Nano-enzyme catalytic activity self-reporting system as well as preparation method and application thereof

Abstract

The invention discloses a nano-enzyme catalytic activity self-reporting system and a preparation method and application thereof, and belongs to the cross technical field of monatomic nano-enzyme and biomedicine, the preparation method comprises the following steps: step 1, preparing monatomic copper nano-enzyme FCuSA by taking flash graphene as a carrier; and step 2, co-encapsulating the photoacoustic reporter molecule EM 1 and the nano enzyme FCuSA by adopting a nano precipitation technology assisted by an amphiphilic polymer DSPE-PEG2000, and constructing a nano enzyme catalytic activity self-reporting system 1-FCuSA. The 1-FCuSA prepared by the invention not only can automatically monitor the peroxidase-like catalytic efficiency of the nano-enzyme FCuSA in a tumor in real time through a ratio photoacoustic imaging signal, but also can be used for the catalytic treatment of the tumor nano-enzyme by accurately regulating and controlling the generation of active oxygen, so that the tumor treatment effect is evaluated in situ in real time, and the 1-FCuSA has a wide application prospect. And a novel molecular imaging tool is provided for early curative effect monitoring and precise treatment intervention of tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the cross - technical field of single - atom nanozymes and biomedicine, and specifically relates to a nanozyme catalytic activity self - reporting system, its preparation method and application. Background Art

[0002] As a type of nanomaterial with natural enzyme catalytic activity, nanozymes have shown broad application prospects in the biomedical field due to their advantages such as low preparation cost, excellent stability, easy large - scale production, and good environmental tolerance. These artificial enzymes can mimic the catalytic characteristics of a variety of natural enzymes, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and oxidase (OXD), etc., thus enabling precise regulation of reactive oxygen species (ROS), which is of great significance in the occurrence and development of various diseases. Among them, POD - like nanozymes have been most widely used in the field of tumor treatment because they can catalyze the generation of highly reactive ·OH. It should be noted that ·OH is considered to be the molecule with the strongest oxidation ability among all reactive oxygen species, which gives it unique advantages in tumor treatment. By real - time monitoring the generation of ·OH in the tumor microenvironment, not only can the treatment effect of POD - like nanozymes be evaluated early, but also important real - time feedback information can be provided for optimizing treatment strategies. However, at present, the detection of POD - like catalytic activity of nanozymes mainly relies on in vitro analysis methods, such as using molecular probes like 2′,7′ - dichlorodihydrofluorescein diacetate (DCFH - DA) and hydroxyphenyl fluorescein (HPF). Due to the extreme complexity of the in - vivo environment, these traditional probes are difficult to achieve non - invasive detection of catalytic activity in vivo.

[0003] Photoacoustic (PA) imaging technology has shown unique value in the field of in - vivo imaging due to its three - dimensional imaging advantages of both high spatial resolution and deep tissue penetration ability (centimeter - level depth). Although several PA probes for in - vivo imaging of ·OH have been developed currently, most still adopt the traditional absolute signal intensity output mode. This mode is easily interfered by non - specific factors such as changes in environmental conditions and fluctuations in imaging parameters, which may lead to false - positive results. To break through these technical bottlenecks, ratio - type PA imaging probes achieve signal self - calibration by monitoring the relative changes in absorption bands at two or more characteristic wavelengths, and can specifically quantify target molecules. Compared with traditional methods, ratio - type PA imaging technology significantly improves the detection reliability by eliminating interference factors, provides more accurate and stable optical signals for in - vivo imaging, and opens up a new way for the activity monitoring and efficacy evaluation of nanozymes in tumor treatment. However, currently, ratio - type PA probes for ·OH detection are still scarce. Except for the two reported ratio - type PA imaging probes, there are no other relevant reports, and there is a lack of ratio - PA imaging probes that can self - report the catalytic activity of POD - like nanozymes. Summary of the Invention

[0004] To provide a ratio PA imaging probe for self-reporting the catalytic activity of POD-like nanozymes, the present invention provides a nanozyme catalytic activity self-reporting system, its preparation method and application.

[0005] Technical solution: A preparation method of a nanozyme catalytic activity self-reporting system includes the following steps:

[0006] Step 1: Prepare single-atom copper nanozyme FCuSA using flash graphene as a carrier;

[0007] Step 2: Adopt the amphiphilic polymer DSPE-PEG 2000 to assist the nano-precipitation method to encapsulate the photoacoustic reporter molecule EM 1 and the nanozyme FCuSA to prepare the nanozyme catalytic activity self-reporting system 1-FCuSA. The structural formula of the EM 1 is as follows:

[0008]

[0009] wherein, R is ethyl.

