A preparation method of a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal performance

Abstract

The application relates to a preparation method of a non-noble metal-noble metal bimetallic nano alloy with high enzyme activity and mild photothermal performance, and relates to a preparation method of a non-noble metal-noble metal bimetallic nano alloy.The problems that the existing nano enzyme only contains a single metal element, the catalytic activity of the nano enzyme is low, and the enzyme reaction conditions provided by the tumor microenvironment are limited are solved.The preparation method comprises the following steps: one, synthesizing a bimetallic nano alloy PdCu; and two, modifying the nano material.The application is used for the preparation of a non-noble metal-noble metal bimetallic nano alloy with high enzyme activity and mild photothermal performance.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing non-noble metal-noble metal bimetallic nanalloy. BACKGROUND

[0002] Malignant tumor seriously endangers human health, traditional tumor treatment methods, such as surgical treatment, chemotherapy and radiotherapy, have poor selectivity, large side effects, large drug resistance produced by continuous drug use, and cannot realize real-time tracking of tumor treatment effect. It is worth noting that the emerging and booming nanoscience and technology in the late 1980s, with the characteristics of multi-disciplinary cross-fusion, provides new technical means and research perspectives for the fields of physics, chemistry, materials and life sciences. The fine adjustable structure and morphology of nanomaterials, and the modifiable surface properties directly determine the interaction mode of nanomaterials with biological molecules, cells, tissues, organs and individuals, and thus produce a unique biological effect-nano biological effect. Nanobiology has become a challenging and hot frontier field by deeply studying the nano biological effect from the individual, cell and molecular levels and elucidating its precise mechanism. In 2004, Pasquato et al. first named the nanomaterial with enzyme catalytic activity as nanenzyme. The special physicochemical properties of nanenzyme endow it with superior catalytic performance, and thus produce a special frontier interdisciplinary-nanocatalytic medicine. At present, in order to achieve tumor targeted therapy and reduce toxic side effects, the construction of tumor microenvironment (TME) specific response treatment strategy has become the most promising tumor treatment method in nanocatalytic medicine.

[0003] Tumor tissues have the characteristics of high interstitial fluid pressure, uneven blood flow, hypoxia, micro-acid, excessive inflammation, high content of glutathione (GSH) and hydrogen peroxide (H2O2) compared with normal tissues. In recent years, a treatment method using the specific microenvironment of tumor lesion area as the reaction condition, using metal nanoscale enzyme as the catalyst to produce hydroxyl radicals and other strong oxidizing active species through enzymatic reaction has attracted widespread attention. This therapy uses the logical response relationship of acid dissociation of metal nanoscale enzyme in tumor microenvironment and then H2O2 dismutation to produce active oxygen efficiently, and has high tumor treatment specificity and low invasiveness. The research groups of Lin Jun of Changchun Institute of Applied Chemistry of Chinese Academy of Sciences, Chen Xiaoyuan of National Institutes of Health of the United States, Bu Wenbo of Shanghai Institute of Silicate of Chinese Academy of Sciences and Liu Zhuang of Suzhou University have made rich research achievements in this direction. The reported nanoscale enzymes are mostly metal oxides (such as iron, manganese, copper, cerium oxides), but the catalytic activity of the nanoscale enzyme containing only a single metal element is low. For example, the common divalent iron material is used as Fenton reagent for chemical kinetic therapy, which has strict requirements for acidic environment, requiring pH to be between 2-4. The tumor microenvironment is weakly acidic, with pH of about 6.4-6.7, and the overexpression of H2O2 can provide limited enzymatic reaction conditions. SUMMARY

[0004] The present application solves the problems that the existing nanoscale enzyme contains only a single metal element, the catalytic activity of the nanoscale enzyme is low, and the enzymatic reaction conditions provided by the tumor microenvironment are limited, and further provides a preparation method of a non-noble metal-noble metal bimetallic nanoscale alloy with high enzymatic activity and mild photothermal performance.

[0005] The preparation method of a non-noble metal-noble metal bimetallic nanoscale alloy with high enzymatic activity and mild photothermal performance is carried out according to the following steps:

[0006] I. Synthesis of bimetallic nanoscale alloy PdCu:

[0007] ①, uniformly ultrasonic the palladium source, copper source and high-temperature resistant solvent to obtain a mixed solution A;

[0008] ②, under the conditions of magnetic stirring and vacuum extraction, the mixed solution A is heated from room temperature to 110-130℃, and then heated at 110-130℃, and then nitrogen is introduced, and then the temperature is increased to 125-145℃, and then the carbonyl compound is added, and the solution changes from transparent to black, to obtain a mixed solution B;

[0009] ③ Under magnetic stirring and nitrogen atmosphere, the mixed solution B is heated to 240℃~250℃ and reacted at 240℃~250℃ for 20min~60min. After the reaction is completed, it is cooled to room temperature, centrifuged, washed and dried to obtain bimetallic nanoparticles PdCu.

[0010] II. Modification of Nanomaterials:

[0011] ① Disperse bimetallic nanoparticles PdCu in a mixed solvent of cyclohexane and ethanol to obtain solution C. Then, NH2-PEG... 2000 Add to ethanol and sonicate until homogeneous to obtain solution D;

[0012] ② Under stirring conditions, solution D is added to solution C to obtain mixed solution E. Mixed solution E is reacted at room temperature and under stirring conditions for 12h to 24h. After centrifugation, the reaction product is washed with deionized water to obtain polyethylene glycol-modified PdCu nano-alloy.

