Titanium-doped / conductive polymer-supported cerium vanadate nanoszyme, and preparation method and application thereof

Abstract

The application discloses a titanium-doped / conductive polymer-loaded cerium vanadate nano-enzyme and a preparation method and application thereof. The titanium-doped cerium vanadate nanorods are synthesized by a hydrothermal method, and polypyrrole small balls are in-situ loaded on the nanorods by an in-situ chemical oxidation polymerization method of pyrrole to construct the titanium-doped / conductive polymer-loaded cerium vanadate nano-enzyme. The titanium-doped / conductive polymer-loaded cerium vanadate nano-enzyme can realize four enzyme activities, i.e., peroxidase-like activity, glutathione peroxidase-like activity, superoxide dismutase-like activity and catalase-like activity. Meanwhile, the titanium-doped / conductive polymer-loaded cerium vanadate nano-enzyme has excellent photothermal stability and photothermal conversion efficiency, can further improve the enzyme-like activity, and realizes synergistic treatment. The titanium-doped / conductive polymer-loaded cerium vanadate nano-enzyme can also be used as a photoacoustic imaging contrast agent to realize photoacoustic imaging-induced early diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a titanium-doped / conductive polymer-supported cerium vanadate nanozyme, its preparation method, and its application. Background Technology

[0002] Chemotherapy, as a widely used clinical treatment, often produces serious side effects, drug resistance, and poor prognosis, necessitating the development of new and highly effective treatment methods to improve treatment efficiency and reduce toxic side effects. In recent years, novel treatment methods, such as photodynamic therapy (PDT), photothermal therapy (PTT), and chemodynamic therapy (CDT), have been widely used in tumor treatment due to their minimally invasive nature, controllability, low drug resistance, low toxicity, and high specificity. PTT, in particular, converts light energy, especially near-infrared light in the second region—a highly biocompatible and tissue-penetrating light source—into heat energy. This heat selectively kills cancer cells through localized heating, offering advantages such as minimal invasiveness, long-lasting effects, and safety. Furthermore, the thermal effect of photothermal therapy can significantly enhance the therapeutic effect of tumors mediated by reactive oxygen species (ROS).

[0003] Enzyme therapy is a promising treatment for tumors, enhancing therapeutic efficacy by modulating the redox state of the tumor microenvironment, catalyzing key biochemical reactions, and increasing local tumor temperature. Compared to natural enzymes, synthetic nanozymes offer advantages such as designability, multifunctionality, and applicability. They can simultaneously possess the activities of peroxidase (POD), oxidase (OXD), catalase (CAT), and superoxide dismutase (SOD), facilitating the controllable regulation of reactive oxygen species (ROS) levels in the tumor microenvironment (TME). However, single-method treatments are still insufficient to effectively eliminate tumor tissue, and low photothermal conversion efficiency and poor heating effects significantly limit the wider clinical application of photothermal therapy (PTT). Furthermore, most artificially synthesized nanozymes exhibit only single catalytic activities in the highly complex tumor microenvironment, and these activities are insufficient to achieve the desired therapeutic effect. Therefore, developing a nanomaterial that combines the ability to mimic multiple enzymes with photothermal properties to enhance anti-tumor efficacy through synergistic effects of photothermal and chemokinetics has significant scientific and clinical value. Summary of the Invention

[0004] To address the shortcomings of existing nanozyme technologies, the purpose of this invention is to design nanomaterials that combine multiple enzyme mimicry capabilities with photothermal properties, specifically a titanium-doped / conductive polymer-supported cerium vanadate nanozyme. Based on biomimetic principles, this invention prepares a composite nanozyme with a plum blossom-like structure, specifically through titanium doping and modification with a conductive polymer. The modified nanozyme can achieve both multiple enzyme activities and photothermal properties, realizing a highly efficient chemokinetic-photothermal synergistic effect.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The nanoenzyme material provided by this invention, which combines multiple enzyme activities and photothermal properties, is constructed by synthesizing cerium vanadate nanorods doped with titanium, and further loading conductive polymers onto the nanorods.

[0007] In a first aspect, the present invention provides a method for preparing titanium-doped cerium vanadate nanomaterials with multiple enzyme activities.

[0008] The method for preparing titanium-doped cerium vanadate nanomaterials provided by the present invention includes the following steps: mixing a titanium source, a cerium source, a vanadium source and a template agent under alkaline conditions to obtain a mixed solution; then subjecting the mixed solution to a hydrothermal reaction, centrifuging and washing to obtain the titanium-doped cerium vanadate nanomaterials.

[0009] As a preferred technical solution of the present invention, all solutions are prepared using deionized water.

