Preparation method and application of Cu-doped W18O49 nano-enzyme with near-infrared two-region photoresponse

Abstract

The invention relates to the field of biological functional materials, and discloses a preparation method and application of a Cu-doped W18O49 nano-enzyme with near-infrared two-region photoresponse. The specific method is as follows: a solvothermal method is adopted, the Cu-doped W18O49 (xCu-W18O49) nano-enzyme is prepared by regulating and controlling the molar doping amount of a Cu element, taking tungsten chloride as a tungsten source, taking copper chloride dihydrate as a copper source and taking n-propyl alcohol as a reaction solvent. The crystallinity of the nano enzyme is good, and the nano enzyme shows remarkable glutathione oxidase (GSHOx) activity in a tumor microenvironment. Compared with W18O49, Cu doping enables enzyme activity to be remarkably improved, the problem of low activity generally existing in current cancer treatment nano-enzymes is effectively solved, and meanwhile the nano-enzyme has application potential in the aspect of CT imaging.

Description

Technical Field

[0001] The present invention relates to the field of biofunctional materials and discloses a Cu-doped W nanostructured ... 18 O 49 The preparation method and application of nanozymes are used to solve the problem of low enzyme activity caused by oxidative stress generated by overexpressed glutathione (GSH) in the tumor microenvironment during cancer treatment. Background Art

[0002] Nanozyme-mediated tumor catalytic therapy is a new type of cancer treatment method that uses the catalytic action of nanozymes to convert hydrogen peroxide (H2O2) in the tumor microenvironment (TME) into toxic hydroxyl radicals (·OH), thereby inducing tumor cell apoptosis or necrosis. Compared with traditional treatment methods, nanozyme catalytic therapy has higher tumor specificity, which can effectively kill tumor cells at low doses and reduce treatment side effects. Nanozymes with near-infrared second region light (NIR-II) response not only show good photothermal therapy effects under near-infrared light excitation, but their own nanozyme catalytic properties can also produce reactive oxygen species in the complex tumor microenvironment (TME), effectively enhancing the synergistic therapeutic effect.

[0003] W 18 O 49 It is a non-stoichiometric tungsten oxide. Its unique crystal structure (monoclinic phase) and abundant oxygen vacancies on the surface give it multiple functional properties. At the same time, its large atomic number and inherent CT imaging function make it a research hotspot in the field of tumor treatment.

[0004] The present invention starts from the dimension of element doping and adopts a solvothermal method to prepare TME-responsive Cu-doped W by changing the molar doping amount of element Cu. 18 O 49 Nanozymes effectively solve the low activity problem that is common in current cancer treatment nanozymes. Summary of the Invention

[0005] In order to solve the shortcomings of the existing technology, the present invention discloses a Cu-doped W-type near-infrared second-region light response 18 O 49 Nanozymes were prepared by solvothermal method by regulating the amount of element doping to obtain xCu-W 18 O 49 (x = 0%, 3%, 5%, 7%, 9%) nanozymes. This nanozyme has good crystallinity and exhibits significant GSHOx activity in the tumor microenvironment, effectively solving the low activity problem commonly found in current cancer treatment nanozymes and showing potential for application in CT imaging.

[0006] The present invention is achieved through the following technical solutions: A Cu-doped W with near-infrared photoresponse in the second region 18 O 49 The preparation method of nanozyme includes the following steps: weighing tungsten chloride and copper chloride dihydrate in different molar ratios, adding them to n-propanol, stirring until a yellow clear solution is obtained, placing the solution in a polytetrafluoroethylene liner, placing the solution in a reactor, and heating the reaction. After cooling to room temperature, the blue sample solution is taken out and washed alternately with anhydrous ethanol and deionized water to prepare xCu-W. 18 O 49 Nanozymes.

[0007] Preferably, the mass of the tungsten chloride is 0.1785 g.

[0008] Preferably, the molar doping ratio of Cu is 0-9%, and more preferably, the molar doping ratio of Cu is 5%.

[0009] Preferably, the solvent thermal reaction conditions are a heating temperature of 180° C. and a reaction time of 24 h.

[0010] Preferably, the washing condition is to wash alternately with anhydrous ethanol and deionized water for 3 times.

[0011] The Cu-doped W nanostructured ... 18 O 49 Applications of nanozymes.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] (1) Based on the dimension of element doping, the present invention adopts a solvothermal method to prepare TME-responsive Cu-doped W by changing the Cu element doping amount. 18 O 49 Nanozymes have simple preparation methods, easy-to-control reaction conditions, and high feasibility.

