Copper-based nanoszyme and preparation method and application thereof

Abstract

The present application relates to the biomedical field, and specifically provides a copper-based nano-enzyme, a preparation method and application thereof, aiming to solve the problems of diagnosis difficulty and lack of effective treatment drugs in the prior art for the treatment of primary sclerosing cholangitis (PSC) in clinical practice. To this end, the copper-based nano-enzyme comprises: a nano-enzyme carrier, which is a two-dimensional nanosheet formed by coordination bonds between copper ions, gallic acid and ursodeoxycholic acid; and a probe molecule, which is a phycobilin dye molecule connected to the surface of the nano-enzyme carrier by a covalent bond; wherein the copper-based nano-enzyme has enhanced fluorescence intensity after the action of alkaline phosphatase. The present application innovatively integrates alkaline phosphatase-responsive diagnosis, nano-enzyme catalytic treatment and liver targeting of ursodeoxycholic acid into one nano-platform, can perform treatment functions while performing specific imaging diagnosis on diseases, and provides a new strategy for the diagnosis and treatment of PSC and other diseases.

Description

Copper-based nanozymes, their preparation methods and applications Technical Field

[0001] This invention relates to the field of biomedicine, specifically providing a copper-based nanozyme, its preparation method, and its application. Background Technology

[0002] Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease whose exact pathophysiological mechanisms are not fully understood. The disease is characterized by progressive inflammation, fibrosis, and destruction of the intrahepatic and extrahepatic bile duct system, manifesting as multifocal bile duct strictures. As the disease progresses, persistent bile excretion impairment can lead to cholestasis, liver fibrosis, and may eventually develop into cirrhosis, liver failure, or even cholangiocarcinoma. Due to its unknown etiology, complex pathophysiology, challenging diagnosis, and high risk of malignancy, PSC is clinically known as the "black box of hepatology." PSC occurs in all age groups, sexes, and ethnicities worldwide. According to a meta-analysis of existing literature, the global reported incidence of PSC is 0 to 1.58 cases per 100,000 people, and the prevalence is 0 to 34.7 cases per 100,000 people, with a significant decreasing trend in prevalence from high to low latitudes worldwide.

[0003] Currently, clinical treatment options for PSC are very limited and ineffective. The treatment challenges mainly manifest in two aspects.

[0004] First, diagnosis is difficult. Currently, magnetic resonance cholangiopancreatography (MRCP), the preferred diagnostic method in clinical practice, is costly, has low sensitivity for early lesions, and cannot provide simultaneous intervention and treatment. Serological tests are cumbersome, endoscopic retrograde cholangiopancreatography (ERCP) is invasive and carries a high risk of complications, and liver biopsy is prone to sampling errors leading to false negatives. Therefore, there is an urgent clinical need to develop new, simple, rapid, and non-invasive diagnostic methods.

[0005] Second, there is a lack of effective treatments. Current treatments mainly focus on relieving symptoms and slowing disease progression, but cannot reverse or stop the disease process. Ursodeoxycholic acid (UDCA), as a first-line drug, can regulate bile secretion, but its efficacy is limited, it cannot target the lesion site, has a short circulation time, and low bioavailability, resulting in very limited long-term therapeutic effects for PSC. Immunosuppressants and biologics may be effective for some patients, but they lack support from large-scale clinical trials and may cause serious side effects. For end-stage patients, liver transplantation is the only effective treatment, but it faces serious problems such as donor shortages, high relapse rates after transplantation, and poor prognosis. At its root, current treatments fail to effectively intervene in the core pathological process of persistent inflammation in the liver parenchyma. This uncontrolled inflammatory response continuously aggravates hepatobiliary cell damage, leading to a vicious cycle of "inflammation-damage-cholestasis-more severe inflammation," which is the key reason for the current treatment difficulties.

[0006] In recent years, nanozymes—a class of nanomaterials with catalytic activity similar to natural enzymes—have provided a novel approach to overcoming the aforementioned challenges. Compared to natural enzymes, nanozymes offer significant advantages such as high catalytic efficiency, good stability, low preparation cost, and ease of functionalization, demonstrating great potential in the treatment of inflammatory diseases. They can directly intervene in the oxidative stress process by catalytically scavenging excess reactive oxygen species (ROS) and other inflammatory mediators, thereby breaking the vicious cycle at its source.