[0010] As a preferred technical solution of the present invention, the titanium source is selected from one or a mixture of several of titanium sulfate, titanium nitrate, titanium tetrachloride, titanium dioxide, and titanium oxysulfate, preferably titanium sulfate.

[0011] As a preferred technical solution of the present invention, the cerium source is selected from one or a mixture of several of cerium nitrate, cerium oxalate, cerium chloride, cerium sulfate, cerium ammonium sulfate, cerium carbonate, cerium acetate, and cerium phosphate, preferably cerium nitrate.

[0012] As a preferred technical solution of the present invention, the vanadium source is selected from one or a mixture of several of sodium orthovanadate, ammonium metavanadate, vanadium oxysulfate, vanadium oxalate, vanadium tetrachloride, and vanadium pentoxide, preferably sodium orthovanadate.

[0013] As a preferred embodiment of the present invention, the template agent is selected from one or a mixture of several of ethylenediaminetetraacetic acid (EDTA), hexadecyltrimethylammonium bromide (CTAB), polyethylene glycol-2000 (PEG-2000), and polyethylene glycol-4000 (PEG-4000), preferably ethylenediaminetetraacetic acid.

[0014] Preferably, in the mixed solution, the concentration of the cerium source is 60-300 mM, the concentration of the vanadium source is 60-300 mM, the concentration of the template agent is 60-300 mM, and the concentration of the titanium source is 10-50 mM.

[0015] As a preferred embodiment of the present invention, the preparation method of the mixed solution includes the following steps:

[0016] 1) Dissolve the cerium source and template agent in deionized water, stir and mix evenly to obtain a cerium source-template agent mixture;

[0017] 2) Dissolve the vanadium source in deionized water and stir to mix evenly to obtain a vanadium source solution;

[0018] 3) Dissolve the titanium source in deionized water and stir to mix evenly to obtain a titanium source solution;

[0019] 4) Slowly add the vanadium source solution dropwise into the cerium source-template agent mixture, and stir thoroughly at room temperature;

[0020] 5) Slowly add the titanium source solution dropwise to the cerium source-template agent-vanadium source mixed solution in step 4), and stir thoroughly at room temperature to obtain the mixed solution;

[0021] In the above steps, steps 1) to 3) are not in any particular order and can be adjusted arbitrarily.

[0022] Preferably, the stirring conditions in step 1) above are: stirring at 20-40℃ for 10-20 minutes;

[0023] Preferably, the stirring conditions in step 2) above are: stirring at 50-80℃ for 10-20 minutes;

[0024] Preferably, the stirring conditions for step 2) above are: stirring at 20-40℃ for 10-20 minutes.

[0025] Preferably, in step 4) above, the dropping rate is 2-4 mL / min;

[0026] Preferably, in step 5) above, the dropping rate is 2-4 mL / min.

[0027] As a preferred technical solution of the present invention, the reagent for adjusting the alkaline conditions is selected from one or a mixture of several of sodium hydroxide, potassium hydroxide, ammonia, ammonium carbonate, and ammonium bicarbonate, preferably ammonia.

[0028] As a preferred technical solution of the present invention, the pH of the mixed solution is adjusted to alkaline, and the pH range is adjusted to pH 8-10, preferably to pH 9.

[0029] As a preferred technical solution of the present invention, the hydrothermal reaction is carried out in a high-temperature and high-pressure resistant reactor, the reaction temperature of the hydrothermal reaction is 160-180℃, and the reaction time is 16-24h.

[0030] As a preferred technical solution of the present invention, after the hydrothermal reaction is completed, the material is washed to remove impurities. The washing solution is water and ethanol, and the washing method is centrifugal washing with a speed of 8000-12000 rpm and a time of 8-15 min.

[0031] The titanium-doped cerium vanadate nanomaterials with multiple enzyme activities prepared by the above method are also within the scope of protection of this invention.

[0032] Secondly, based on the first aspect, the present invention provides a method for preparing titanium-doped conductive polymer-supported cerium vanadate nanomaterials that possess both multiple enzyme activities and photothermal properties.

[0033] The method for preparing titanium-doped cerium vanadate nanomaterials supported by conductive polymers provided by the present invention includes the following steps: adding conductive polymer monomers to the titanium-doped cerium vanadate nanomaterial solution obtained in the first aspect, and loading conductive polymers onto titanium-doped cerium vanadate nanorods by in-situ chemical polymerization with an oxidant to obtain titanium-doped cerium vanadate nanomaterials supported by conductive polymers.

[0034] As a preferred technical solution of the present invention, all are prepared using deionized water.

