temperature of PDA@MXene under light irradiation, leading to the accumulation of intracellular reactive oxygen species (H2O2 and ROS) and the destruction of mitochondrial activity, ultimately inducing apoptosis.

[0121] 2.6 In vivo tumor preservation and photothermal conversion

[0122] This embodiment first investigated the tumor retention behavior and photothermal conversion efficiency of PDA@MXene@GOX in tumors. To explore the tumor retention behavior of PDA@MXene@GOX, PDA@MXene was labeled with the red fluorescent dye Cy5 (PDA@MXene-Cy5) and then injected into the tumors of Balb / c mice carrying 4T1 breast cancer. This embodiment analyzed the fluorescence intensity of Cy5 in the tumor region at different time points (0 hours, 0.5 hours, 2 hours, 6 hours, and 24 hours) after PDA@MXene injection. Figure 8 As shown in (a), compared with the control group, PDA@MXene@Cy5 exhibited significantly enhanced fluorescence intensity and remained within the tumor for up to 6 hours, which is sufficient to support the role of GOX within the tumor. Figure 8 As shown in b), 6 hours post-injection, the fluorescence intensity of PDA@MXene@Cy5 within the tumor was higher than that of free Cy5, indicating that loading PDA@MXene can effectively enhance the retention of the drug-loaded molecule within the tumor. Subsequently, the photothermal conversion capacity of PDA@MXene@GOX in mice was evaluated using infrared imaging, and the results are as follows: Figure 8 As shown in c) and d). By Figure 8 As shown in c) and d), under laser activation, the local tumor temperature in mice injected with PDA@MXene@GOX rapidly increased from 32°C to 54°C in just 5 minutes. In contrast, under the same conditions, the local tumor temperature in mice treated with PBS showed only a slight change, increasing from 33.2°C to 39.6°C. These results indicate that PDA@MXene@GOX can be effectively maintained within the tumor and exhibits significant photothermal conversion capabilities in vivo.

[0123] 2.7 In vivo synergistic tumor starvation and photothermal therapy

[0124] Next, the effects of PDA@MXene@GOX in synergistic tumor starvation and photothermal therapy in vivo were systematically investigated. The tumor volume reached 100 mm. 3 4T1 tumor-bearing mice were randomly divided into six groups: (1) control group (PBS), (2) PBS + laser, (3) PDA@MXene, (4) PDA@MXene@GOX, (5) PDA@MXene + laser, and (6) PDA@MXene@GOX + laser. In the laser irradiation group, the tumor was irradiated with an 808nm laser 4 hours after injection, and only one irradiation was performed. Figure 9 As shown in (a), all six groups of mice did not experience significant weight loss during treatment, indicating that PDA@MXene@GOX has good biocompatibility. Figure 9 As shown in b), tumors in mice in the control and PBS+laser groups grew rapidly within 18 days, reaching 7.5-fold and 8.6-fold their initial volume, respectively. Furthermore, PDA@MXene alone showed no tumor-inhibiting effect, indicating its non-toxicity. In contrast, when photothermal therapy was applied with laser, PDA@MXene partially inhibited the growth of 4T1 tumors, reaching 4.1-fold their initial volume. Additionally, tumor volume in mice in the PDA@MXene@GOX group without laser treatment was also partially inhibited (to 4.7-fold of the initial volume), revealing the effect of GOX-induced starvation therapy. For the PDA@MXene@GOX+laser group, tumor volume was inhibited to 1.8-fold of the initial volume (***p<0.001), representing the best treatment effect among these groups. Notably, in one mouse, the tumor was completely inhibited and eliminated after 18 days. The favorable anti-tumor effects of PDA@MXene@GOX were further supported by tumor weight, images of resected tumors, and representative images of tumor-bearing mice at the start and end of treatment, specifically as follows: Figure 9 As shown in c)-e). It is noteworthy that this remarkable antitumor efficacy was achieved with only a single administration and irradiation session, suggesting that the potential of this combined therapy system could be further enhanced with multiple treatments. To verify the catalytic activity of GOX in vivo, tumor tissue was excised and glucose concentrations were quantitatively analyzed. (See Figure c)-e). Figure 9 As shown in f), 12 hours after treatment with GOX and PDA@MXene@GOX, the intratumoral glucose concentration was significantly reduced. Furthermore, the PDA@MXene@GOX + laser group exhibited the lowest glucose concentration, consistent with previous results, indicating that high temperature can enhance GOX activity.

[0125] Subsequently, tumor sections were extracted from each group, and tumor proliferation and damage were analyzed by hematoxylin and eosin (H&E) staining, TUNEL assay, and anti-heat shock protein (HSP-70) staining. Figure 9 As shown in (g), mouse tumors treated with PDA@MXene@GOX+ laser exhibited significant cellular damage and numerous voids, representing tumor necrosis. In contrast, no abnormal cells were observed in the control and PDA@MXene groups. Furthermore, TUNEL staining revealed that the PDA@MXene@GOX+ laser group induced the most severe apoptosis, characterized by marked cell shrinkage and nuclear disappearance. In contrast, apoptosis was less severe in the PDA@MXene+ laser group and the PDA@MXene@GOX group. Figure 9 As shown in (g), HSP-70 immunofluorescence staining results revealed higher levels of HSP-70 expression in the PDA@MXene+laser group, attributed to the self-protective mechanism of tumor cells under photothermal stimulation. However, in the GOX-containing group (PDA@MXene@GOX, regardless of laser irradiation), HSP-70 expression was significantly downregulated, consistent with previous reports. The results of HSP-70 expression and intratumoral glucose concentration indicate that GOX-induced starvation therapy effectively overcomes the heat tolerance of tumor cells by reducing glucose levels and inhibiting protein expression. In summary, these immunohistochemical results demonstrate the powerful antitumor effect of this multimorphic nanodevice through the synergistic effect of tumor starvation and photothermal therapy.