[0007] It is noteworthy that alkaline phosphatase (ALP), a hydrolase significantly overexpressed in pneumocystic sclerosis (PSC), has been confirmed by numerous studies not only as a key biomarker of cholestasis, but also as having a close correlation with the severity and prognosis of PSC due to its serum levels. Therefore, developing smart probes that specifically respond to ALP is an effective approach to achieving accurate diagnosis of PSC. However, currently, there is no integrated nanoplatform targeting ALP that combines therapeutic (e.g., ROS scavenging for anti-inflammatory effects) and diagnostic (e.g., fluorescence imaging) functions.

[0008] Accordingly, a new technical solution is needed in this field to solve the above-mentioned technical problems. Summary of the Invention

[0009] The present invention aims to address the aforementioned deficiencies in the prior art and at least partially provide an integrated solution that can simultaneously achieve highly sensitive diagnosis and efficient treatment of diseases such as primary sclerosing cholangitis.

[0010] In a first aspect, the present invention provides a copper-based nanozyme, wherein the copper-based nanozyme comprises: a nanozyme carrier, which is a two-dimensional nanosheet formed by coordination bonds of copper ions, gallic acid, and ursodeoxycholic acid; and a probe molecule, which is an anthocyanin dye molecule covalently linked to the surface of the nanozyme carrier; wherein the probe molecule is configured to be specifically activated by alkaline phosphatase as a substrate of alkaline phosphatase through its carried phosphate group.

[0011] In the preferred embodiment of the above copper-based nanozyme, the two-dimensional nanosheet is CuGA-UDCA NSs; and / or, the cyanine dye molecule is a CyP probe, the structural formula of which is shown in Formula 1:

[0012] .

[0013] In the preferred embodiment of the above copper-based nanozyme, the covalent bond is an amide bond.

[0014] In a second aspect, the present invention provides a method for preparing copper-based nanozymes, comprising the following steps: S1, providing an aqueous dispersion of a nanozyme carrier; S2, activating the carboxyl groups on the surface of the nanozyme carrier in the presence of a coupling agent; S3, adding a probe molecule to perform a coupling reaction; S4, after the reaction is completed, separating and purifying the obtained product to obtain the copper-based nanozyme; wherein, in step S1, the nanozyme carrier is a two-dimensional nanosheet formed by copper ions, gallic acid, and ursodeoxycholic acid through coordination bonds; and in step S3, the probe molecule is anthocyanin dye molecule.

[0015] In the preferred embodiment of the above preparation method, in step S1, the concentration of the aqueous dispersion of the nanozyme carrier is 0.5-2.0 mg / mL; and / or, in step S2, the coupling agent is a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) / N-hydroxysuccinimide (NHS); the amount of the EDCI / NHS mixture, calculated as EDCI, is 1.5-5.0 equivalents relative to the carboxyl groups on the surface of the nanozyme carrier; the molar ratio of EDCI to NHS in the EDCI / NHS mixture is 1:(1-1.5); and / or, in step S2, the activation temperature is 0-5℃ and the time is 0.5-1.5 hours; and / or, in step S3, the coupling reaction is carried out at room temperature for 12-36 hours; and / or, in step S3, the amount of the probe molecule added is 0.5-2.0 equivalents relative to the carboxyl groups on the surface of the nanozyme carrier.

[0016] In the preferred embodiment of the above preparation method, in step S1, the two-dimensional nanosheets are CuGA-UDCANSs.

[0017] In the preferred embodiment of the above preparation method, the preparation of CuGA-UDCA NSs includes the following steps: S11, dissolving gallic acid and ursodeoxycholic acid in an organic solvent and adding an alkali; S12, providing an aqueous solution of copper salt; S13, mixing the solutions from steps S11 and S12 and carrying out a solvothermal reaction; S14, after the reaction is completed, separating, washing and drying the obtained solid to obtain the CuGA-UDCA NSs.

[0018] In the preferred embodiment of the above preparation method, in step S11, the mass ratio of gallic acid to ursodeoxycholic acid is (8.0-8.8):1; and / or, in step S11, the organic solvent is N,N-dimethylformamide (DMF), and the base is triethylamine; and / or, in step S13, the temperature of the solvothermal reaction is 105-115℃, and the reaction time is 17-19 hours.

[0019] In the preferred embodiment of the above preparation method, in step S3, the cyanine dye molecule is a CyP probe, and its structural formula is shown in Formula 1:

[0020] .