[0035] As a preferred embodiment of the present invention, the conductive polymer monomer is selected from one or a mixture of several of pyrrole, aniline, thiophene, acetylene, and dopamine, preferably pyrrole.

[0036] As a preferred embodiment of the present invention, the oxidant is selected from one or a mixture of several of ferric chloride, ammonium persulfate, potassium permanganate, and potassium persulfate, preferably ferric chloride.

[0037] As a preferred embodiment of the present invention, the concentration of the titanium-doped cerium vanadate solution is 0.5-5 mg / mL, the concentration of the conductive polymer monomer is 2-7 mM, and the concentration of the oxidant is 6-21 mM.

[0038] As a preferred technical solution of the present invention, the reaction temperature of the in-situ chemical polymerization of the oxidant is 2-10℃ and the reaction time is 4-12h.

[0039] As a preferred technical solution of the present invention, after the reaction of in-situ chemical polymerization of oxidant is completed, the material is washed to remove impurities. The washing solution is water and ethanol, and the washing method is centrifugal washing with a speed of 8000-12000 rpm and a time of 8-15 min.

[0040] The titanium-doped conductive polymer-supported cerium vanadate nanomaterials prepared by the above method are also within the scope of protection of this invention.

[0041] Thirdly, based on the second aspect, the present invention provides the application of the above-mentioned titanium-doped conductive polymer-supported cerium vanadate nanomaterial that possesses multiple enzyme activities and photothermal properties.

[0042] The applications of the titanium-doped conductive polymer-supported cerium vanadate nanomaterials provided by this invention are selected from at least one of the following: 1) application in the preparation of drugs for tumor treatment; 2) application in the preparation of materials for tumor treatment; 3) application in the preparation of photoacoustic imaging contrast agents.

[0043] The materials used for tumor treatment include: materials for tumor photothermal therapy, materials for tumor chemokinetic therapy, and tumor treatment materials that combine the effects of chemokinetics and photothermal therapy.

[0044] The titanium-doped, conductive polymer-supported cerium vanadate nanomaterials provided by this invention simultaneously possess peroxidase-like (POD-like), catalase-like (CAT-like), superoxide dismutase-like (SOD-like), and glutathione peroxidase-like (GP) properties. X It exhibits catalytic activity similar to that of a glutathione-like enzyme. This enzyme-like activity can scavenge overexpressed glutathione in the tumor microenvironment and decompose endogenous hydrogen peroxide into oxygen, alleviating tumor microenvironment hypoxia. Simultaneously, through a Fenton-like reaction, it converts excess hydrogen peroxide into highly toxic free radicals, inducing tumor cell death. Furthermore, the combination with conductive polymers endows this composite nanozyme with excellent photothermal conversion capabilities and photothermal stability. It can kill cells by generating heat, achieving image-guided photothermal therapy (PTT) and photothermally enhanced chemokinetic therapy (CDT).

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. Compared with the single catalytic activity of natural enzymes, the present invention can simultaneously possess the activities of four enzymes: peroxidase-like, glutathione peroxidase-like, superoxide dismutase-like, and catalase-like.

[0047] 2. This invention improves the photothermal properties of materials and enables photoacoustic imaging by doping with conductive polymers, which is of great significance for the early diagnosis and treatment of tumors.

[0048] 3. Most natural enzymes have very low content and are easily denatured and lose their function when exposed to non-physiological conditions such as heat, acid, and alkali. This invention has the characteristics of being minimally invasive, controllable, having low drug resistance, low toxicity, and high specificity.

[0049] 4. The method of the present invention is simple in process, uses inexpensive raw materials, and can be mass-produced. Attached Figure Description

[0050] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0051] Figure 1 This is a schematic diagram illustrating the synthesis of the nanoenzyme with multi-enzyme activity and photothermal properties of the present invention.

[0052] Figure 2 Scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the composite nanozyme: (a)(d)CeVO4, (b)(e)CeVO4-Ti, (c)(f)CeVO4-Ti@PPy.

[0053] Figure 3 XPS images of the composite nanozymes: (a) CeVO4, (b) CeVO4-Ti, (c) CeVO4-Ti@PPy.

[0054] Figure 4 XPS images of Ce3d composite nanozymes: (a) CeVO4, (b) CeVO4-Ti, (c) CeVO4-Ti@PPy.

[0055] Figure 5 The absorption spectra and absorption coefficients of the composite nanozymes are: (a)(d)CeVO4, (b)(e)CeVO4-Ti, (c)(f)CeVO4-Ti@PPy.