[0010] Further, the specific operation of Step 1 is as follows:

[0011] Step 1.1: Disperse the carbon source in deionized water, add the CuCl solution, and mix evenly; wherein, the mass ratio of the carbon source to CuCl is 200:60.

[0012] Step 1.2: Centrifuge the mixture in Step 1.1, and then place the centrifuged product in a vacuum drying oven to dry to obtain a black powder;

[0013] Step 1.3: Put the black powder obtained in Step 1.2 into a sample holder, transfer it to a Joule flash heating system for flash heating treatment to obtain single-atom copper nanozyme FCuSA; the conditions for the flash heating treatment are: the flash heating temperature is 2200 °C, the flash heating time is 10 s, and the chamber pressure is 10 -1 ~10 Pa.

[0014] Further, the specific operation of Step 2 is as follows:

[0015] Step 2.1: Dissolve FCuSA and DSPE-PEG 2000 in a mixed solution composed of tetrahydrofuran and ethanol to form a homogeneous solution;

[0016] Step 2.2: Dissolve EM 1 in dimethyl sulfoxide;

[0017] Step 2.3: Under continuous ultrasonic treatment, inject the solutions in Step 2.1 and Step 2.2 into deionized water, and ultrasonically treat the obtained mixed solution in an ice-water bath for 5-20 min;

[0018] Step 2.4. Remove tetrahydrofuran, ethanol, and dimethyl sulfoxide under vacuum. Transfer the resulting aqueous solution to a centrifugal filter, and wash it with deionized water under centrifugation to remove free compounds, obtaining 1-FCuSA.

[0019] Furthermore, the mass ratio of the FCuSA, DSPE-PEG 2000 , and EM 1 is 0.45:10:0.52.

[0020] The nanozyme catalytic activity self-reporting system prepared by the method of the present invention can be used for real-time self-prediction of the catalytic activity of nanozymes. The nanozyme catalytic activity self-reporting system can not only monitor the POD-like catalytic efficiency in tumors in real time through the ratio PA imaging signal, but also be used for tumor catalytic therapy.

[0021] Beneficial effects:

[0022] (1) The copper single-atom nanozyme (FCuSA) prepared in the present invention adopts a Joule flash heating system, which can enhance the POD-like activity of the nanozyme to generate more ·OH.

[0023] (2) The present invention uses diene EM 1 as the ·OH-responsive chromophore, and utilizes the generated near-infrared (NIR) photoacoustic signal (808 nm) and the "constant" photoacoustic signal of the FCuSA nanozyme at 1064 nm to successfully achieve ratiometric PA imaging of ·OH in vivo.

[0024] (3) Based on the extremely high sensitivity and selectivity of diene EM 1 to ·OH, the prepared nanozyme catalytic activity self-reporting system 1-FCuSA can reliably monitor the ·OH generated during the nanozyme catalytic process, and then be used to evaluate the POD-like catalytic activity of the nanozyme, realizing real-time monitoring and analysis of the nanozyme catalytic activity.

[0025] (3) The 1-FCuSA self-reporting system developed in the present invention exhibits excellent specific POD-like catalytic activity in in vivo tumor therapy, and achieves a significant tumor suppression effect by efficiently generating therapeutic ·OH. Description of the drawings

[0026] Figure 1 is the design flow chart of the nanozyme catalytic activity self-reporting system 1-FCuSA of the present invention;

[0027] Figure 2 is the in vitro characterization of FCuSA and 1-FCuSA prepared in the examples of the present invention;

[0028] Figure 3 is the performance characterization of 1-FCuSA prepared in the examples of the present invention;

[0029] Figure 4 It is a schematic diagram of the experimental results of the cytotoxicity of HUVEC and HCT116 tumor cells in the test example;

[0030] Figure 5 It is a schematic diagram of the ratio PA imaging results of the uptake of 1-FCuSA by tumor cells and the POD-like activity in the test example;

[0031] Figure 6 It is a schematic diagram of the fluorescence image results of ·OH of HPF in cells after incubation with 1-FCuSA, 1-FCuSA + NAC or 1-FCuSA + H2O2;

[0032] Figure 7 It is a schematic diagram of the photoacoustic image results of subcutaneous injection of 1-FCuSA in mice;

[0033] Figure 8 It is a schematic diagram of the ratio photoacoustic imaging results of tracking the POD-like activity of 1-FCuSA during cancer treatment;

[0034] Figure 9 It is a schematic diagram of the tumor treatment image results of mice injected with 1-FCuSA. Detailed implementation manners

[0035] The technical solution of the present invention will be described in detail below through examples, but the protection scope of the present invention is not limited to the described examples. In the examples of the present invention, unless otherwise specified, the raw materials used can be purchased from the market or prepared conventionally.