[0013] The beneficial effects of this invention are:

[0014] This invention successfully synthesized copper-doped palladium-based PdCu nanoalloys using a high-temperature pyrolysis method. Compared with conventional synthesis methods, the reaction raw materials are simpler, and the synthesized nanomaterials have uniform and controllable morphology. The tumor microenvironment is weakly acidic, with a pH of approximately 6.4–6.7. The copper-doped palladium-based PdCu nanoalloy of this invention can utilize the weak acidity of the tumor microenvironment to undergo the Fenton reaction. The prepared non-noble metal-noble metal bimetallic nanoalloy with high catalytic activity utilizes endogenous H2O2 in the tumor microenvironment to trigger a palladium / copper dual-cycle enzymatic reaction. The bimetallic valence change reaction of the non-noble metal-noble metal and its synergistic effect reduced the enzyme reaction constant Km from 133.55 mM to 80.13 mM, resulting in a significant improvement in catalytic efficiency, exhibiting superior catalytic performance compared to palladium or copper alone. Furthermore, the mild photothermal effect of the synergistic palladium-based material can increase the local reaction temperature, further enhancing the enzyme reaction efficiency. At a reaction temperature of 48°C, the enzyme reaction efficiency is increased by up to 74.7% compared to the room temperature reaction. Furthermore, the divalent copper component can consume excess reducing substances such as glutathione (GSH) within tumors, ensuring the effectiveness of enzymatic reactions and reducing the consumption of reactive oxygen species by GSH. Attached Figure Description

[0015] Figure 1 The XRD pattern of the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1;

[0016] Figure 2 The image shows a TEM image of the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1.

[0017] Figure 3High resolution TEM image of bimetallic nanoparticles PdCu prepared in step 1-3 of Example 1;

[0018] Figure 4 Electron diffraction image of bimetallic nanoparticles PdCu prepared in step 1-3 of Example 1;

[0019] Figure 5 Particle size distribution histogram of bimetallic nanoparticles PdCu prepared in step 1-3 of Example 1;

[0020] Figure 6 NH2-PEG modified in Example 1 2000 Zeta potential comparison of bimetallic nanoparticles PdCu before and after modification, a is bimetallic nanoparticles PdCu prepared in step 1-3 of Example 1, b is PEGylated PdCu nanalloy prepared in Example 1;

[0021] Figure 7 XPS spectrum of bimetallic nanoparticles PdCu prepared in step 1-3 of Example 1;

[0022] Figure 8 Fine scanning spectrum of Pd3d subpeak of bimetallic nanoparticles PdCu prepared in step 1-3 of Example 1;

[0023] Figure 9 Fine scanning spectrum of Cu2p subpeak of bimetallic nanoparticles PdCu prepared in step 1-3 of Example 1;

[0024] Figure 10 UV-Vis absorption spectrum of oxidation of PEGylated PdCu nanalloy prepared in Example 1 with TMB as dye, 1 is TMB, 2 is PEGylated PdCu nanalloy + TMB, 3 is H2O2+ TMB, 4 is PEGylated PdCu nanalloy + H2O2+ TMB, 5 is PEGylated PdCu nanalloy + H2O2+ TMB + 37℃, 6 is PEGylated PdCu nanalloy + H2O2+ TMB + 48℃;

[0025] Figure 11 UV-Vis absorption spectrum of oxidation of H2O2+ TMB under conditions containing different concentrations of PEGylated PdCu nanalloy, 1 is 6 μg / mL, 2 is 12.5 μg / mL, 3 is 25 μg / mL, 4 is 50 μg / mL, 5 is 100 μg / mL;

[0026] Figure 12UV absorption spectra of PEGylated PdCu nanalloy + H2O2 + TMB over time, 1 is 1 min reaction, 2 is 2 min reaction, 3 is 3 min reaction, 4 is 4 min reaction, 5 is 5 min reaction, 6 is 6 min reaction, 7 is 7 min reaction, 8 is 8 min reaction, 9 is 9 min reaction;

[0027] Figure 13 ESR spectra of ROS production catalyzed by PEGylated PdCu nanalloy prepared in Example 1, 1 is DMPO + H2O2, 2 is DMPO + H2O2 + PEGylated PdCu nanalloy;

[0028] Figure 14 Absorbance curves of H2O2 + PEGylated PdCu nanalloy + TMB at 612 nm wavelength under different H2O2 concentrations and reaction times;

[0029] Figure 15 Fitting curves of the corresponding rates of different concentrations of H2O2 and PEGylated PdCu nanalloy or pure palladium nanoparticles, · is PEGylated PdCu nanalloy, ■ is pure palladium nanoparticles;

[0030] Figure 16 Double-reciprocal fitting curves of the corresponding reaction rates of different concentrations of H2O2 and PEGylated PdCu nanalloy or pure palladium nanoparticles, · is PEGylated PdCu nanalloy, ■ is pure palladium nanoparticles;

[0031] Figure 17 Thermal infrared imaging pictures of water and different concentrations of PEGylated PdCu solution under 1064 nm laser irradiation of 0.9 W / cm 2 ;

[0032] Figure 18 Temperature rise curves of different concentrations of PEGylated PdCu solution and deionized water under 1064 nm light irradiation of 1.7 W / cm 2 , 1 is 100 μg / mL PEGylated PdCu solution, 2 is 50 μg / mL PEGylated PdCu solution, 3 is 25 μg / mL PEGylated PdCu solution, 4 is 12.5 μg / mL PEGylated PdCu solution, 5 is deionized water;

[0033] Figure 19 Temperature rise column chart of 100 ug / mL PEGylated PdCu solution excited by lasers of different powers, a is the material temperature at room temperature, b is the material temperature after 10 min of laser irradiation;

[0034] Figure 20 Temperature variation curve of single cycle of the PdCu solution with polyethylene glycol for heating and cooling;

[0035] Figure 21 Efficiency diagram of photo-thermal conversion of the PdCu solution with polyethylene glycol;

[0036] Figure 22 Temperature variation curve obtained by four cycles of laser switching measurement of the PdCu solution with polyethylene glycol. DETAILED DESCRIPTION

[0037] Embodiment 1: A method for preparing a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photo-thermal performance, which is carried out according to the following steps:

[0038] I. Synthesis of bimetallic nanoalloy PdCu:

[0039] ①, ultrasonic homogenize the palladium source, copper source and high-temperature resistant solvent to obtain a mixed solution A;

[0040] ②, under the conditions of magnetic stirring and vacuum pumping, the mixed solution A is heated from room temperature to 110-130°C, and then heated at 110-130°C, and then nitrogen is introduced, and then the temperature is raised to 125-145°C, and then the carbonyl compound is added, and the solution changes from transparent to black, to obtain a mixed solution B;