[0056] Figure 6 Photothermal effect diagram of composite nanozymes: (a) using 808nm with a power of 0.8W / cm 2 Temperature rise of three materials under laser irradiation at a concentration of 200 μg / mL; (bd) using 808 nm laser with a power of 0.8 W / cm². 2 (e) Temperature rise, temperature histogram, and infrared thermogram of CeVO4-Ti@PPy at different concentrations under laser irradiation; using 808nm laser with a power of 0.8W / cm². 2 Temperature curves of five heating-cooling cycles after laser irradiation of CeVO4-Ti@PPy at a concentration of 200 μg / mL; (fg) using 1064 nm and 0.8 W / cm². 2The temperature rise of CeVO4-Ti@PPy under different concentrations under laser irradiation and the temperature rise bar graph; (h) Photothermal conversion efficiency of CeVO4-Ti@PPy.

[0057] Figure 7 (a) A schematic diagram of a biomimetic plum blossom of composite nanozymes; (b) A schematic diagram of the detection of multiple enzyme activities.

[0058] Figure 8 Enzyme-like activities of composite nanozymes: (a) peroxidase-like activities of the three materials at different concentrations at room temperature; (b) peroxidase-like activities of the three materials at different concentrations at 43°C; (c) glutathione peroxidase-like activities of the three materials at different concentrations; (d) the activity of the three materials against superoxide anion radicals (·O2) at different concentrations. - (e) The scavenging ability of the three materials under the action of H2O2; (f) The H2O2 scavenging curves of the three materials in PBS.

[0059] Figure 9 Cytotoxicity of CeVO4-Ti@PPy nanozymes: (a) Effect of CeVO4-Ti@PPy on the survival rate of NIH-3T3 cells; (b) Effect of CeVO4-Ti@PPy on the survival rate of 4T1 cells.

[0060] Figure 10 In vitro photoacoustic imaging of composite nanozymes: (a) In vitro photoacoustic signal images of the three materials; (b) In vitro photoacoustic signal intensity of the three materials.

[0061] Figure 11 In vivo photoacoustic imaging of CeVO4-Ti@PPy nanozymes: (a) Time-resolved cumulative PA image of CeVO4-Ti@PPy nanozymes at the tumor site; (b) Quantitative analysis of PA signal of CeVO4-Ti@PPy nanozymes. Detailed Implementation

[0062] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

[0063] Example 1: Preparation of multifunctional composite nanozymes

[0064] like Figure 1 As shown, it includes the following steps:

[0065] Step 1: Synthesis of Cerium Vanadate Nanozymes

[0066] Weigh 1 mmol of cerium nitrate hexahydrate and 1 mmol of ethylenediaminetetraacetic acid into a beaker, add 15 mL of deionized water, and stir thoroughly at room temperature for 15 min to obtain a cerium nitrate hexahydrate-ethylenediaminetetraacetic acid solution.

[0067] Weigh 1 mmol of sodium orthovanadate into a beaker, add 15 mL of deionized water, heat and stir for 15 min until the solution becomes clear and transparent, and obtain an aqueous solution of sodium orthovanadate.

[0068] Sodium orthovanadate aqueous solution was slowly added dropwise to cerium nitrate hexahydrate-ethylenediaminetetraacetic acid solution, and stirred thoroughly at room temperature for 15 minutes.

[0069] Adjust the pH of the mixed aqueous solution to 9.0 using ammonia solution, and stir thoroughly at room temperature for 15 minutes.

[0070] The mixed aqueous solution was transferred to a 100 mL polytetrafluoroethylene-lined high-temperature and high-pressure reactor and reacted at 180 °C for 18 h. After the reaction was completed, the mixture was washed twice by alternating centrifugation with water and ethanol at 10,000 rpm to remove the hydrothermal reaction solvent and impurities, thus synthesizing cerium vanadate nanozymes.

[0071] The synthesized cerium vanadate nanozyme was dissolved in 30 mL of deionized water, sonicated for 30 min for better dispersion, and stored at room temperature for later use.

[0072] Step 2: Synthesis of titanium-doped cerium vanadate nanozymes

[0073] Weigh 1 mmol of cerium nitrate hexahydrate and 1 mmol of ethylenediaminetetraacetic acid into a beaker, add 15 mL of deionized water, and stir thoroughly at room temperature for 15 min to obtain a cerium nitrate hexahydrate-ethylenediaminetetraacetic acid solution.

[0074] Weigh 0.9 mmol of sodium orthovanadate into a beaker, add 15 mL of deionized water, heat and stir for 15 min until the solution becomes clear and transparent, and obtain an aqueous solution of sodium orthovanadate.

[0075] Weigh 0.1 mmol of titanium sulfate into a beaker, add 5 mL of deionized water, and stir thoroughly at room temperature for 15 min to obtain an aqueous solution of titanium sulfate.