[0021] In the preferred embodiment of the above preparation method, the preparation of the CyP probe includes the following steps: S31, Synthesis of compound 2: 2,3,3-trimethylindole reacts with 3-bromopropylamine hydrobromide in methanol to obtain compound 2, as shown in the following reaction formula:

[0022] ;

[0023] S32. Synthesis of Compound 3: Compound 2 is reacted with 2-chloro-3-(hydroxymethylene)-cyclohexenaldehyde in a 1-butanol / benzene mixed solvent by dehydration condensation to generate cyanine dye skeleton compound 3, as shown in the following reaction formula:

[0024] ;

[0025] S33. Synthesis of compound 4: The amino group of compound 3 is protected with Boc2O (di-tert-butyl dicarbonate) to generate compound 4, as shown in the following reaction formula:

[0026] ;

[0027] S34. Synthesis of compound 5: Compound 4 is reacted with 4-chloro-1,3-dihydroxybenzene under alkaline conditions to produce compound 5, as shown in the following reaction formula:

[0028] ;

[0029] S35. Synthesis of Compound 6: Compound 5 was phosphorylated using POCl3 (phosphorus oxychloride) to generate the phosphorylated intermediate compound 6, as shown in the following reaction formula:

[0030] ;

[0031] S36. Synthesis of CyP probe: Under acidic conditions, the Boc (tert-butyloxycarbonyl) protecting group of compound 6 was removed to obtain the CyP probe, as shown in the following reaction formula:

[0032] .

[0033] In the preferred embodiment of the above preparation method, in step S31, the molar ratio of 2,3,3-trimethylindole to 3-bromopropylamine hydrobromide is 1:(1.1-1.3), and the reaction temperature is 95-105℃; and / or, in step S32, the molar ratio of compound 2 to 2-chloro-3-(hydroxymethylene)-cyclohexenecarbaldehyde is (2.0-2.2):1; the dehydration condensation reaction is first refluxed at 130-140℃ until the dehydration is completed, and then the temperature is lowered to 70-80℃ and stirring is continued for 5-7 hours; and / or, in step S34, the molar ratio of compound 4 to 4-chloro-1,3-dihydroxybenzene is 1:(2.8-3.2), and the reaction temperature is 70-80℃; and / or, in step S35, the phosphorylation reaction is carried out at 0-5℃.

[0034] In a third aspect, the present invention provides the use of the copper-based nanozyme described in the first aspect or the copper-based nanozyme prepared by the preparation method described in the second aspect in the preparation of products for any of the following purposes: (a) a diagnostic reagent for detecting alkaline phosphatase activity in in vitro solutions or isolated organs; (b) a diagnostic reagent for assessing the severity or treatment progress of diseases related to alkaline phosphatase overexpression at the in vivo level by fluorescence imaging; and (c) a medicament for treating diseases related to alkaline phosphatase overexpression.

[0035] In the preferred embodiment of the above application, the disease is a hepatobiliary disease.

[0036] In the preferred embodiment of the above application, the hepatobiliary disease is primary sclerosing cholangitis.

[0037] With the above technical solution, the present invention has the following beneficial effects: (1) Functional integration: It innovatively integrates three major functions, namely alkaline phosphatase (ALP) responsive diagnosis, nanozyme catalytic therapy and ursodeoxycholic acid (UDCA) liver targeting, into a nanoplatform, which can perform therapeutic functions while performing specific imaging diagnosis of diseases, providing a new strategy for the diagnosis and treatment of diseases such as primary sclerosing cholangitis (PSC); (2) Advanced diagnostic mechanism: Based on the specific activation of CyP probe by ALP, a significant fluorescence on-off signal is generated. This signal amplification mechanism lays the foundation for achieving high sensitivity and high specificity optical imaging detection of lesions; (3) Strong therapeutic targeting: The nanozyme carrier itself has catalytic therapeutic function. At the same time, the introduction of UDCA not only improves the biocompatibility of the material, but more importantly, it endows it with inherent liver targeting ability, thereby hoping to improve treatment efficiency and reduce systemic side effects; (4) Optimized and controllable preparation process: The present invention has systematically optimized the preparation method of nanozyme carrier material and final product, and established a series of key process parameters. The method has clear steps and good reproducibility, providing a reliable guarantee for the quality control and large-scale preparation of the copper-based nanozyme. Attached Figure Description

[0038] The preferred embodiments of the present invention are described below with reference to the accompanying drawings, in which:

[0039] Figure 1 is a process flow diagram of the preparation method of the copper-based nanozyme of the present invention;

[0040] Figure 2 is a flowchart of the preparation process of CuGA-UDCA NSs of the present invention;

[0041] Figure 3 is a flowchart of the preparation process of the CyP probe of the present invention;

[0042] Figure 4 shows the infrared spectrum of CuGA-UDCA NSs;

[0043] Figure 5 is the proton NMR spectrum of the ALP-responsive fluorescent probe CyP.