[0036] Example 1: Preparation of a nanozyme catalytic activity self-reporting system

[0037] The design flow chart of the nanozyme catalytic activity self-reporting system (1-FCuSA) of the present invention is as Figure 1 shown, where (a) represents the process of preparing 1-FCuSA by encapsulating EM 1 and FCuSA with the assistance of DSPE-PEG 2000 During the POD-like catalytic reaction process, the EM 1 probe in 1-FCuSA can specifically respond to the hydroxyl radicals (·OH) generated by the nanozyme catalysis, undergo a molecular structure transformation to generate 2-FCuSA, and be accompanied by PA 808 / PA 1064 ratio enhancement. (b) represents the chemical structure of diene EM 1 and the chemical structure of the cationic EM 2 transformed during ·OH oxidation, as well as the chemical structure of FCuSA. (c) represents a schematic diagram of predicting the anti-cancer effect by ratio PA imaging related to nanozyme activity.

[0038] The preparation method of 1-FCuSA in this example is as follows:

[0039] Step 1. Prepare single-atom copper nanozyme (FCuSA) using flash graphene (FG) as a carrier. The specific operation steps are as follows:

[0040] Step 1.1. Obtain a carbon source by pyrolyzing biomass (rice straw and wheat straw) at 900 °C, 200 sccm, and an Ar gas flow rate for 3 hours; disperse 200 mg of the carbon source in 200 mL of deionized water, and then combine it with 3 mL of a 20 mg / mL CuCl solution.

[0041] Step 1.2. Subsequently, centrifuge the mixture in Step 1.1 (9000 rpm, 5 min), and then dry it under vacuum drying conditions to obtain a black powder.

[0042] Step 1.3. Put 100 mg of the black powder obtained in Step 1.2 into a sample holder and transfer it to a Joule flash heating system. Conduct flash heating treatment under the following conditions: flash heating temperature is 2200 °C, flash heating time is 10 s, chamber pressure: 10 -1 ~10 Pa, to obtain a monodisperse modified Cu light black powder, abbreviated as FCuSA, for further use.

[0043] Step 2. Use an amphiphilic polymer-assisted nanoprecipitation method to prepare a nanozyme catalytic activity self-reporting system (1-FCuSA). The specific operation steps are as follows:

[0044] Step 2.1. Dissolve the FCuSA (0.45 mg) prepared in Step 1 and DSPE-PEG 2000 (10 mg) in a mixed solution composed of tetrahydrofuran (THF, 0.5 mL) and ethanol (EtOH, 0.5 mL) to form a homogeneous solution.

[0045] Step 2.2. Dissolve EM 1 (0.52 mg) in dimethyl sulfoxide (DMSO, 0.25 mL); the structural formula of EM 1 is as follows:

[0046]

[0047] Among them, R is an ethyl group.

[0048] EM 1 is prepared by the method in the patent with the application number 201811189682.X.

[0049] Step 2.3. Under continuous ultrasound, quickly inject the solutions obtained in Step 2.1 and Step 2.2 into secondary water (D.I. water, 9 mL); ultrasonicate the mixed solution in an ice-water bath (power: 150 W, frequency: 33 KHZ, temperature: 20 °C) for 30 minutes.

[0050] Step 2.4: Remove THF, EtOH, and DMSO under vacuum. Transfer the resulting aqueous solution to a centrifugal filter (Millipore, 10 kDa), and wash with deionized water under centrifugation (2040×g) to remove free compounds, obtaining 1-FCuSA.

[0051] The in vitro characterization of FCuSA and 1-FCuSA prepared in Example 1 is as Figure 2 shown. Among them, (a) is the dynamic light scattering (DLS) result of FCuSA; (b) is the low-resolution STEM image (dark field) of FCuSA; (c) is the high-resolution STEM image of FCuSA, with single copper atoms marked by red circles and flash graphene marked by red parallel lines; (d) is the atomic-resolution STEM image of FCuSA. (e) The ultraviolet absorption spectrum result of 1-FCuSA; (f) The dynamic light scattering (DLS) and high-resolution TEM image results of 1-FCuSA. It can be Figure 2 seen that 1-FCuSA can be well dispersed in water and has a spherical morphology, with an average hydrodynamic size of 147.2 ± 2.5 nm.