[0076] Sodium orthovanadate aqueous solution was slowly added dropwise to cerium nitrate hexahydrate-ethylenediaminetetraacetic acid solution, and stirred thoroughly at room temperature for 15 min. Further, titanium sulfate aqueous solution was slowly added dropwise to cerium nitrate hexahydrate-ethylenediaminetetraacetic acid-sodium orthovanadate solution, and stirred thoroughly at room temperature for 15 min to obtain a mixed solution.

[0077] Adjust the pH of the mixture to 9.0 using ammonia solution and stir thoroughly at room temperature for 15 minutes.

[0078] The mixed solution was transferred to a 100 mL polytetrafluoroethylene-lined reactor and reacted at 180 °C for 18 h. After the reaction, the mixture was washed twice by alternating centrifugation with water and ethanol at 10,000 rpm to remove the hydrothermal reaction solvent and impurities, thus synthesizing titanium-doped cerium vanadate nanozymes.

[0079] The synthesized titanium-doped cerium vanadate nanozyme was dissolved in 30 mL of deionized water, sonicated for 30 min for better dispersion, and stored at room temperature for later use.

[0080] Step 3: Synthesis of titanium-doped polypyrrole-supported cerium vanadate nanozymes.

[0081] The concentration of the titanium-doped cerium vanadate nanozyme solution prepared in step two was determined.

[0082] Add 40 mL of deionized water to the beaker, and then add a certain amount of nanoenzyme aqueous solution using a pipette according to the concentration of the titanium-doped cerium vanadate nanoenzyme solution obtained in step two, to obtain a final volume of 50 mL and a concentration of 1 mg / mL of titanium-doped cerium vanadate nanoenzyme aqueous solution.

[0083] Stir the nanozyme aqueous solution thoroughly in ice water (2-10℃) for 20 min. Use a pipette to take 20 μL of pyrrole (AR grade, concentration above 99.5%) and slowly add it dropwise to the nanozyme aqueous solution. Stir thoroughly in ice water (2-10℃) for 20 min.

[0084] Weigh 0.23 g of ferric chloride hexahydrate and slowly add it to the nanozyme aqueous solution. Stir thoroughly in ice water (2-10℃) for 8 hours. After the reaction is complete, wash twice with water and ethanol alternately at 10000 rpm to remove the solvent and impurities in the reaction.

[0085] The synthesized titanium-doped polypyrrole-supported cerium vanadate nanozyme was dissolved in 30 mL of deionized water, sonicated for 30 min for better dispersion, and stored at room temperature for later use.

[0086] Characterization and performance:

[0087] See results Figure 2 Characterized by scanning electron microscopy and transmission electron microscopy, the three cerium vanadate nanozymes prepared in this embodiment were rod-shaped, among which... Figure 2 As can be seen from f, polypyrrole is loaded onto nanorods in the form of polypyrrole microspheres, such as Figure 7Its morphology resembles plum blossoms; the polypyrrole microspheres are like plum blossom flowers, and the titanium-doped cerium vanadate nanorods are like plum blossom branches. The CeVO4 nanozyme has a particle size of 112.67±32.15 nm, the CeVO4-Ti nanozyme has a particle size of 116.67±21.98 nm, and the CeVO4-Ti@PPy nanozyme has a particle size of 116.65±11.25 nm. The X-ray photoelectron spectra of the three nanozymes are shown below. Figure 3 As shown, the X-ray photoelectron spectra of the three nanozymes Ce3d are as follows: Figure 4 As shown, the peaks at 880.5 (V') and 884.4 (V') eV are Ce3d 5 / 2 The transition peaks at 899.1 (U') and 903 (U”) eV are Ce3d. 3 / 2 The transition peak proves the presence of Ce in the material. +3 Oxidation state. 914 eV (U 0 The peak at ) corresponds to Ce +4 The presence of characteristic peaks in the valence state confirms Ce 3+ and Ce 4+ Coexistence in nanozymes, and Ce 3+ The proportion is greater than Ce 4+ .

[0088] The multifunctional composite nanozyme prepared in this embodiment is obtained through, as shown in... Figure 5 As shown in the UV-Vis-NIR absorption spectrum, compared with CeVO4 and CeVO4-Ti, the polypyrrole-loaded nanozyme CeVO4-Ti@PPy has a stronger absorption in the 700-900 nm range. Figure 5 df calculated the absorption coefficients of three composite nanozymes, among which the polypyrrole-loaded nanozyme CeVO4-Ti@PPy had the highest absorption coefficient at 808 nm.