[0044] Figure 6 is the carbon NMR spectrum of the ALP-responsive fluorescent probe CyP.

[0045] Figure 7 shows the mass spectrum of the ALP-responsive fluorescent probe CyP.

[0046] Figure 8 is a verification diagram of the in vitro response of probe CyP to ALP;

[0047] Figure 9 shows the results of HPLC in vitro verification of the ability of the probe CyP to respond to ALP.

[0048] Figure 10 shows the hydrated particle size and potential of CuGA NSs;

[0049] Figure 11 is an electron microscope image of CuGA NSs;

[0050] Figure 12 is the EDS spectrum of CuGA NSs;

[0051] Figure 13 is the infrared spectrum of CuGA NSs;

[0052] Figure 14 shows the XPS spectrum of CuGA NSs;

[0053] Figure 15 shows the results of in vitro validation of CyP-CuGA-UDCA NSs' response to ALP;

[0054] Figure 16 is a small animal in vivo imaging result that verifies the ability of CyP-CuGA-UDAC NSs to luminesce in mice that respond to PSC treatment and ALP levels.

[0055] Figure 17 shows the fluorescence intensity results of various organs in PSC mice after tail vein injection of CyP-CuGA-UDAC NSs;

[0056] Figure 18 shows the liver treatment effects of PSC mice after different treatments;

[0057] Figure 19 shows the changes in AST, ALT, ALP, TBA, and TBIL levels in PSC mice after different treatments.

[0058] Figure 20 shows the MASSON staining results of the livers of PSC mice after different treatments;

[0059] Figure 21 shows the K19 / NIK immunofluorescence images of intrahepatic bile duct injury in PSC mice after different treatments;

[0060] Figure 22 shows the HE staining results of the livers of PSC mice after different treatments;

[0061] Figure 23 shows the changes in serum inflammatory factor levels in PSC mice after different treatments;

[0062] Figure 24 shows the immunohistochemical results of F4 / 80 liver cells in PSC mice after different treatments;

[0063] Figure 25 shows the results of DHE staining of the liver of PSC mice after different treatments;

[0064] Figure 26 shows the TUNEL staining results of the livers of PSC mice after different treatments. Detailed Implementation

[0065] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0066] CuGA-UDCA NSs: This refers to the copper-gallic acid-ursodeoxycholic acid nanosheets synthesized by the solvothermal method in this invention. "Cu" represents copper ions, "GA" represents gallic acid, "UDCA" represents ursodeoxycholic acid, and "NSs" is an abbreviation for nanosheets. This material is a two-dimensional nanostructure formed by the self-assembly of the three components through coordination bonds.

[0067] CyP probe: refers to a cyanine dye probe synthesized in this invention that responds to alkaline phosphatase (ALP) and has the structure shown in Formula 1.

[0068] Based on the problems of diagnostic difficulties and lack of effective treatment drugs in the clinical treatment of PSC as pointed out in the background art, the present invention provides an integrated solution that can simultaneously achieve highly sensitive diagnosis and efficient treatment of diseases such as primary sclerosing cholangitis.

[0069] Specifically, in a first aspect, the present invention provides a copper-based nanozyme, wherein the copper-based nanozyme comprises: a nanozyme carrier, which is a two-dimensional nanosheet formed by coordination bonds of copper ions, gallic acid, and ursodeoxycholic acid; and a probe molecule, which is an anthocyanin dye molecule covalently linked to the surface of the nanozyme carrier; wherein the probe molecule is configured to be specifically activated by alkaline phosphatase as a substrate of alkaline phosphatase through its carried phosphate group.