[0041] ③, under the conditions of magnetic stirring and nitrogen atmosphere, the mixed solution B is heated to 240-250°C, and reacted at 240-250°C for 20-60 min, and then cooled to room temperature, centrifuged, washed and dried to obtain the bimetallic nanoparticles PdCu;

[0042] II. Modification of nanomaterials:

[0043] ①, disperse the bimetallic nanoparticles PdCu in a mixed solvent of cyclohexane and ethanol to obtain a solution C, and add NH2-PEG 2000 to ethanol and ultrasonic homogenize to obtain a solution D;

[0044] ②, under stirring conditions, the solution D is added to the solution C to obtain a mixed solution E, and the mixed solution E is reacted at room temperature and under stirring conditions for 12-24 h, and then the reaction product is collected by centrifugation and washed with deionized water to obtain the PdCu nanoalloy with polyethylene glycol.

[0045] The bimetallic nanoparticles PdCu synthesized by the high-temperature pyrolysis method in step I of the embodiment have uniform size and obvious spherical structure.

[0046] The high-temperature pyrolysis method is used in step one of the embodiment, and the product synthesized by this method is smaller than the particles synthesized by the solvent thermal method and has better dispersibility than the material synthesized by the coprecipitation method, and thus is more suitable for application in vivo.

[0047] In step one ② of the embodiment, the solution is vacuumized before the reaction, so as to remove water and oxygen in the solution.

[0048] In step one ② and step one ③ of the embodiment, the temperature is raised at a rate of 3-10 ℃ / min, which is beneficial to avoid the phenomenon of explosive boiling, the reaction can be fully carried out, and it is also beneficial to timely observation and control.

[0049] In step one ② of the embodiment, nitrogen is used as a protective gas to avoid the introduction of external oxygen to generate impurities and avoid the interference of air on the reaction system, so as to improve the purity of the product.

[0050] In step one ③ of the embodiment, the product is washed in anhydrous ethanol, so as to more effectively and stably obtain a pure product.

[0051] In the embodiment, the stirring is used to better disperse the reagents, so as to achieve the purposes of complete reaction and uniform temperature rise.

[0052] In step two ② of the embodiment, the polyethylene glycolized PdCu nanometer alloy is prepared and used as a tumor treatment nanometer preparation with good biocompatibility, and under controllable near-infrared two-zone light irradiation, the mild photothermal enhancement of the PdCu nanometer alloy is used to trigger the chemical dynamic treatment.

[0053] In the embodiment, the nanometer enzyme based on double metal elements uses a two-cycle catalytic system to enhance the oxidative damage of cells, and at the same time, uses the multi-element enzyme activity of the nanometer enzyme to in-situ adjust the tumor microenvironment TME and optimize the enzyme reaction conditions, so as to maximize the use of the endogenous power of the tumor microenvironment, which is of great significance to improve the treatment efficiency of the nanometer enzyme.

[0054] The process of the embodiment is stable and mature, the reaction system is not polluting to the environment, the proposed process route can successfully synthesize the target nanometer alloy material, and has a good application prospect. The small-size PdCu nanometer alloy synthesized by high-temperature pyrolysis has a uniform and controllable morphology, and the small-size effect makes the PdCu nanometer alloy beneficial to cell uptake. After modification, the material has good biocompatibility. The embodiment discloses a synthesis method of a double-metal nanometer alloy with high-efficiency catalytic activity and mild photothermal effect, and the double-metal cycle catalytic treatment is triggered by external near-infrared light and endogenous tumor environment; the local photothermal effect of the PdCu nanometer alloy generated by the external near-infrared light stimulation can further enhance the enzyme activity of the nanometer alloy, so as to realize the mild photothermal enhancement of the nanometer enzyme catalytic treatment and the synergistic treatment strategy.

[0055] The method described in this embodiment can yield PdCu nanoalloy materials, providing a new design concept and process flow. It rationally designs a noble metal-non-noble metal bimetallic nanoalloy material with anti-tumor effects and the ability to regulate the tumor microenvironment. The polyethylene glycol-modified PdCu nanoalloy can reduce excess hydrogen peroxide in the tumor microenvironment through the Fenton reaction: Pd + 2H₂O₂ → Pd 2+ +2OH ﹣ +2·OH, Cu + +H₂O₂→Cu 2+ +OH ﹣+ +·OH; Copper in nanomaterials exists in two states: zero valence and +2 valence. The +2 valence copper can be reduced by glutathione to +1 valence copper using Fenton's reagent, thus reducing excess glutathione. Cu 2+ +GSH→Cu + +GSSH, Cu + +H₂O₂→Cu 2+ +OH ﹣ The addition of ·OH, through the Fenton reaction, generates reactive oxygen species. These reactive oxygen species can turn TMB blue and produce a characteristic absorption peak at 652 nm. This demonstrates that polyethylene glycol-modified PdCu nanoalloys possess antitumor effects and can regulate the tumor microenvironment. This embodiment demonstrates a novel research approach and application prospects through its excellent therapeutic efficiency and biocompatibility.

[0056] The beneficial effects of this specific implementation method are:

[0057] This specific embodiment successfully synthesized copper-doped palladium-based PdCu nanoalloys using a high-temperature pyrolysis method. Compared with conventional synthesis methods, the reaction raw materials are simpler, and the synthesized nanomaterials have uniform and controllable morphology. The tumor microenvironment is weakly acidic, with a pH of approximately 6.4–6.7. The copper-doped palladium-based PdCu nanoalloy of this embodiment can utilize the weak acidity of the tumor microenvironment to undergo the Fenton reaction. The prepared non-noble metal-noble metal bimetallic nanoalloy with high catalytic activity utilizes endogenous H2O2 in the tumor microenvironment to trigger a palladium / copper dual-cycle enzymatic reaction. The bimetallic valence change reaction of the non-noble metal-noble metal and its synergistic effect reduced the enzyme reaction constant Km from 133.55 mM to 80.13 mM, resulting in a significant improvement in catalytic efficiency, exhibiting superior catalytic performance compared to palladium or copper alone. Furthermore, the mild photothermal effect of the synergistic palladium-based material can increase the local reaction temperature, further enhancing the enzyme reaction efficiency. At a reaction temperature of 48°C, the enzyme reaction efficiency is increased by up to 74.7% compared to room temperature. Furthermore, the divalent copper component can consume excess reducing substances such as glutathione (GSH) within tumors, ensuring the effectiveness of enzymatic reactions and reducing the consumption of reactive oxygen species by GSH.