[0089] Application Example 1: Photothermal Properties of Multifunctional Composite Nanoenzymes

[0090] The photothermal heating performance of the three composite nanozymes prepared above was tested. Figure 6 a. Using 808nm, the power is 0.8W / cm². 2 Near-infrared laser light was used to irradiate water and three composite nanozymes at a concentration of 200 μg / mL, and their photothermal temperature rise was recorded. The data showed that the polypyrrole-loaded nanozyme CeVO4-Ti@PPy achieved the highest photothermal performance. Figure 6 b-6e, further investigation was conducted on CeVO4-Ti@PPy nanozymes at 808 nm with a power of 0.8 W / cm². 2The photothermal properties of CeVO4-Ti@PPy nanozymes were observed. With increasing CeVO4-Ti@PPy nanozyme concentration, the photothermal heating effect became increasingly pronounced, and the excellent photothermal stability of CeVO4-Ti@PPy nanozymes was demonstrated through five heating-cooling temperature changes. Further changes in laser wavelength, such as... Figure 6 fg, using 1064nm, has a power consumption of 0.8W / cm. 2 The photothermal properties of CeVO4-Ti@PPy nanozymes were studied using near-infrared lasers. The photothermal heating effect became more and more obvious with the increase of CeVO4-Ti@PPy nanozyme concentration. Figure 6 h calculated the photothermal conversion efficiency of CeVO4-Ti@PPy to be 36.54%.

[0091] Application Example 2: Enzyme-like Activity of Multifunctional Composite Nanozymes

[0092] The three composite nanozymes prepared above were tested for four types of enzyme activity: peroxidase-like, glutathione peroxidase-like, superoxide dismutase-like, and catalase-like. Figure 7 b is a schematic diagram of the detection of four types of enzyme activities. For example... Figure 8 a. 3,3′,5,5′-Tetramethylbenzidine (TMB) was selected to evaluate the peroxidase-like activity catalyzing the conversion of H₂O₂ to ·OH. Changes in solution absorbance were recorded using a UV-Vis spectrophotometer. At room temperature, under the conditions of pH 5.5, 0.1 mM H₂O₂, and 8 mM TMB, all three nanozymes exhibited peroxidase-like activity at the same material concentration, showing a concentration-dependent increase in peroxidase-like activity. This is based on the high correlation between photothermal temperature changes and catalytic activity. Figure 8 ab evaluated the peroxidase-like activities at room temperature and 43 °C. It was confirmed that the three nanozymes exhibited higher peroxidase-like activities at higher temperatures, with the CeVO4-Ti@PPy nanozyme showing the most significant enhancement, demonstrating that increasing temperature can improve the catalytic activity of the enzyme.

[0093] like Figure 8c. The glutathione depletion capacity of the three composite nanozymes was investigated using a 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reduction assay. Nanozymes of different concentrations were mixed with glutathione (4 mM) in PBS solution (pH 7.4), and then DTNB was added to a final concentration of 0.2 mM to detect the -SH group in the glutathione. The absorbance change at 412 nm was recorded using a UV-Vis spectrophotometer. All three nanozymes exhibited excellent glutathione depletion capacity, showing a concentration-dependent increase in glutathione peroxidase-like activity. Simultaneously, at the same material concentration, the CeVO4-Ti@PPy nanozyme exhibited the strongest glutathione peroxidase-like activity.

[0094] like Figure 8 d. The inhibition of photoreduction by nitroblue tetrazolium (NBT) functionally mimics the activity of superoxide dismutase to evaluate the superoxide anion radical (·O2) activity of the nanozyme. - The scavenging activity of NBT (0.75 mM) was assessed. NBT was mixed with L-methionine (130 mM), riboflavin (200 μM), and different concentrations of nanozymes in PBS buffer (pH 7.4), and irradiated with an LED for 5 minutes. The decrease in absorbance at 560 nm was measured using a UV-Vis spectrophotometer, and the quantitative result was expressed as the percentage of inhibition of formazan production, i.e., superoxide dismutase-like activity. The three nanozymes at a concentration of 200 μg / mL scavenged more than 50% of ·O2. - This indicates that the three nanozymes exhibit excellent superoxide dismutase-like activities, and that the superoxide dismutase-like activities gradually increase in a concentration-dependent manner.