[0070] The ingenious design of the copper-based nanozyme provided by this invention lies in:

[0071] The innovation of the nanozyme carrier: The nanozyme carrier is a two-dimensional nanosheet formed by coordination bonds between copper ions, gallic acid (GA), and ursodeoxycholic acid. The two-dimensional nanosheet formed by the coordination of copper ions and gallic acid (GA) is itself a highly efficient copper-based nanozyme, possessing catalytic activities such as peroxidase-like activity, and can be used for catalytic therapy. The introduction of ursodeoxycholic acid (UDCA) improves the biocompatibility of the material.

[0072] The probe's intelligence lies in its covalently linked cyanine dye probe (CyP) responsive to alkaline phosphatase (ALP) to the surface of the nanozyme carrier. This probe can be specifically activated by ALP, and the hydrolysis of its phosphate group enables a switching of fluorescence from "off" to "on," resulting in a significant enhancement of fluorescence intensity. This "on-off" imaging mechanism ensures a high signal-to-noise ratio and high sensitivity at the lesion site.

[0073] Synergistic Functions: This invention integrates three major functions: catalytic therapy of nanozymes, liver targeting of UDCA, and ALP response diagnosis of CyP. It can target the lesion area and simultaneously achieve precise diagnosis of ALP-triggered disease and efficient treatment mediated by catalysis, effectively overcoming the technical bottleneck of the disconnect between diagnosis and treatment in traditional diagnostic and treatment strategies.

[0074] It is understood that, based on the concept of this invention, the nanoenzyme carrier is not strictly limited to gallic acid and ursodeoxycholic acid. Those skilled in the art will readily conceive of using other polyphenolic acids (such as tannic acid, ellagic acid, etc.) or bile acids (such as chenodeoxycholic acid, cholic acid, etc.) with similar coordination capabilities for substitution or partial substitution, and these obvious substitutions should all be considered to fall within the scope of this invention.

[0075] Similarly, the core of a probe molecule lies in the presence of a phosphate group that can be recognized by alkaline phosphatase. Therefore, any fluorescent dye molecule containing such a group, including but not limited to cyanine, rhodamine, and fluorescein, can be considered as a potential probe molecule suitable for this invention based on the same detection mechanism.

[0076] In some specific embodiments, the nanozyme carrier is CuGA-UDCA NSs; and / or, the cyanine dye molecule is a CyP probe with the structural formula shown in Formula 1:

[0077] .

[0078] In some embodiments, the covalent bond is an amide bond.

[0079] Furthermore, in a second aspect, the present invention provides a method for preparing the copper-based nanozyme, as shown in Figure 1. The preparation method includes the following steps: S1, providing an aqueous dispersion of the nanozyme carrier; S2, activating the carboxyl groups on the surface of the nanozyme carrier in the presence of a coupling agent; S3, adding probe molecules to perform a coupling reaction; S4, after the reaction is completed, separating and purifying the obtained product to obtain the copper-based nanozyme; wherein, the nanozyme carrier is a two-dimensional nanosheet formed by copper ions, gallic acid, and ursodeoxycholic acid through coordination bonds; and the probe molecule is an anthocyanin dye molecule.

[0080] The method for preparing the copper-based nanozyme provided by this invention, through careful step design and optimization of key process parameters, as well as a modular "nanozyme carrier synthesis-probe synthesis-controlled coupling" strategy, has outstanding advantages such as clear steps, mild conditions, good reproducibility, and ease of scale-up, creating favorable conditions for the large-scale preparation and clinical application of this nanozyme material. The key steps and preferred parameters in the preparation method will be described in detail below.

[0081] In some specific embodiments, in step S1, the concentration of the aqueous dispersion of the nanozyme carrier is 0.5-2.0 mg / mL. For example, it can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2.0 mg / mL, or any value within the range.

[0082] In some specific embodiments, in step S2, the coupling agent is an EDCI / NHS mixture. EDCI is the main coupling agent; NHS is the activating additive. This combination can efficiently convert carboxyl groups into active esters for subsequent coupling reactions with amino groups.

[0083] In some specific embodiments, in step S2, the amount of the EDCI / NHS mixture, calculated as EDCI, is 1.5-5.0 equivalents relative to the carboxyl groups on the surface of the nanozyme carrier. For example, it can be 1.5 equivalents, 2 equivalents, 3 equivalents, 4 equivalents, 5 equivalents, or any value within the range.

[0084] In some specific embodiments, in step S2, the molar ratio of EDCI to NHS in the EDCI / NHS mixture is 1:(1-1.5). For example, it can be 1:1, 1:1.2, 1:1.5, or any value within the range.