[0058] Specific embodiment two: the difference between this embodiment and specific embodiment one is that: the palladium source in step 1 1 is palladium acetylacetonate; the copper source in step 1 1 is copper chloride; the high-temperature resistant solvent in step 1 1 is a mixed solvent of oleylamine and oleic acid. The others are the same as specific embodiment one.

[0059] Specific embodiment three: the difference between this embodiment and one of specific embodiment one or two is that: the mass ratio of the palladium source to the copper source in step 1 1 is 1 :(0.3-0.7); the mass ratio of the total mass of the palladium source and the copper source to the mass of the high-temperature resistant solvent in step 1 1 is 1 :(300-400). The others are the same as specific embodiment one or two.

[0060] Specific embodiment four: the difference between this embodiment and one of specific embodiment one to three is that: the temperature in step 1 2 is 110-130℃, and the holding time is 10-30min; the temperature of the mixed solution A in step 1 2 is increased from room temperature to 110-130℃ at a rate of 3-10℃ / min; the temperature is increased to 125-145℃ at a rate of 3-10℃ / min; the rotating speed of the magnetic stirring in steps 1 2 and 1 3 is 200-400r / min. The others are the same as specific embodiment one to three.

[0061] Specific embodiment five: the difference between this embodiment and one of specific embodiment one to four is that: the carbonyl compound in step 1 2 is tungsten carbonyl; the mass ratio of the mass of the carbonyl compound to the total mass of the palladium source and the copper source in step 1 2 is 1 :(1-1.5). The others are the same as specific embodiment one to four.

[0062] Specific embodiment six: the difference between this embodiment and one of specific embodiment one to five is that: the temperature of the mixed solution B in step 1 3 is increased to 240-250℃ at a rate of 3-10℃ / min; the centrifugal washing and drying in step 1 3 is carried out by the following steps: centrifuging at a speed of 4000-8000r / min for 5-15min, then washing the solid substance with anhydrous ethanol and cyclohexane alternately for 2-4 times, and finally drying at a temperature of 50-80℃ for 8-12h. The others are the same as specific embodiment one to five.

[0063] Specific embodiment seven: the difference between this embodiment and one of specific embodiment one to six is that: the particle size of the bimetallic nanoparticles PdCu in step 1 3 is 10-25nm. The others are the same as specific embodiment one to six.

[0064] Specific embodiment eight: different from one of the specific embodiments one to seven is that: the volume ratio of cyclohexane to ethanol in the mixed solvent of cyclohexane and ethanol in step two ① is (100-200) μL:1 mL; the mass to volume ratio of NH2-PEG 2000 in step two ① is (1-5) mg:1 mL. The others are the same as the specific embodiments one to seven.

[0065] Specific embodiment nine: different from one of the specific embodiments one to eight is that: the mass ratio of bimetallic nanoparticles PdCu to NH2-PEG 2000 in the mixed solution E in step two ② is 1:(5-10). The others are the same as the specific embodiments one to eight.

[0066] Specific embodiment ten: different from one of the specific embodiments one to nine is that: the stirring speed in step two ② is 200 r / min-300 r / min; the centrifugal collection in step two ② is specifically under the condition of centrifugal speed of 6000 r / min-8000 r / min for 5 min-10 min. The others are the same as the specific embodiments one to nine.

[0067] The beneficial effects of the present application are verified by the following examples:

[0068] Example one:

[0069] A preparation method of a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal performance, which is carried out according to the following steps:

[0070] I. Synthesis of bimetallic nanoalloy PdCu:

[0071] ①, ultrasonic homogenization of palladium source, copper source and high-temperature resistant solvent to obtain mixed solution A;

[0072] The palladium source is palladium acetylacetone; the copper source is copper chloride; the high-temperature resistant solvent is a mixed solvent of oleylamine and oleic acid with a volume ratio of 4:1;

[0073] The mass ratio of the palladium source to the copper source is 1:0.5; the total mass of the palladium source and the copper source to the mass of the high-temperature resistant solvent is 1:300;

[0074] ②、Under the conditions of magnetic stirring speed of 300 r / min, vacuumizing and temperature increasing speed of 10 ℃ / min, the mixed solution A is heated from room temperature to 130 ℃, and then kept at 130 ℃ for 20 min, and then nitrogen is introduced, and then the temperature is increased to 130 ℃ at a temperature increasing speed of 10 ℃ / min, and then the carbonyl compound is added at 130 ℃, and the solution changes from transparent to black, and then a mixed solution B is obtained;

[0075] The carbonyl compound is tungsten carbonyl, and the mass ratio of the carbonyl compound to the total mass of the palladium source and the copper source is 1:1.

[0076] ③、Under the conditions of magnetic stirring speed of 300 r / min, nitrogen atmosphere and temperature increasing speed of 3 ℃ / min-10 ℃ / min, the mixed solution B is heated to 240 ℃, and then kept at 240 ℃ for 50 min, and then cooled to room temperature, and then centrifuged, washed and dried to obtain the bimetallic nanoparticles PdCu.

[0077] II. Modification of the nanomaterial:

[0078] ①、The bimetallic nanoparticles PdCu are dispersed in a mixed solvent of cyclohexane and ethanol to obtain a solution C, and NH2-PEG 2000 is added to ethanol and ultrasonically mixed to obtain a solution D.