[0095] like Figure 8 e-8f tested the catalase-like activities of three nanozymes. Nanomaterials with catalase-like activity can catalyze the decomposition of H2O2 into H2O and O2. The catalase-like activities of the three nanozymes were evaluated by monitoring changes in O2 and H2O2. Figure 8 e. The time-dependent evolution of O2 was recorded, among which CeVO4-Ti@PPy nanozyme showed the fastest O2 generation rate, increasing from 0 mg / mL to 11.5 mg / mL within 25 minutes. Figure 8 f. The decomposition of H2O2 was measured by monitoring the change in H2O2 absorbance at 240 nm. Among them, CeVO4-Ti@PPy nanozyme had the fastest decomposition rate of H2O2, and it only took 15 min to catalyze the decomposition of 50% of H2O2, indicating that CeVO4-Ti@PPy nanozyme has the strongest catalase-like activity.

[0096] Application Example 3: Cytotoxicity of CeVO4-Ti@PPy Nanozymes

[0097] The CeVO4-Ti@PPy nanozyme prepared above was subjected to cytotoxicity testing. The CeVO4-Ti@PPy nanozyme was co-incubated with NIH-3T3 and 4T1 cells for 24 h and 48 h, respectively, and cytotoxicity was assessed using the CCK-8 assay. Figure 9 a. As the concentration of CeVO4-Ti@PPy nanozyme gradually increased, no significant cytotoxicity was observed in normal cells (NIH-3T3). Figure 9 b. As the concentration of CeVO4-Ti@PPy nanozyme gradually increased, its activity against mouse breast cancer cells (4T1) rapidly decreased. When the concentration of CeVO4-Ti@PPy nanozyme was 200 μg / mL, only 28% of the 4T1 cells survived after co-incubation for 24 h. This indicates that CeVO4-Ti@PPy nanozyme can generate toxic reactive oxygen species through various enzyme activities, converting hydrogen peroxide in tumor cells into highly cytotoxic hydroxyl radicals and ultimately inducing tumor cell death. The cytotoxicity test concludes that CeVO4-Ti@PPy nanozyme can specifically kill tumor cells without producing toxic side effects on normal cells.

[0098] Application Example 4: Photoacoustic Imaging of Multifunctional Composite Nanoenzymes

[0099] The three nanozymes prepared above were subjected to in vitro / in vivo photoacoustic imaging tests. Different concentrations of the three nanozymes were used to evaluate their in vitro photoacoustic imaging capabilities. Figure 10 a. CeVO4 and CeVO4-Ti nanozymes did not show obvious photoacoustic signals, while CeVO4-Ti@PPy nanozymes showed obvious photoacoustic signals, and the signal intensity increased with increasing sample concentration. In particular, the intensity of the photoacoustic signal was directly proportional to the concentration of the CeVO4-Ti@PPy aqueous solution, indicating that CeVO4-Ti@PPy possesses ideal photoacoustic imaging capabilities. Figure 11 In vivo photoacoustic imaging (PA) was performed on the tumor region. The intensity of the tumor PA signal gradually increased over time, reaching a maximum at 10 hours. These results confirm that, thanks to its excellent photothermal effect and photothermal conversion efficiency, CeVO4-Ti@PPy nanozyme can serve as a photoacoustic imaging contrast agent, enabling photoacoustic imaging-induced early diagnosis and treatment.

Claims

1. A method for preparing a titanium-doped conductive polymer supported cerium vanadate nanomaterial, comprising the following steps: adding a conductive polymer monomer to a solution of the titanium-doped cerium vanadate nanomaterial, in-situ chemical polymerization by an oxidizing agent, loading the conductive polymer on the titanium-doped cerium vanadate nanorod, and obtaining the titanium-doped conductive polymer supported cerium vanadate nanomaterial. The conductive polymer monomer is selected from pyrrole. The method for preparing the titanium-doped cerium vanadate nanomaterial comprises the following steps: mixing a titanium source, a cerium source, a vanadium source, and a template agent under alkaline conditions to obtain a mixed solution; and then performing hydrothermal reaction on the mixed solution to obtain the titanium-doped cerium vanadate nanomaterial.

2. The method of claim 1, wherein: The solution is prepared using deionized water.

3. The method of claim 1, wherein: The oxidizing agent is selected from one or a mixture of several of iron chloride, ammonium persulfate, potassium permanganate, and potassium persulfate.

4. The method of claim 1, wherein: The concentration of the solution of the titanium-doped cerium vanadate nanomaterial is 0.5-5 mg / mL, the concentration of the conductive polymer monomer is 2-7 mM, and the concentration of the oxidizing agent is 6-21 mM.

5. The method of claim 1, wherein: The reaction temperature of the in-situ chemical polymerization by the oxidizing agent is 2-10 °C, and the reaction time is 4-12 h.

6. The method of any one of claims 1-5, wherein: After the reaction of the in-situ chemical polymerization by the oxidizing agent is completed, the material is further washed to remove impurities, the solution used for the washing is water and ethanol, the washing mode is centrifugal washing, the rotation speed is 8000-12000 rpm, and the time is 8-15 min.