[0085] In some specific embodiments, in step S2, the activation temperature is 0-5℃ and the time is 0.5-1.5 hours. For example, the activation temperature can be 0℃, 1℃, 2℃, 3℃, 4℃, 5℃ or any value within the range; the time can be 0.5 hours, 1 hour, 1.5 hours or any value within the range.

[0086] In some specific embodiments, in step S3, the coupling reaction is carried out at room temperature for a reaction time of 12-36 hours. For example, the reaction time is 12 hours, 20 hours, 24 hours, 30 hours, 36 hours, or any value within the range.

[0087] In some specific embodiments, in step S3, the amount of the probe molecule added is 0.5-2.0 equivalents relative to the carboxyl groups on the surface of the nanozyme carrier. For example, it can be 0.5 equivalents, 1 equivalent, 1.0 equivalent, 1.5 equivalent, 2 equivalents, or any value within the range.

[0088] In some specific embodiments, in step S1, the two-dimensional nanosheets are CuGA-UDCA NSs.

[0089] In some preferred embodiments, please refer to Figure 2. The preparation of CuGA-UDCA NSs includes the following steps: S11, dissolving gallic acid and ursodeoxycholic acid in an organic solvent and adding a base; S12, providing an aqueous solution of copper salt; S13, mixing the solutions from steps S11 and S12 and carrying out a solvothermal reaction; S14, after the reaction is completed, separating, washing and drying the obtained solid to obtain CuGA-UDCA NSs.

[0090] In some specific embodiments, in step S11, the mass ratio of gallic acid to ursodeoxycholic acid is (8.0-8.8):1. For example, it can be 8.0:1, 8.2:1, 8.5:1, 8.4:1, 8.6:1, 8.8:1, or any value within the range.

[0091] In some specific embodiments, in step S11, the organic solvent is DMF and the base is triethylamine (TEA).

[0092] In some specific embodiments, in step S13, the temperature of the solvothermal reaction is 105-115°C, and the reaction time is 17-19 hours. For example, the temperature of the solvothermal reaction can be 105°C, 110°C, 115°C, or any value within the range; the reaction time can be 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19 hours, or any value within the range.

[0093] In some specific embodiments, in step S3, the cyanine dye molecule is a CyP probe, the structural formula of which is shown in Formula 1 above.

[0094] In some preferred embodiments, referring to Figure 3, the preparation of the CyP probe includes the following steps: S31, Synthesizing compound 2: Reacting 2,3,3-trimethylindole with 3-bromopropylamine hydrobromide in methanol to obtain compound 2; S32, Synthesizing compound 3: Performing a dehydration condensation reaction of compound 2 with 2-chloro-3-(hydroxymethyl)-cyclohexenecarboxaldehyde in a 1-butanol / benzene mixed solvent to generate cyanine dye skeleton compound 3; S33, Synthesizing compound 4: Protecting the amino group of compound 3 with Boc2O to generate compound 4; S34, Synthesizing compound 5: Reacting compound 4 with 4-chloro-1,3-dihydroxybenzene under alkaline conditions to generate compound 5; S35, Synthesizing compound 6: Phosphorylating compound 5 with POCl3 (phosphorus oxychloride) to generate phosphorylated intermediate compound 6; S36, Synthesizing CyP probe: Removing the Boc protecting group of compound 6 under acidic conditions to obtain the CyP probe.

[0095] Its synthetic route is as follows:

[0096] .

[0097] This invention successfully constructed the CyP probe via the aforementioned modular synthetic route: Steps S31-S32 constructed the cyanine dye core framework, serving as a fluorescent signal reporter group, through a condensation reaction; Step S33 employed a Boc protecting group strategy to ensure the selectivity and efficiency of the subsequent nucleophilic substitution reaction (S34), thereby precisely introducing a phenolic hydroxyl structure that can serve as an ALP recognition and response site; finally, the phosphorylation reaction in Step S35 converted the phenolic hydroxyl group into a phosphate ester, making the probe itself a specific substrate for ALP and realizing its "activated" function. This route is rationally designed, with each step working synergistically to ensure the structural correctness and functional integrity of the final CyP probe.

[0098] In some specific embodiments, in step S31, the molar ratio of 2,3,3-trimethylindole to 3-bromopropylamine hydrobromide is 1:(1.1-1.3), and the reaction temperature is 95-105°C. For example, the molar ratio can be 1:1.1, 1:1.2, 1:1.3, or any value within the range; the reaction temperature can be 95°C, 100°C, 105°C, or any value within the range.