[0079] The volume ratio of cyclohexane to ethanol in the mixed solvent of cyclohexane and ethanol is 100 μL:900 μL, and the mass ratio of NH2-PEG 2000 to ethanol is 5 mg:1 mL.

[0080] ②、Under the conditions of stirring speed of 300 r / min, the solution D is added to the solution C to obtain a mixed solution E, and the mixed solution E is reacted at room temperature and stirring speed of 300 r / min for 24 h, and then the reaction product is collected by centrifugation and washed with deionized water to obtain the PEGylated PdCu nanoscale alloy.

[0081] The mass ratio of the bimetallic nanoparticles PdCu to NH2-PEG 2000 in the mixed solution E is 1:5.

[0082] The centrifugation, washing and drying in step one ③ are carried out according to the following steps: centrifugation at a speed of 4000 r / min for 10 min, then the solid material is washed with anhydrous ethanol and cyclohexane alternately for 3 times, and finally dried at a temperature of 60 ℃ for 10 h.

[0083] The centrifugal collection described in step 2② specifically involves centrifuging at a speed of 6000 r / min for 10 min.

[0084] Figure 1 The image shows the XRD pattern of the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1. As can be seen from the image, the diffraction peaks of the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1 are at 2θ values ​​of 40.90°, 46.86°, and 68.80°, corresponding to the (111), (200), and (220) crystal planes, respectively. Compared with the diffraction peaks (40.12°, 46.67°, and 68.12°) corresponding to the standard PDF card, there is a slight shift to the right overall. This is due to the reduction in interplanar spacing d caused by the substitution of palladium atoms by the relatively smaller copper atoms. XRD characterization confirms that the synthesized bimetallic nanoparticles PdCu have a single-phase structure, space group Fm3m, and cell parameter a. It is basically consistent with the standard PDF card (JCPDS No.: 46-1043).

[0085] Figure 2 The image shows a TEM image of the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1. As can be seen from the image, the bimetallic nanoparticles PdCu have nanoscale size, with a particle size of about 17 nm, and good dispersibility, uniform size, and obvious spherical structure.

[0086] Figure 3 The image shows a high-resolution TEM image of the bimetallic nanoparticles PdCu prepared in step 3 of Example 1. As can be seen from the image, the spacing of the visible lattice stripes in the high-resolution TEM image is 0.220 nm, which mainly corresponds to the (111) crystal plane.

[0087] Figure 4 The image shows the electron diffraction pattern of the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1. As can be seen from the image, the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1 are polycrystalline.

[0088] Figure 5 The figure shows the particle size distribution of the bimetallic nanoparticles PdCu prepared in step 1③ of Example 1. As can be seen from the figure, the average diameter of the nanoparticles is 17nm to 19nm.

[0089] Figure 6 Example 1: Modified NH2-PEG 2000 Comparison of Zeta potentials of bimetallic PdCu nanoparticles before and after preparation: a) PdCu bimetallic nanoparticles prepared in step 1.3 of Example 1; b) PEGylated PdCu nanoalloy prepared in Example 1. As shown in the figure, the surface electrical properties measured by Zeta potential indicate that the initial negative potential of the material is -0.78 mV. NH2-PEG 2000The higher negative potential of -5.12 mV after connection proves that the modification of the process is successful.

[0090] Figure 7 XPS energy spectrum of the bimetallic nanoparticles PdCu prepared in step one ③ of the example; the existence of Pd and Cu in the PdCu sample is confirmed.

[0091] Figure 8 Fine scanning spectrum of Pd3d peak in the bimetallic nanoparticles PdCu prepared in step one ③ of the example; from the figure, Pd3d 3 / 2 (340.85 eV and 341.71 eV) and Pd3d 1 / 2 (335.55 eV and 336.1 eV) in PdCu have right-shifted characteristic peaks compared with the characteristic peaks of pure Pd Pd3d 3 / 2 (340.4 eV) and Pd3d 1 / 2 (335.2 eV), which is caused by the alloying of Cu and Pd, and the fine scanning spectrum also proves the existence of multi-valence Pd in PdCu.

[0092] Figure 9 Fine scanning spectrum of Cu2p peak in the bimetallic nanoparticles PdCu prepared in step one ③ of the example; from the figure, Cu2p 3 / 2 (951.83 eV and 952.76 eV) and Cu2p 1 / 2 (932.84 eV, 932.03 eV and 931.36 eV) characteristic peaks prove the existence of multi-valence Cu in PdCu.

[0093] UV absorption spectrum of the polyethylene glycolized PdCu nanometer alloy prepared in example one when oxidizing TMB dye, grouped as: TMB, PdCu+TMB, H2O2+TMB, PdCu+H2O2+TMB, PdCu+H2O2+TMB+37℃ and PdCu+H2O2+TMB+48℃, the specific operation process is as follows:

[0094] The polyethylene glycolized PdCu nanometer alloy prepared in example one is added to a phosphate buffer solution with a pH of 6.6 to obtain a polyethylene glycolized PdCu solution with a pH of 6.6;

[0095] The TMB group has a characteristic absorption peak at 652 nm;

[0096] PEG-PdCu alloy + TMB group: 100 μL of TMB dye dimethyl sulfoxide solution with a concentration of 12.5 mmol / L was added to 2.4 mL of PEG-PdCu solution with a concentration of 100 μg / mL and pH of 6.6, and reacted at room temperature for 5 min, and the characteristic absorption peak at 652 nm was tested;

[0097] H2O2+TMB group: 100 μL of TMB dye dimethyl sulfoxide solution with a concentration of 12.5 mmol / L and 100 μL of H2O2 solution with a concentration of 200 mmol / L were added to 2.3 mL of phosphate buffer solution with pH of 6.6, and reacted at room temperature for 5 min, and the characteristic absorption peak at 652 nm was tested;

[0098] PEG-PdCu alloy + H2O2+TMB group: 100 μL of TMB dye dimethyl sulfoxide solution with a concentration of 12.5 mmol / L and 100 μL of H2O2 solution with a concentration of 200 mmol / L were added to 2.3 mL of PEG-PdCu solution with a concentration of 100 μg / mL and pH of 6.6, and reacted at room temperature for 5 min, and the characteristic absorption peak at 652 nm was tested;