[0052] Test Example 1: Performance test of 1-FCuSA prepared in Example 1

[0053] Perform a performance test on 1-FCuSA prepared in Example 1. The test items, methods, and results are as follows:

[0054] (I) Measure the sensitivity of 1-FCuSA to H2O2. The specific steps are as follows:

[0055] (1) Incubate 1-FCuSA (56 μM EM 1) in PBS (pH 6.8) with H2O2 (0, 2, 5, 10, 20, 30, 50, 80, 100, 150, 200 μM) at room temperature for 30 min;

[0056] (2) Collect PA images at 808 nm and 1064 nm on a LOIS-3D PA imaging system, and calculate the PA 808 / PA 1064 ratio by dividing the PA intensity at 808 nm by the PA intensity at 1064 nm;

[0057] (3) Normalize the PA intensities of 808 nm, 1064 nm, and PA 808 / PA 1064 values to the value of 1-FCuSA incubated with 200 μM H2O2;

[0058] (4) The normalized PA 808 / PA1064 The graph of the ratio versus the H2O2 concentration provides a linear regression between 0 - 20 μM H2O2. The slope (k) and standard deviation (δ) of the linear graph are obtained by performing 11 measurements on the blank solution. The detection limit is calculated by the 3δ / k method.

[0059] (5) The test results are as Figure 3 shown in (a)-(c) and (e) below.

[0060] Figure 3 Among them, (a) shows the PA 808 image, PA 1064 image, and ratio PA 808 / PA 1064 image of 1-FCuSA when different H2O2 concentrations (0, 2, 5, 10, 20, 30, 50, 80, 100, 150, 200 μM) are used as substrates and the POD activity is measured after reacting with 1-FCuSA for 30 minutes; (b) shows the PA 808 / PA 1064 ratio of 1-FCuSA when different H2O2 concentrations are used as substrates to measure the POD activity; (c) shows the linear relationship graph of the standardized PA 808 / PA 1064 ratio versus the H2O2 concentration (1 - 20 μM); (e) shows the correlation fitting curve between the nanozyme activity (%SE maximum signal enhancement) and the PA 808 / PA 1064 ratio of 1-FCuSA.

[0061] Figure 3 The results in it show that as the H2O2 concentration increases, the photoacoustic signal at 808 nm of the self-reporting system gradually increases, and the photoacoustic ratio signal PA 808 / PA 1064 gradually increases, and the detection limit is 0.45 μM. The relevant POD activity (%SE maximum signal enhancement) of 1-FCuSA at different H2O2 concentrations is fitted with the PA Figure 3 in (c) 808 / PA 1064 ratio to obtain the relevant curve, and the results are as Figure 3 shown in (e) below, indicating that 1-FCuSA can self-monitor the catalytic activity of 1-FCuSA through the ratio PA signal.

[0062] (II) Measure the HPF fluorescence spectrum of 1-FCuSA after adding different concentrations of H2O2. The specific steps are as follows:

[0063] (1) Incubate 1-FCuSA (56 μM EM 1) in PBS (pH 6.8) with H2O2 (0, 2, 5, 10, 20, 30 μM) at room temperature for 30 min;

[0064] (2) Dissolve 1 mg of HPF in DMF to prepare a 5 mM stock solution, and dilute the 5 mM stock solution 500-fold to 10 μM with a phosphate solution (0.1 M, pH 7.4);

[0065] (3) Add 10 μM HPF to the sample in (1) and incubate for 1 min, then measure the fluorescence value (Ex / Em, 490 / 515 nm), and plot the measurement results as a fluorescence spectrum;

[0066] (4) The test results are as shown in Figure 3 (d) in, Figure 3 (d) in is the fluorescence spectrum of HPF when 1-FCuSA is incubated with different concentrations of the substrate H2O2.

[0067] Figure 3 (d) in indicates that the fluorescence intensity of HPF gradually increases with the increase in the concentration of H2O2, indicating that 1-FCuSA has excellent POD-like activity.

[0068] (III) Measure the selectivity of 1-FCuSA for different ROS, and the specific steps are as follows:

[0069] (1) Incubate 1-FCuSA (56 μM EM 1) in PBS (pH 6.8) with 9 representative ROS at room temperature for 10 minutes, where 1: GSH (5 mM), 2: L-Cys (200 μM), 3: Hcy (300 μM), 4: ClO - (200 μM), 5: O 2- (100 μM xanthine + 22 mU xanthine oxidase XO), 6: ONOO - (200 μM), 7: control, 8: H2O2 (200 μM), 9: ·OH (200 μM Fe 2+ + 200 μM H2O2), 10: H2O2 (200 μM) + NAC (200 μM);

[0070] (2) Collect PA images at 808 nm and 1064 nm on a LOIS-3D PA imaging system, and calculate the PA 808 / PA 1064 ratio by dividing the PA intensity at 808 nm by the PA intensity at 1064 nm;

[0071] (3) For 808 nm and 1064 nm and PA 808 / PA1064 The PA intensities were all normalized to the value of 1-FCuSA incubated with 200 μM H2O2;

[0072] (4) The test results are shown in Figure 3 (f)-(g) below.