7. The method of claim 1, wherein: In the method for preparing the titanium-doped cerium vanadate nanomaterial, all the solutions are prepared using deionized water.

8. The method of claim 1, wherein: The titanium source is selected from one or a mixture of several of titanium sulfate and titanyl sulfate.

9. The method of claim 8, wherein: The titanium source is titanium sulfate.

10. The method of claim 1, wherein: The cerium source is selected from one or a mixture of several of cerium nitrate, cerium chloride, cerium sulfate, cerium ammonium sulfate, and cerium acetate.

11. The method of claim 10, wherein: The cerium source is cerium nitrate.

12. The method of claim 1, wherein: The vanadium source is selected from one or a mixture of several of sodium orthovanadate, ammonium metavanadate, vanadyl sulfate, and oxovanadium oxalate.

13. The method of claim 1, wherein: The vanadium source is sodium orthovanadate.

14. The method of claim 1, wherein: The template agent is selected from one or a mixture of several of ethylenediaminetetraacetic acid, cetyltrimethylammonium bromide, polyethylene glycol-2000, and polyethylene glycol-4000.

15. The method of claim 14, wherein: The template agent is ethylenediaminetetraacetic acid.

16. The method of claim 1, wherein: In the mixed solution, the concentration of the cerium source is 60-300 mM, the concentration of the vanadium source is 60-300 mM, the concentration of the template agent is 60-30 mM, and the concentration of the titanium source is 10-50 mM.

17. The method of claim 1, wherein: In the method for preparing the titanium-doped cerium vanadate nanomaterial, the reagent for adjusting the alkaline conditions is selected from one or a mixture of several of sodium hydroxide, potassium hydroxide, ammonia, ammonium carbonate, and ammonium bicarbonate.

18. The method of claim 17, wherein: The reagent for adjusting the alkaline conditions is ammonia.

19. The method of claim 1, wherein: The pH of the mixed solution is adjusted to alkaline, and the adjusted pH range is pH 8-10.

20. The method of claim 19, wherein: The pH of the mixed solution is adjusted to alkaline, and the adjusted pH is 9.

21. The method of claim 1, wherein: The hydrothermal reaction is performed by placing in a high-temperature and high-pressure resistant reaction kettle, the reaction temperature of the hydrothermal reaction is 160-180 °C, and the reaction time is 16-24 h.

22. The method of claim 1, wherein: In the method for preparing the titanium-doped cerium vanadate nanomaterial, the preparation mode of the mixed solution comprises the following steps: 1) Dissolve the cerium source and template agent in deionized water, stir and mix evenly to obtain a cerium source-template agent mixture; 2) Dissolve the vanadium source in deionized water and stir to mix evenly to obtain a vanadium source solution; 3) Dissolve the titanium source in deionized water and stir to mix evenly to obtain a titanium source solution; 4) Slowly add the vanadium source solution dropwise into the cerium source-template agent mixture, and stir thoroughly at room temperature; 5) Slowly add the titanium source solution dropwise to the cerium source-template agent-vanadium source mixed solution in step 4), and stir thoroughly at room temperature to obtain the mixed solution; In the above steps, steps 1) to 3) are not in any particular order and can be adjusted arbitrarily.

23. The method of claim 22, wherein: The stirring conditions for step 1) are: stirring at 20-40 °C for 10-20 minutes; The stirring conditions for step 2) are: stirring at 50-80 °C for 10-20 minutes; The stirring conditions for step 3) are: stirring at 20-40 °C for 10-20 minutes; In step 4), the dropping rate is 2-4 mL / min; In step 5), the dripping rate is 2-4 mL / min.

24. The method of claim 1, wherein: In the preparation method of the titanium-doped cerium vanadate nanomaterial, after the hydrothermal reaction, the material is further washed to remove impurities. The washing solution is water and ethanol, and the washing method is centrifugal washing at a speed of 8000-12000 rpm for 8-15 min.

25. Titanium-doped conductive polymer-supported cerium vanadate nanomaterials prepared by the method of any one of claims 1-24.

26. The application of the titanium-doped conductive polymer-supported cerium vanadate nanomaterial according to claim 25, wherein the application is selected from at least one of the following: 1) application in the preparation of medicaments for tumor treatment; 2) application in the preparation of materials for tumor treatment; 3) application in the preparation of photoacoustic imaging contrast agents.

27. The use according to claim 26, characterized in that: The materials for tumor treatment include: materials for tumor photothermal therapy, materials for tumor chemokinetic therapy, and tumor treatment materials that combine the effects of chemokinetics and photothermal therapy.

Images