[0099] PEG-PdCu alloy + H2O2+TMB+37℃ group: 100 μL of TMB dye dimethyl sulfoxide solution with a concentration of 12.5 mmol / L and 100 μL of H2O2 solution with a concentration of 200 mmol / L were added to 2.3 mL of PEG-PdCu solution with a concentration of 100 μg / mL and pH of 6.6, and reacted at 37℃ water bath heating for 5 min, and the characteristic absorption peak at 652 nm was tested;

[0100] PEG-PdCu alloy + H2O2+TMB+48℃ group: 100 μL of TMB dye dimethyl sulfoxide solution with a concentration of 12.5 mmol / L and 100 μL of H2O2 solution with a concentration of 200 mmol / L were added to 2.3 mL of PEG-PdCu solution with a concentration of 100 μg / mL and pH of 6.6, and reacted at 48℃ water bath heating for 5 min, and the characteristic absorption peak at 652 nm was tested.

[0101] The above ultraviolet absorption spectrum is shown in detail in Figure 10 , Figure 10For TMB as a dye, the UV absorption spectrum of the polyethylene glycolized PdCu nanometer alloy oxidation prepared in Example One is shown in the figure, 1 is TMB, 2 is polyethylene glycolized PdCu nanometer alloy + TMB, 3 is H2O2 + TMB, 4 is polyethylene glycolized PdCu nanometer alloy + H2O2 + TMB, 5 is polyethylene glycolized PdCu nanometer alloy + H2O2 + TMB + 37℃, 6 is polyethylene glycolized PdCu nanometer alloy + H2O2 + TMB + 48℃; as can be seen from the figure, when TMB is mixed with polyethylene glycolized PdCu nanometer alloy or H2O2 alone, the negligible absorbance value indicates that no oxidation reaction occurs in the mixed solution. After adding polyethylene glycolized PdCu nanometer alloy to the mixed solution of TMB and H2O2, the maximum absorbance value increases, confirming that the polyethylene glycolized PdCu nanometer alloy can catalyze H2O2 to generate reactive oxygen species (ROS). TMB + H2O2 + polyethylene glycolized PdCu nanometer alloy + 37℃ and TMB + H2O2 + polyethylene glycolized PdCu nanometer alloy + 48℃ are comparative verification using 37℃ and 48℃ water bath heating respectively, among which 37℃ simulates the natural temperature of the human body and 48℃ simulates the temperature reached by the photothermal effect, and the ROS generation capacity is enhanced with the increase of temperature. The absorbance peak value of the color reaction of ROS at room temperature is 0.684, and the absorbance peak value of the color reaction of ROS at 48℃ is 1.195, and the ROS production efficiency is increased by 74.7%. These results show that the mild photothermal effect of polyethylene glycolized PdCu nanometer alloy indeed promotes the production of ROS, that is, the increase of temperature promotes the activity of polyethylene glycolized PdCu nanometer alloy as a nano-enzyme.

[0102] The polyethylene glycolized PdCu nanometer alloy prepared in Example One was added to a phosphate buffer solution with a pH of 6.6 to obtain a polyethylene glycolized PdCu solution with a pH of 6.6; 100 μL of a 200 mmol / L H2O2 solution and 100 μL of a 12.5 mmol / L TMB dye dimethyl sulfoxide solution were added to 2.3 mL of a polyethylene glycolized PdCu solution with a concentration of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL and 6 μg / mL and a pH of 6.6, respectively, and reacted at room temperature for 5 min. The UV absorption spectrum is shown in Figure 11

[0103] Figure 11 The UV spectrum of H2O2 + TMB oxidation under the condition of containing different concentrations of polyethylene glycolized PdCu nanometer alloy is shown in the figure, 1 is 6 μg / mL, 2 is 12.5 μg / mL, 3 is 25 μg / mL, 4 is 50 μg / mL, 5 is 100 μg / mL; as can be seen from the figure, the ROS content increases with the increase of the concentration of polyethylene glycolized PdCu nanometer alloy. ​

[0104] The polyethylene glycol-modified PdCu nanoalloy prepared in Example 1 was added to a phosphate buffer solution with a pH of 6.6 to obtain a polyethylene glycol-modified PdCu solution with a pH of 6.6. 100 μL of a 200 mmol / L H₂O₂ solution and 100 μL of a 12.5 mmol / L TMB dye dimethyl sulfoxide solution were added to 2.3 mL of the 100 μg / mL polyethylene glycol-modified PdCu solution with a pH of 6.6. The reaction was carried out at room temperature for 1 min to 9 min. The UV absorption spectra at different reaction times are shown in [Figure showing UV absorption spectra]. Figure 12 As shown;

[0105] Figure 12 The UV absorption spectra of polyethylene glycol-modified PdCu nanoalloy + H2O2 + TMB over time are shown in the figure. 1 represents 1 min of reaction, 2 represents 2 min of reaction, 3 represents 3 min of reaction, 4 represents 4 min of reaction, 5 represents 5 min of reaction, 6 represents 6 min of reaction, 7 represents 7 min of reaction, 8 represents 8 min of reaction, and 9 represents 9 min of reaction. As can be seen from the figure, the ROS content in the mixed solution gradually increases with the extension of reaction time.

[0106] And by Figure 10 It is known that polyethylene glycol-modified PdCu nanoalloys can reduce excess hydrogen peroxide in the tumor microenvironment through the Fenton reaction: Pd + 2H₂O₂ → Pd 2+ +2OH ﹣ +2·OH, Cu + +H₂O₂→Cu 2+ +OH ﹣+ +·OH; by Figure 9 It is known that copper in nanomaterials exists in two states: zero valence and +2 valence. The +2 valence copper can be reduced by glutathione to +1 valence copper using Fenton's reagent, thus reducing excess glutathione. 2+ +GSH→Cu + +GSSH, Cu + +H₂O₂→Cu 2+ +OH ﹣ +·OH, the Fenton reaction produces reactive oxygen species, which can turn TMB blue and has a characteristic absorption peak at 652 nm. Figure 12 This demonstrates that polyethylene glycol-modified PdCu nanoalloys have anti-tumor effects and can regulate the tumor microenvironment.