[0126] Finally, the biocompatibility of PDA@MXene@GOX in vivo was investigated by hematological tests and H&E staining of major mouse organs (heart, liver, spleen, lung, and kidney). Figure 10 Figure a) shows the whole blood analysis of mice after receiving PBS or PDA@MXene@GOX+ laser treatment, and the results showed no significant abnormalities in various blood cells. Figure 10 Figure b) shows that even at high concentrations (100 μg / mL), the hemolysis rate of PDA@MXene@GOX is almost negligible, indicating its good blood biocompatibility. Furthermore, as... Figure 10 As shown in c), no significant physiological damage was observed in the H&E staining images of the PDA@MXene group and the PDA@MXene+laser group. The results at both the cellular and tissue levels collectively demonstrate the good biocompatibility of PDA@MXene@GOX under laser irradiation.

[0127] 3. Conclusion

[0128] This invention utilizes the biocompatible polymer molecule PDA, with its inherent photothermal conversion capability, to modify two-dimensional MXene. The modified MXene exhibits excellent stability and enhanced photothermal conversion capability, achieving synergistic tumor therapy when combined with the GOX biocatalyst. Both in vivo and in vitro experiments demonstrate that the enzymatic activity of GOX can be significantly enhanced under high-temperature conditions induced by the PDA@MXene photothermal effect. Simultaneously, the enhanced glucose depletion capacity of GOX effectively alleviates tumor heat tolerance by downregulating metabolic activity and inhibiting the expression of heat shock proteins (HSPs).

[0129] The synergistic therapy provided by PDA@MXene@GOX demonstrated superior tumor suppression in 4T1 tumor-bearing mice, achieving a tumor inhibition rate of 76%, significantly higher than photothermal therapy (PTT) and starvation therapy alone, without any obvious toxicity. Therefore, the PDA@MXene@GOX prepared in this invention has great potential for combined PTT / starvation therapy in cancer and shows promising opportunities for clinical translation. Due to the excellent loading capacity and high photothermal conversion efficiency of two-dimensional nanomaterials, this bifunctional nanoformulation can timely and precisely trigger simultaneous and localized photothermal therapy and starvation therapy effects.

[0130] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A bifunctional nanoformulation for tumor starvation and photothermal therapy, characterized in that, The bifunctional nanoformulation comprises: PDA@MXene, and glucose oxidase loaded on the PDA@MXene; the PDA@MXene comprises: MXene nanosheets, and polydopamine modified on the MXene nanosheets.

2. The bifunctional nanoformulation according to claim 1, characterized in that, In the bifunctional nanoformulation, the mass ratio of PDA@MXene to glucose oxidase is (800-1200):

1.

3. The bifunctional nanoformulation according to claim 1, characterized in that, In the PDA@MXene, the mass ratio of MXene nanosheets to polydopamine is 1:(2.5-5).

4. The bifunctional nanoformulation according to claim 1, characterized in that, The average size of the bifunctional nanoformulation is 50-200 nm.

5. The bifunctional nanoformulation according to claim 1, characterized in that, The average size of the MXene nanosheets is 20-100 nm.

6. A method for preparing a bifunctional nano-formulation as described in any one of claims 1-5, characterized in that, Including the following steps: Solution A containing MXene nanosheets and solution B containing dopamine hydrochloride are provided; Solution B was added dropwise to solution A, and after stirring, PDA@MXene was obtained. The glucose oxidase solution was added dropwise to the dispersion of PDA@MXene, and the mixture was stirred to obtain the bifunctional nano-formulation.

7. The preparation method according to claim 6, characterized in that, In solution A, the concentration of MXene nanosheets is 0.1-5 mg / mL; in solution B, the concentration of dopamine hydrochloride is 0.5-10 mg / mL; the volume ratio of solution A to solution B is 1:(0.625-1.25). And / or, in the PDA@MXene dispersion, the concentration of PDA@MXene is 0.1-5 mg / mL; in the glucose oxidase solution, the concentration of glucose oxidase is 50-500 μg / mL; and the volume ratio of the PDA@MXene dispersion to the glucose oxidase solution is 1:(3-8).

8. The preparation method according to claim 6, characterized in that, The stirring conditions are: temperature 10-50℃, rotation speed 200-600rpm / min, and time 8-24h.

9. The preparation method according to claim 6, characterized in that, The method for preparing the MXene nanosheets includes the following steps: Ti3C2 nanosheets were dispersed in N-methylpyrrolidone, sonicated, and then centrifuged for the first time to collect the supernatant. Acetone was added to the supernatant, and a second centrifugation was performed to collect the precipitate and obtain the MXene nanosheets.

10. The use of a bifunctional nanoformulation as described in any one of claims 1-4 in the preparation of an antitumor drug.

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