[0099] In some specific embodiments, in step S32, the molar ratio of compound 2 to 2-chloro-3-(hydroxymethylene)-cyclohexenecarboxaldehyde is (2.0-2.2):1; the dehydration condensation reaction is first refluxed at 130-140°C until dehydration is complete, then the temperature is lowered to 70-80°C and stirring is continued for 5-7 hours. For example, the molar ratio can be 2:1, 2.1:1, 2.2:1, or any value within the range; the dehydration condensation reaction is first refluxed at 135°C until dehydration is complete, then the temperature is lowered to 75°C and stirring is continued for 6 hours.

[0100] In some specific embodiments, in step S34, the molar ratio of compound 4 to 4-chloro-1,3-dihydroxybenzene is 1:(2.8-3.2), and the reaction temperature is 70-80°C. For example, the molar ratio can be 1:2.8, 1:3.0, 1:3.2, or any value within the range mentioned above; the reaction temperature can be 70°C, 75°C, 80°C, or any value within the range mentioned above.

[0101] In some specific embodiments, the phosphorylation reaction in step S35 is carried out at 0-5°C. For example, it can be 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, or any value within the range.

[0102] In a third aspect, the present invention provides the use of the copper-based nanozyme described in the first aspect or the copper-based nanozyme prepared by the preparation method described in the second aspect in the preparation of products for any of the following purposes: (a) a diagnostic reagent for detecting alkaline phosphatase activity in in vitro solutions or isolated organs; (b) a diagnostic reagent for assessing the severity or treatment progress of diseases related to alkaline phosphatase overexpression at the in vivo level by fluorescence imaging; and (c) a medicament for treating diseases related to alkaline phosphatase overexpression.

[0103] In some preferred embodiments, the disease associated with alkaline phosphatase overexpression is a hepatobiliary disease.

[0104] In some specific embodiments, the hepatobiliary disease is primary sclerosing cholangitis (PSC).

[0105] The copper-based nanozyme provided by this invention achieves an organic integration of diagnostic and therapeutic functions. As shown in the following embodiments and figures, this copper-based nanozyme not only exhibits a sensitive fluorescent response to alkaline phosphatase activity at both in vitro and in vivo levels, enabling real-time monitoring of disease progression, but also demonstrates significant therapeutic effects in a mouse model of primary sclerosing cholangitis (PSC). Its application covers the entire medical process from in vitro detection and in vivo assessment to in vivo treatment, providing a novel and clinically promising solution for the precision diagnosis and treatment of alkaline phosphatase-related diseases, especially PSC.

[0106] The copper-based nanozyme of the present invention, its preparation method, and its application are described below through several specific embodiments.

[0107] Example 1 Preparation of CyP-CuGA-UDCA NSs

[0108] I. Preparation and Characterization of CuGA-UDCA NSs

[0109] S11. Weigh gallic acid (170 mg) and ursodeoxycholic acid (20 mg) and dissolve them separately in 15 mL of DMF. Add 20 μL of triethylamine and mix. Sonicate in a water bath (300 W, 10 minutes).

[0110] S12. Weigh out 250 mg of anhydrous copper sulfate and dissolve it in 10 mL of ultrapure water;

[0111] S13. Slowly mix the solutions from steps S11 and S12 into a 100mL round-bottom flask, stir at room temperature for half an hour, transfer to a high-pressure reactor, and place in an oven at 110℃ for 18 hours.

[0112] S14. After the reaction is complete, centrifuge and discard the supernatant. Wash the supernatant three times with water and anhydrous ethanol respectively, and then freeze-dry under vacuum to obtain CuGA-UDCA NSs.

[0113] The infrared spectrum of CuGA-UDCA NSs is shown in Figure 4. A symmetric stretching vibration peak of the methylene group (2926 cm⁻¹) appeared in the infrared spectrum of CuGA-UDCA NSs. -1 ) and antisymmetric extension vibration peak (2865cm) -1 The methylene group is derived from UDCA, demonstrating the successful loading of UDCA onto CuGA-UDCA NSs.