[0107] 10 μL of a 200 mmol / L H₂O₂ solution was reacted with 10 μL of a 1 mmol / mL DMPO solution at room temperature for 5 min. The ESR spectrum is shown below. Figure 13ESR spectra of ROS produced by polyethylene glycolylated PdCu nanoscale alloy prepared in Example 1, 1 is DMPO+H2O2, 2 is DMPO+H2O2+polyethylene glycolylated PdCu nanoscale alloy; it can be seen from the figure that the intensity ratio of ·OH detected using DMPO as a trapping agent is 1:2:2:1 characteristic peak. Figure 13 ESR spectra of ROS produced by polyethylene glycolylated PdCu nanoscale alloy prepared in Example 1, 1 is DMPO+H2O2, 2 is DMPO+H2O2+polyethylene glycolylated PdCu nanoscale alloy; it can be seen from the figure that the intensity ratio of ·OH detected using DMPO as a trapping agent is 1:2:2:1 characteristic peak.

[0108] Figure 13 ESR spectra of ROS produced by polyethylene glycolylated PdCu nanoscale alloy prepared in Example 1, 1 is DMPO+H2O2, 2 is DMPO+H2O2+polyethylene glycolylated PdCu nanoscale alloy; it can be seen from the figure that the intensity ratio of ·OH detected using DMPO as a trapping agent is 1:2:2:1 characteristic peak.

[0109] Polyethylene glycolylated PdCu nanoscale alloy or pure palladium nanoparticles prepared in Example 1 were added to deionized water to obtain polyethylene glycolylated PdCu solution or pure palladium nanoparticle solution, 10 μL of polyethylene glycolylated PdCu solution or pure palladium nanoparticle solution with a concentration of 100 μg / mL, 200 μL of H2O2 solution with different concentrations (25 mmol / L, 50 mmol / L, 100 mmol / L, 200 mmol / L and 400 mmol / L) and 50 μL of TMB dye dimethyl sulfoxide solution with a concentration of 12.5 mmol / L were mixed in a 96-well plate, and the absorbance at 612 nm was tested every 20 s, the ultraviolet absorption spectra of polyethylene glycolylated PdCu nanoscale alloy under different concentrations of H2O2 solution and different reaction times are shown in Figure 14 and the fitting curves of the corresponding rates of different concentrations of H2O2 and polyethylene glycolylated PdCu nanoscale alloy or the fitting curves of the corresponding rates of different concentrations of H2O2 and pure palladium nanoparticles were obtained Figure 15 , the double-reciprocal fitting curves of the corresponding reaction rates of different concentrations of H2O2 and polyethylene glycolylated PdCu nanoscale alloy or the double-reciprocal fitting curves of the corresponding reaction rates of different concentrations of H2O2 and pure palladium nanoparticles Figure 16 ;

[0110] Figure 14 Absorbance curves of H2O2+polyethylene glycolylated PdCu nanoscale alloy+TMB at different H2O2 concentrations and reaction times at a wavelength of 612 nm; the concentrations of H2O2 from low to high are 25 mmol / L, 50 mmol / L, 100 mmol / L, 200 mmol / L and 400 mmol / L. It can be seen from the figure that the ROS content in the mixed solution increases with the increase of the content of H2O2, and the ROS content in the mixed solution changes linearly with time.

[0111] Figure 15 The fitting curves of the corresponding rates of different concentrations of H2O2 and polyethylene glycolized PdCu nanoalloy or pure palladium nanoparticles, · for polyethylene glycolized PdCu nanoalloy, ■ for pure palladium nanoparticles; as can be seen from the figure, the change of the catalytic rate of polyethylene glycolized PdCu nanoalloy and pure palladium nanoparticles under different concentrations of H2O2 conforms to the Michaelis equation.

[0112] Figure 16 The double-reciprocal fitting curves of the corresponding reaction rates of different concentrations of H2O2 and polyethylene glycolized PdCu nanoalloy or pure palladium nanoparticles, · for polyethylene glycolized PdCu nanoalloy, ■ for pure palladium nanoparticles; as can be seen from the figure, from the vertical intercept of the fitting curve, the maximum reaction rate of polyethylene glycolized PdCu nanoalloy catalyzing H2O2 is 5.56 x 10 - 8 M / s, the Michaelis constant is 80.13 mM, and the maximum reaction rate of pure palladium nanoparticles catalyzing H2O2 is 3.45 x 10 -8 M / s, and the Michaelis constant is 133.55 mM.

[0113] The polyethylene glycolized PdCu nanoalloy prepared in Example 1 was added to deionized water to obtain polyethylene glycolized PdCu solutions with concentrations of 12.5 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL; Figure 17 The thermal infrared imaging pictures obtained every 2 min under the irradiation of 0.9 W / cm 2 of 1064 nm laser; as can be seen from the figure, the polyethylene glycolized PdCu nanoalloy prepared in Example 1 generates local high temperature under the excitation of 1064 nm near-infrared light, and can be used as a photothermal conversion preparation. With the increase of the concentration of the PdCu solution, the warming effect is improved. Under the conditions of a laser power of 0.9 W / cm 2 and a concentration of 100 μg / mL, the infrared imaging temperature tracking cursor shows that the temperature can be raised to 48°C.

[0114] Figure 18 The thermal infrared imaging pictures obtained every 2 min under the irradiation of 1.7 W / cm 2The temperature rising curve of the PdCu nanometer alloy under the 1064nm light irradiation, 1 is 100ug / mL of the PdCu solution, 2 is 50ug / mL of the PdCu solution, 3 is 25ug / mL of the PdCu solution, 4 is 12.5ug / mL of the PdCu solution, 5 is deionized water; it can be seen from the figure that after 600s light irradiation, with the increase of the concentration of the PdCu solution, the temperature can be increased from room temperature to 38.8℃, 41.9℃, 43.3℃, 47℃ and 48℃ in turn.