[0014] (2) Cu-doped W with near-infrared second-region light response according to the present invention 18 O 49 Nanozymes effectively solve the low activity problem that is common in current cancer treatment nanozymes, and also have application potential in CT imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 W in Example 1 18 O 49 XRD pattern of nanozyme.

[0016] Figure 2 is xCu-W in Example 1 18 O 49(x=3%, 5%, 7%, 9%) XRD patterns of nanozymes.

[0017] Figure 3 5% Cu-W in Example 1 18 O 49 SEM image of nanozyme.

[0018] Figure 4 is W at different concentrations in Example 2 18 O 49 GSHOx activity diagram of nanozyme.

[0019] Figure 5 is W at different times in Example 2 18 O 49 GSHOx activity diagram of nanozyme.

[0020] Figure 6 5% Cu-W at different concentrations in Example 3 18 O 49 GSHOx activity diagram of nanozyme.

[0021] Figure 7 5% Cu-W at different times in Example 3 18 O 49 GSHOx activity diagram of nanozyme.

[0022] Figure 8 5% Cu-W in Example 4 18 O 49 Comparison of CT imaging of nanozymes. DETAILED DESCRIPTION

[0023] The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0024] Example 1 A near-infrared second-region photoresponsive xCu-W 18 O 49 The preparation method of nanozyme comprises the following steps: 0.1785g of WCl6 and CuCl2·2H2O with molar doping ratios of 0%, 3%, 5%, 7%, and 9% were weighed and added to 30mL of n-propanol. After stirring until a yellow clear solution was obtained, the solution was placed in a polytetrafluoroethylene liner and placed in a reactor and reacted at 180°C for 24 hours. After cooling to room temperature, the blue sample solution was removed and washed alternately with anhydrous ethanol and deionized water three times to prepare xCu-W. 18 O 49 Nanozymes.

[0025] Figure 1 W was prepared by the solvent thermal method in Example 1.18 O 49 The XRD pattern of nanozyme shows that the obtained sample can well match the monoclinic W 18 O 49 (JCPDS: 71-2450) corresponds to the standard spectrum.

[0026] Figure 2 The xCu-W was prepared by the solvent thermal method in Example 1. 18 O 49 (x=3%, 5%, 7%, 9%) XRD patterns of nanozymes are shown in the figure. It can be clearly seen from the XRD patterns that the obtained xCu-W 18 O 49 The XRD pattern of the monoclinic W 18 O 49 (JCPDS: 71-2450) corresponds to the standard spectrum, and the diffraction peak intensity is compared with that of pure W 18 O 49 The results show that xCu-W 18 O 49 The basic crystal structure of the nanomaterial still remains the monoclinic W 18 O 49 The doping of Cu element has no effect on W 18 O 49 The monoclinic phase structure of the sample is affected by Cu doping, and no new diffraction peaks appear. Two narrow and sharp diffraction peaks appear at 23.3° and 47.7°, corresponding to the crystal planes (010) and (020), respectively. The diffraction peaks of the sample are relatively left-shifted. This is because Cu doping into W 18 O 49 In the lattice, part of W is replaced, due to the Cu 2+ The radius is greater than W 6+ and W 5+ , resulting in lattice expansion.

[0027] Figure 3 5% Cu-W prepared in Example 1 18 O 49 As shown in the SEM image, when the Cu doping ratio is 5%, in addition to nanowires and nanospheres, a small amount of nanoclusters also appear in the sample. This phenomenon may be attributed to the effect of Cu doping on W 18 O 49 The regulation effect of crystal structure. 5% Cu doping may lead to W 18 O 49 The local lattice is deformed, which in turn promotes the formation of nanoclusters.

[0028] Example 2W 18 O 49Study on GSHOx activity of nanozymes DTNB was used to evaluate the GSHox activity of the samples. 18 O 49 The cells were mixed with PBS (pH 7.4) and then 50 μL of 4 M GSH solution was added, resulting in final sample concentrations of 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg / mL, respectively. After a period of reaction, 50 μL of ethanolic DTNNB solution was added. After incubation for 5 minutes, the absorbance of the supernatant was recorded using a UV-Vis-NIR spectrophotometer. The effect of 1064 nm laser irradiation on GSHox activity was also tested using DTNB.

[0029] Figure 4 is W at different concentrations in Example 2 18 O 49 The GSHOx activity diagram is shown in the figure. The experimental results show that with the increase of W 18 O 49 As the nanozyme concentration gradually increased from 0 to 1 mg / mL, the absorbance at a specific wavelength of 412 nm showed a clear concentration-dependent decrease trend. This phenomenon fully confirmed that W 18 O 49 Nanozymes have the ability to oxidize GSH, and their oxidative ability increases with increasing concentration.