[0073] Figure 3 In (f), the photoacoustic images at 808 nm and the photoacoustic images at 1064 nm and the PA 808 / PA 1064 ratio photoacoustic images of 1-FCuSA incubated with 9 representative ROSs at room temperature for 10 minutes are shown, where 1: GSH (5 mM), 2: L-Cys (200 μM), 3: Hcy (300 μM), 4: ClO - (200 μM), 5: O2 ·- (100 μM xanthine + 22 mU xanthine oxidase (XO)), 6: ONOO - (200 μM), 7: control, 8: H2O2 (200 μM), 9: ·OH (200 μM Fe 2+ + 200 μM H2O2), 10: H2O2 (200 μM) + NAC (200 μM); (g) shows the specific numerical values quantified from the PA 808 / PA 1064 ratio photoacoustic images of 1-FCuSA incubated with 9 representative ROSs at room temperature for 10 minutes.

[0074] Figure 3 (f)-(g) below illustrate the excellent selectivity of 1-FCuSA towards ·OH and H2O2.

[0075] (IV) Cytotoxicity (MTT) assay. The cytotoxicity experiment of 1-FCuSA was carried out on HUVEC and HCT116 tumor cells. The specific steps are as follows:

[0076] (1) HUVEC and HCT116 tumor cells were incubated in 96-well plates (5000 cells per well), and treated with DMEM (Jiangsu KeyGen Biotech Co., Ltd.) (100 μL) culture medium for 24 h;

[0077] (2) Subsequently, 1-FCuSA (0, 2, 5, 10, 20, 50, 100, 200, 300 μg / mL) was added to the above wells. After 3 h, the cells were washed 3 times with PBS;

[0078] (3) After co-incubation for 24 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 1 mg / mL, 50 μL) was added to each well;

[0079] (4) Incubate HCT116 cells with 1-FCuSA (FCuSA concentration: 300 μg / mL) for 3 h, and wash the cells three times with PBS. After co-incubation for 2, 14, 16, 18, 20, and 24 hours, add MTT (1 mg / mL, 50 μL) to each well;

[0080] (5) Subsequently, carefully remove the solution in each well, and add 150 μL of DMSO to each well to dissolve the purple formazan crystals; obtain the absorbance (OD) at 550 nm in each well on a Tcan microplate reader;

[0081] (6) The test results are as Figure 4 shown; Figure 4 Among them, (a) is the cell viability of HUVEC cells after treatment with different concentrations of 1-FCuSA (0, 2, 5, 10, 20, 50, 100, 200, 300 μg / mL); (b) is the cell viability of HCT116 cells after treatment with different concentrations of 1-FCuSA (0, 2, 5, 10, 20, 50, 100, 200, 300 μg / mL).

[0082] Figure 4 It shows that 1-FCuSA has significant cytotoxicity to tumor cells but has little toxicity to HUVEC cells.

[0083] (V) Ratio PA imaging of the POD-like activity of 1-FCuSA on tumor cells, and the specific steps are as follows:

[0084] (1) Incubate the cells with 1-FCuSA (300 μg / mL) for 3 h;

[0085] (2) To inhibit the POD-like activity of 1-FCuSA in HCT116 cells, incubate the cells with NAC (200 μM) for 1 h. Subsequently, incubate the cells with 1-FCuSA (300 μg / mL) together for 3 h;

[0086] (3) To enhance the POD-like activity of 1-FCuSA, add exogenous H2O2 (200 μM) to HCT116 cells for 1 h, and incubate the cells with 1-FCuSA (300 μg / mL) for 3 h;

[0087] (4) To further verify that the activation of the ratio PA signal is initiated by ·OH, add DMEM medium containing 200 μM Fe 2+ and 200 μM H2O2 to HCT116 cells for 30 min, and incubate the cells with 1-FCuSA (300 μg / mL) for 3 h;

[0088] (5) Discard the above-mentioned culture medium, wash the above-mentioned cells twice with PBS (1×, pH 7.4), and add trypsin to dissociate the cells. Collect the cell pellet and centrifuge at 4°C (161×g, 4 min);

[0089] (6) Collect and analyze the PA images and intensities at 808 nm and 1064 nm on the LOIS-3D photoacoustic imaging system, calculate the corresponding PA 808 / PA 1064 values, and construct a ratio image using Image J software;

[0090] (7) The test results are shown in (a)-(d) of Figure 5 ; Figure 5 Among them, (a) is the photoacoustic image of HCT116 cells (~5×10 5 cells) at 808 nm after treatment with 1-FCuSA for 3 hours under different conditions. The conditions in the figure are: I: PBS, II: NAC (200 μM), III: 200 μM H2O2, IV: 200 μM Fe 2+ + 200 μM H2O2; (b) is the photoacoustic image of HCT116 cells (~5×10 5 cells) at 1064 nm after treatment with 1-FCuSA for 3 hours under different conditions. The conditions in the figure are: I: PBS, II: NAC (200 μM), III: 200 μM H2O2, IV: 200 μM Fe 2+ + 200 μM H2O2; (c) is the ratio image of PA 5 / PA 808 of HCT116 cells (~5×10 1064 cells) after treatment with 1-FCuSA for 3 hours under different conditions. The conditions in the figure are: I: PBS, II: NAC (200 μM), III: 200 μM H2O2, IV: 200 μM Fe 2+ + 200 μM H2O2; (d) is the specific numerical value of the quantification of the ratio image of PA 808 / PA 1064 of HCT116 cells in (c).