[0115] The PdCu nanometer alloy prepared in Example One is added into deionized water to obtain a PdCu solution; Figure 19 The temperature rising column chart of the PdCu solution with a concentration of 100ug / mL under the excitation of lasers with different powers, a is the material temperature at room temperature, b is the material temperature after 10min laser irradiation; it can be seen from the figure that the PdCu solution can be heated to 35.5℃ after 10min irradiation under the irradiation power of 0.3W / cm 2 , to 42.7℃ under the irradiation power of 0.5W / cm 2 , to 50℃ under the irradiation power of 0.9W / cm 2 , and to 56.3℃ under the irradiation power of 1.4W / cm 2 , and the heating effect is significantly improved with the increase of the 1064nm irradiation light power.

[0116] The PdCu nanometer alloy prepared in Example One is added into deionized water to obtain a PdCu solution; under the 1064nm light irradiation with a power of 0.9W / cm 2 , the single cycle temperature rising and falling curve of the PdCu solution with a concentration of 100ug / mL, the laser is turned off after 10min laser irradiation, the temperature change curve with time is shown in Figure 20 , and the photo-thermal conversion efficiency relationship chart is obtained.

[0117] Figure 20 The temperature change curve of the PdCu solution single cycle temperature rising and falling; it can be seen from the figure that the solution is naturally cooled to the initial temperature.

[0118] Figure 21The graph of the photo-thermal conversion efficiency of the PEGylated PdCu solution; as can be seen from the graph, the correlation coefficient of the photo-thermal conversion curve of the PEGylated PdCu nanometer alloy and the first function fitting curve is 0.9944>0.99, the fitting effect is good, and the sampling system time constant τs of the PEGylated PdCu nanometer alloy is calculated as 330.4s, and the photo-thermal conversion efficiency is 52.4%.

[0119] Under the irradiation of 1064nm light with a power of 0.9W / cm 2 , the temperature-time curve of the PEGylated PdCu solution of 100μg / mL is shown in FIG. 4. Figure 22

[0120] Figure 22 The graph of the temperature-time curve obtained by measuring the laser on-off of the PEGylated PdCu solution for 4 cycles; as can be seen from the graph, there is no obvious temperature rise or decay during the photo-thermal conversion cycle of the PEGylated PdCu solution, which indicates that the photo-thermal stability of the material is good.​

Claims

1. A method for preparing a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal properties, characterized in that... It is done in the following steps: I. Synthesis of bimetallic nanoalloy PdCu: ① The palladium source, copper source, and high-temperature resistant solvent are ultrasonically homogenized to obtain a mixed solution A; The palladium source is palladium acetylacetone; the copper source is copper chloride; and the high-temperature resistant solvent is a mixture of oleylamine and oleic acid. The mass ratio of the palladium source to the copper source is 1:(0.3-0.7); the mass ratio of the total mass of the palladium source and the copper source to the mass of the high-temperature resistant solvent is 1:(300-400). ② Under magnetic stirring speed of 200 r / min to 400 r / min and vacuum conditions, the mixed solution A is heated from room temperature to 110°C to 130°C at a heating rate of 3°C to 10°C / min, and kept at 110°C to 130°C for 10 min to 30 min. Then nitrogen gas is introduced, and the temperature is raised to 125°C to 145°C at a heating rate of 3°C to 10°C / min. At 125°C to 145°C, a carbonyl compound is added, and the solution changes from transparent to black, yielding mixed solution B. The carbonyl compound is carbonyl tungsten; the mass ratio of the carbonyl compound to the total mass of the palladium and copper sources is 1:(1-1.5). ③ Under a magnetic stirring speed of 200 r / min to 400 r / min and a nitrogen atmosphere, the mixed solution B is heated to 240°C to 250°C at a heating rate of 3°C / min to 10°C / min. The reaction is carried out at 240°C to 250°C for 20 min to 60 min. After the reaction is completed, the temperature is lowered to room temperature, centrifuged, washed and dried to obtain bimetallic nanoparticles PdCu. II. Modification of Nanomaterials: ① Disperse bimetallic nanoparticles PdCu in a mixed solvent of cyclohexane and ethanol to obtain solution C. Then, NH2-PEG... 2000 Add to ethanol and sonicate until homogeneous to obtain solution D; ② Under stirring conditions, solution D is added to solution C to obtain mixed solution E. Mixed solution E is reacted at room temperature and under stirring conditions for 12h to 24h. After centrifugation, the reaction product is washed with deionized water to obtain polyethylene glycol-modified PdCu nano-alloy.

2. The method for preparing a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal properties according to claim 1, characterized in that... The centrifugation washing and drying described in step 1③ is carried out in the following steps: centrifuge at a speed of 4000r / min to 8000r / min for 5min to 15min, then wash the solid material alternately with anhydrous ethanol and cyclohexane 2 to 4 times, and finally dry at a drying temperature of 50℃ to 80℃ for 8h to 12h.

3. The method for preparing a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal properties according to claim 1, characterized in that... The particle size of the bimetallic nanoparticles PdCu mentioned in step 1③ is 10nm to 25nm.

4. The method for preparing a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal properties according to claim 1, characterized in that... In step 2①, the volume ratio of cyclohexane to ethanol in the mixed solvent is (100-200) μL:1 mL; The NH2-PEG mentioned in step 2① 2000 The mass ratio of ethanol to its volume is (1-5) mg: 1 mL.

5. The method for preparing a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal properties according to claim 1, characterized in that... In step 2②, the mixed solution E contains bimetallic nanoparticles PdCu and NH2-PEG 2000 The mass ratio is 1:(5~10).

6. The method for preparing a non-noble metal-noble metal bimetallic nanoalloy with high enzyme activity and mild photothermal properties according to claim 1, characterized in that... The stirring speed mentioned in step 2② is 200 r / min to 300 r / min; the centrifugal collection mentioned in step 2② is specifically carried out at a centrifugal speed of 6000 r / min to 8000 r / min for 5 min to 10 min.

Images