[0030] Figure 5 is W at different times in Example 2 18 O 49 The GSHOx activity diagram is shown in the figure. The time gradient experimental results further reveal that W 18 O 49 The dynamic process of the reaction between nanozymes and GSH. As the reaction time continues to extend, from the initial 0 min to 3 h, it can be clearly observed that the content of GSH continues to decrease. This indicates that during this period, W 18 O 49 Nanozymes react continuously and stably with GSH, continuously consuming GSH, thereby gradually reducing the concentration of GSH in the system.

[0031] Example 35% Cu-W 18 O 49 The study on the enhancement of GSHox activity of nanozymes includes the following steps: DTNB is used to evaluate the GSHox activity of the sample. 18 O 49The cells were mixed with PBS (pH 7.4) and then 50 μL of 4 M GSH solution was added, resulting in final sample concentrations of 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg / mL, respectively. After a period of reaction, 50 μL of DTNB ethanol solution was added. After incubation for 5 minutes, the absorbance of the supernatant was recorded using a UV-Vis-NIR spectrophotometer. The effect of 1064 nm laser irradiation on GSHox activity was also tested using DTNB.

[0032] Figure 6 5% Cu-W at different concentrations in Example 3 18 O 49 The GSHOx activity diagram is shown in the figure. The experimental results show that with the increase of 5% Cu-W 18 O 49 As the concentration increased (0-1 mg / mL), the absorbance at 412 nm decreased in a concentration-dependent manner, confirming that 5% Cu-W 18 O 49 Nanozymes have efficient GSH oxidation ability.

[0033] Figure 7 5% Cu-W at different times in Example 3 18 O 49 The GSHOx activity diagram is shown in the figure. The experimental results show that with the increase of 5% Cu-W 18 O 49 The concentration increased (0-1 mg / mL), time gradient experiment (0-60 min) showed that the GSH content continued to decrease with the extension of reaction time, and a large amount of GSH was consumed at 15 min, confirming that 5% Cu-W 18 O 49 Nanozymes have efficient GSH oxidation ability.

[0034] Example 45% Cu-W 18 O 49 The specific steps for the CT imaging test of nanozymes are as follows: 0.5 mL of 100 mg / mL 5% Cu-W 18 O 49 The nanozyme sample solution was transferred to a 2mL microcentrifuge tube and placed in an orderly manner on a scanning frame. Next, the scanning frame carrying the microcentrifuge tube was pushed into the CT chamber, and the corresponding CT image was obtained by operating the computer.

[0035] Figure 8 5% Cu-W in Example 4 18 O 49 CT imaging test of nanozyme, as shown in the figure, shows 5% Cu-W prepared by n-propanol 18O 49 The CT imaging effect of the sample, the light and dark contrast diagram shows that the sample has excellent CT imaging performance. This result is xCu-W 18 O 49 It provides important experimental basis for further research in multimodal imaging and integrated diagnosis and treatment applications.

Claims

1. A Cu-doped W with near-infrared light response in the second region 18 O 49 The application of nanozymes in the preparation of nanozymes for cancer treatment is characterized by The Cu-doped W 18 O 49 The preparation method of nanozyme comprises the following steps: Weigh tungsten chloride and copper chloride dihydrate in different molar ratios and add them to n-propanol. Stir until a yellow clear solution is obtained. Place the solution in a polytetrafluoroethylene liner and heat the reactor for reaction. After cooling to room temperature, remove the blue sample solution and wash it alternately with anhydrous ethanol and deionized water to prepare xCu-W. 18 O 49 Nanozymes.

2. The Cu-doped W with near-infrared second-region light response according to claim 1 18 O 49 The use of nanozymes in preparing nanozymes for cancer treatment is characterized in that: The mass of the tungsten chloride is 0.1785 g.

3. The Cu-doped W with near-infrared second-region light response according to claim 1 18 O 49 The use of nanozymes in preparing nanozymes for cancer treatment is characterized in that: The molar doping ratio of Cu is 0-9%.

4. The Cu-doped W with near-infrared second-region light response according to claim 1 18 O 49 The use of nanozymes in preparing nanozymes for cancer treatment is characterized in that: The solvent thermal reaction conditions are a heating temperature of 180° C. and a reaction time of 24 h.

5. The Cu-doped W with near-infrared second-region light response according to claim 1 18 O 49 The use of nanozymes in preparing nanozymes for cancer treatment is characterized in that: The sample was washed alternately with anhydrous ethanol and deionized water for 3 times.

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