[0091] Figure 5 (a)-(d) in

[0092] (VI) Photoacoustic imaging of tumor cell uptake, the specific steps are as follows:

[0093] (1) Incubate HCT116 cells with 1-FCuSA (300 μg / mL) based on the FCuSA concentration for 3 h. Wash the cells three times with PBS and incubate for 2, 14, 16, 18, 20, and 24 hours in total;

[0094] (2) Before PA imaging, wash the above cells twice with PBS (1×, pH 7.4), add trypsin to dissociate the cells, and centrifuge to collect the cell pellet at 4 °C (161×g, 4 min);

[0095] (3) Collect and analyze the PA images and intensities at 808 nm and 1064 nm on the LOIS-3D photoacoustic imaging system, calculate the corresponding PA 808 / PA 1064 value, and construct a ratio image using Image J software;

[0096] (4) The test results are shown in Figure 5 as (e)-(j). Figure 5 Among them, (e) is the photoacoustic image of HCT116 cells treated with 1-FCuSA for 2, 14, 16, 18, 20, and 24 hours at 808 nm; (f) is the photoacoustic image of HCT116 cells treated with 1-FCuSA for 2, 14, 16, 18, 20, and 24 hours at 1064 nm; (g) is the PA 808 / PA 1064 image of HCT116 cells treated with 1-FCuSA for 2, 14, 16, 18, 20, and 24 hours; (h) is the specific value of the PA 808 / PA 1064 ratio photoacoustic image quantification of HCT116 cells treated with 1-FCuSA for 2, 14, 16, 18, 20, and 24 hours; (i) is the cell inhibition rate of HCT116 treated with 1-FCuSA for 2, 14, 16, 18, 20, and 24 hours; (j) is the correlation fitting curve between the cell inhibition rate in (i) and the ratio value of PA 808 / PA 1064 in (h), and the values are mean ± standard deviation (n = 3, ***P < 0.001).

[0097] Figure 5 In (e)-(j), it shows a good correlation between the cell inhibition rate and the PA ratio, indicating that 1-FCuSA can monitor its anti-tumor efficacy through the ratio PA signal output in cells.

[0098] (VII) Detection of ROS in cells, the specific steps are as follows:

[0099] (1) Place HCT116 tumor cells in a glass-bottomed culture dish and culture at 37 °C for 24 h;

[0100] (2) The cells were incubated with four different conditions: I: 1-FCuSA (10 mg / kg); II: 1-FCuSA + NAC (1 mM); III: 1-FCuSA + H2O2 (1 mM), and the untreated cells served as the control group. After incubation for 3 h, the cells were washed twice with PBS;

[0101] (3) HPF was added to the culture dish in (2), and the cells were incubated for 60 min and then washed twice with PBS;

[0102] (4) Hoechst was added to the culture dish in (3), and the cells were incubated for 60 min and then washed twice with PBS;

[0103] (5) Fluorescence images of the cells were captured using an Olympus IX73 fluorescence inverted microscope;

[0104] (6) The test results are as Figure 6 shown.

[0105] Figure 6 Fluorescence images of ·OH of HPF in cells after incubation with 1-FCuSA, 1-FCuSA + NAC, or 1-FCuSA + H2O2. Ctrl in the figure is the control group. Figure 6 It shows that the use of HPF staining for hydroxyl radical detection confirms that hydroxyl radicals generated by the POD-like activity of nanoenzymes activate 1-FCuSA.

[0106] (8) In vivo photoacoustic imaging of the POD-like activity of 1-FCuSA, and the specific steps are as follows:

[0107] (1) PBS buffer (pH 6.8) containing 1-FCuSA (1 mg / mL based on the FCuSA concentration, 50 μL), with or without the addition of other substances, was subcutaneously injected into the thigh of mice. The injection substances were grouped as follows: 1-FCuSA (without the addition of other substances), 1-FCuSA + H2O2 (1 mM), 1-FCuSA + 1 mM H2O2 + 200 μM NAC, 1-FCuSA + 1 mM Fe 2+ + 1 mM H2O2.

[0108] (2) After 30 min, PA images and intensities at 808 nm and 1064 nm were collected and analyzed on a LOIS-3D photoacoustic imaging system;

[0109] (3) Calculate the corresponding PA 808 / PA 1064 value and use Image J software to make a ratio image;

[0110] (4) The test results are as Figure 7As shown; Figure 7 Schematic diagram of the photoacoustic image results of subcutaneous injection of 1-FCuSA in mice, where: (a) PA images and ratio PA at 808 nm and 1064 nm of live mice subcutaneously injected with 1-FCuSA 808 / PA 1064 images. The injection sites are marked with white circles as I: 1-FCuSA (1 mg / kg), II: 1-FCuSA + H2O2 (1 mM), III: 1-FCuSA + H2O2 (1 mM) + NAC (200 μM), IV: 1-FCuSA + Fe 2+ (1 mM) + H2O2 (1 mM); (b) is the photoacoustic quantification data at 808 nm; (c) is the photoacoustic quantification data at 808 nm; (d) is the photoacoustic quantification data of PA 808 / PA 1064 , and the values are mean ± standard deviation (n = 3, ***P < 0.001).

[0111] Figure 7 It shows that 1-FCuSA can perform ratio-type photoacoustic imaging of ·OH in live mice, providing a basis for the self-monitoring of the POD-like catalytic activity of 1-FCuSA.

[0112] (IX) Ratio PA imaging of the POD-like activity of 1-FCuSA in tumors, and the specific steps are as follows:

[0113] (1) Intratumor injection of normal saline (0.9%), NAC (1 mM), H2O2 (1 mM), and H2O2 (1 mM) and Fe 2+ (1 mM) into mice subcutaneously inoculated with HCT116 xenograft tumors;

[0114] (2) Intravenous injection of 1-FCuSA (10 mg / kg FCuSA, 200 μL) into the mice in different treatment groups in (1) at 0, 2, 6, 10, 12, and 24 h;

[0115] (3) Collect and analyze the PA images and intensities at 808 nm and 1064 nm on a LOIS-3D photoacoustic imaging system, and make ratio images using Image J software.

[0116] (4) The test results are as Figure 8 shown; Figure 8Schematic diagram of tumor photoacoustic image results for in vivo cancer treatment of 1-FCuSA monitored by ratio-type photoacoustic imaging. Among them, (a) depicts the ratio photoacoustic imaging illustration for tracking the POD-like activity of 1-FCuSA during cancer treatment; (b) is the ratio map made for mice subcutaneously inoculated with HCT116 xenograft tumors after receiving the following treatments: (I) intravenous injection of 1-FCuSA (10 mg / kg) combined with injection of normal saline group; (II) 1-FCuSA combined with intratumoral injection of NAC (1 mM); (III) 1-FCuSA combined with intratumoral injection of H2O2 (1 mM); (IV) 1-FCuSA combined with intratumoral injection of Fe 2+ (1 mM) and H2O2 (1 mM). After injection, photoacoustic imaging scanning was performed, and PA images and intensities at 808 nm and 1064 nm were collected and analyzed on a LOIS-3D photoacoustic imaging system, and a ratio map was made using Image J software; (c) is the photoacoustic signal intensity of tumors in different treatment groups in (b) at 808 nm; (d) is the photoacoustic signal intensity of tumors in different treatment groups in (b) at 1064 nm; (e) is the normalized photoacoustic ratio (PA 808 / PA 1064 ) of tumors in each treatment group. Data are expressed as mean ± standard deviation (n = 3, ***P < 0.001).

[0117] Figure 8 It shows that 1-FCuSA exhibits a strong "always-on" near-infrared second window (NIR-II) photoacoustic signal at 1064 nm, realizing real-time monitoring of the drug delivery process to tumors. When exogenous ·OH is injected into tumors, the photoacoustic signal at 808 nm and the PA 808 / PA 1064 ratio value both show a significant increase, indicating that 1-FCuSA can visually monitor the POD-like activity in tumors.

[0118] (X) Tumor treatment by injecting 1-FCuSA into mice, and the specific steps are as follows:

[0119] (1) Mice subcutaneously inoculated with HCT116 xenograft tumors were intratumorally injected with normal saline (0.9%), NAC (1 mM), H2O2 (1 mM) and H2O2 (1 mM) and Fe 2+ (1 mM);

[0120] (2) Mice in different treatment groups in (1) were intravenously injected with 1-FCuSA (10 mg / kg FCuSA, 200 μL) via the tail vein, and the changes in the tumor volume of mice were monitored every two days within 20 days.

[0121] (3) After 20 days of treatment, tumor sections of each group were subjected to representative hematoxylin-eosin (H&E) staining.

[0122] (4) The test results are as Figure 9 shown; Figure 9 (a) shows the tumor growth curves of mice in different treatment groups: (I) normal saline group, (II) 1-FCuSA group, (III) 1-FCuSA combined with intratumoral injection of NAC (200 μM) group, (IV) 1-FCuSA combined with intratumoral injection of H2O2 (200 μM) group; Data are expressed as mean ± standard deviation (200 μM) (n = 4, ***P < 0.001); (b) shows representative hematoxylin-eosin (H&E) staining images of tumor sections in each group 20 days after treatment; (c) shows the correlation fitting curve between the relative change in tumor volume and the normalized photoacoustic ratio (PA 808 / PA 1064 )(Pearson r = -0.996).

[0123] Figure 9 It shows that 1-FCuSA has good anti-tumor effects, and there is a significant negative correlation between tumor volume and the ratio-type photoacoustic signal value, indicating that the ratio-type photoacoustic imaging technology can early monitor the therapeutic response of tumors to the peroxidase-like activity of 1-FCuSA.

[0124] From the above tests and Figures 3 to 9 the results, it can be known that:

[0125] (1) The 1-FCuSA self-reporting system exhibits excellent specific POD-like catalytic activity in in vivo tumor treatment, and achieves significant tumor suppression effects by efficiently generating therapeutic ·OH.

[0126] (2) The 1-FCuSA self-reporting system uses diene EM 1 as the ·OH-responsive chromophore, and utilizes the near-infrared (NIR) photoacoustic signal (808 nm) generated by it and the "constant bright" photoacoustic signal of FCuSA nanozyme at 1064 nm to successfully achieve ratio-type PA imaging of ·OH in vivo.

[0127] (3) The 1-FCuSA self-reporting system can not only achieve tumor treatment through specific POD-like activity, but also monitor the treatment effect in real time through ratio-type photoacoustic imaging technology, maximizing the treatment effect while minimizing the risk of side effects, providing a new idea for the integration of tumor diagnosis and treatment.

[0128] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as a limitation of the present invention itself. Various changes can be made in its form and details without departing from the spirit and scope of the present invention.

Claims

1. A preparation method of a nanozyme catalytic activity self-reporting system, characterized in that, It includes the following steps: Step 1: Prepare single-atom copper nanozyme FCuSA using flash graphene as a carrier; Step 2: Use amphiphilic polymer DSPE-PEG 2000 to encapsulate EM 1 and FCuSA by the assisted nanoprecipitation method to prepare the nanozyme catalytic activity self-reporting system 1-FCuSA. The structural formula of EM 1 is as follows: Among them, R is ethyl.

2. The preparation method according to claim 1, characterized in that, The specific operation of Step 1 is as follows: Step 1.1: Disperse the carbon source in deionized water, add CuCl solution, and mix evenly; Step 1.2: Centrifuge the mixture in Step 1.1, and dry the centrifuged product under vacuum conditions to obtain a black powder; Step 1.3: Put the black powder obtained in Step 1.2 into a sample holder, transfer it to a Joule flash heating system for flash heating treatment to obtain single-atom copper nanozyme FCuSA; the conditions for the flash heating treatment are as follows: the flash heating temperature is 2200 °C, the flash heating time is 10 s, and the chamber pressure is 10 -1 ~10 Pa.

3. The preparation method according to claim 1, characterized in that, In Step 1.1, the mass ratio of the carbon source to CuCl is 200:

60.

4. The preparation method according to claim 1, wherein The specific operation of Step 2 is as follows: Step 2.

1. Dissolve FCuSA and DSPE-PEG 2000 in a mixed solution composed of tetrahydrofuran and ethanol to form a homogeneous solution; Step 2.2: Dissolve EM 1 in dimethyl sulfoxide; Step 2.3: Under continuous ultrasonic treatment, inject the solutions in Step 2.1 and Step 2.2 into deionized water, and ultrasonically treat the obtained mixed solution in an ice-water bath for 5 - 20 min; Step 2.4: Remove tetrahydrofuran, ethanol, and dimethyl sulfoxide under vacuum, transfer the obtained aqueous solution to a centrifugal filter, and wash it with deionized water under centrifugation to remove free compounds to obtain a deionized aqueous solution of 1-FCuSA.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the FCuSA and DSPE-PEG 2000 to EM 1 is 0.45:10:0.

52.

6. A nanozyme catalytic activity self-reporting system prepared by the preparation method according to any one of claims 1 - 5.

7. Application of the nanozyme catalytic activity self-reporting system prepared by the preparation method according to any one of claims 1 - 5 in in vivo photoacoustic imaging analysis.

8. The application according to claim 7, wherein The nanozyme catalytic activity self-reporting system monitors the POD-like catalytic efficiency in tumors in real time through a ratio photoacoustic imaging signal.

9. Application of the nanozyme catalytic activity self-reporting system prepared by the preparation method according to any one of claims 1 - 5 in the preparation of tumor drugs.

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