[0114] II. Synthesis of alkaline phosphatase-responsive CyP probes

[0115] S31. Synthesis of Compound 2: 2,3,3-Trimethylindole (3.18 g, 20.0 mmol) was dissolved in an appropriate amount of methanol, followed by the addition of 1.2 equivalents of 3-bromopropylamine hydrobromide (5.25 g, 24.0 mmol). The mixture was stirred at 100 °C for 4 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation to obtain the crude product. The crude product was recrystallized in a cold acetone / methanol mixed solvent (V / V = 5:1), precipitating a pink solid. The obtained solid was filtered, washed, and dried to obtain Compound 2 (2.86 g), with a yield of 65.8%.

[0116] S32. Synthesis of Compound 3: A mixture of compound 2 (2 g, 9.2 mmol) and 2-chloro-3-(hydroxymethylene)-cyclohexenaldehyde (794 mg, 4.6 mmol) was dissolved in 70 mL of a 1-butanol / benzene mixed solvent (V / V = 7:3). Water generated during the reaction was continuously removed using a water separator under reflux and stirring at 135 °C. After water removal, the reaction temperature was lowered to 75 °C and maintained at 75 °C with stirring for another 6 h. After the reaction was complete, the solvent was removed, and the mixture was purified by silica gel column chromatography to obtain a gold-green solid compound 3 (1757 mg), with a yield of 66.9%.

[0117] S33. Synthesis of Compound 4: Di-tert-butyl dicarbonate (920 mg, 4.2 mmol) and TEA (310 μL, 4.2 mmol) were added to an ethanol solution of Compound 3 (1 g, 1.75 mmol). The reaction mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC. After the reaction was complete, the solvent was removed by vacuum distillation, and the residual solid was dissolved in EA, washed three times with saturated brine, dried over anhydrous Na2SO4, filtered, and the organic solvent was removed by rotary evaporation to obtain Compound 4 (1232 mg), with a yield of 91.4%, which can be used directly in subsequent reactions.

[0118] S34. Synthesis of Compound 5: 4-Chloro-1,3-dihydroxybenzene (451 mg, 3.12 mmol) and TEA (440 μL, 3.12 mmol) were dissolved in 3 mL of anhydrous DMF, and the mixture was stirred at room temperature for 15 min under a nitrogen atmosphere. Then, compound 4 (800 mg, 1.04 mmol) was dissolved in 5 mL of anhydrous DMF and added dropwise to the mixture. The reaction was heated to 75 °C and stirred at 75 °C for 5 h. Purification by silica gel column chromatography yielded a blue-green solid compound 5 (402 mg), with a yield of 68.7%.

[0119] S35. Synthesis of Compound 6: POCl3 (456 mg, 3 mmol) and anhydrous CH2Cl2 (20 mL) were added sequentially to the reaction flask. Compound 5 (562 mg, 1 mmol) and N,N-diisopropylethylamine (DIEA) (650 mg, 5 mmol) were dissolved in 10 mL of CH2Cl2 and added to the reaction flask. The mixture was stirred at 0 °C for 3 h. After TLC showed that the reaction was basically complete, the reaction solution was concentrated to 1 / 3 under reduced pressure at 0 °C. Then, 20 mL of a mixed solution of acetonitrile and water (V / V = 3:1) was added to the reaction solution, and the reaction system was heated to 20 °C and stirred for 24 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent. The residue was dissolved in CH2Cl2, and purified by silica gel column chromatography using a wet loading method to finally obtain the target product compound 6, which was directly used in the next reaction step.

[0120] S36. Synthesis of CyP probe: Compound 6 and 4M HCl / 1,4-dioxane (5 mL) were added to the reaction flask. The mixture was stirred at 20 °C for 1 h. After the reaction was completed, the reaction solution was directly evaporated under reduced pressure at low temperature to obtain compound 7, i.e., the CyP probe. The two-step yield was 48.3%.

[0121] The CyP probe structure was characterized by proton NMR, carbon NMR, and mass spectrometry. The results are shown in Figures 5, 6, and 7, confirming the correctness of the CyP structure.

[0122] The ability of the CyP probe to emit fluorescence in response to ALP in vitro was tested. The method was as follows: 100 U / L of ALP was added to 2 mL of 10 μM CyP solution, mixed thoroughly, and the fluorescence intensity change at 710 nm was immediately monitored using a fluorescence spectrophotometer over 1 hour. Additionally, 0, 2, 4, 8, 16, 32, 64, 128, and 256 U / L of ALP were added to 2 mL of 10 μM CyP solution, respectively. After mixing thoroughly and reacting for half an hour, the fluorescence intensity change was immediately monitored using a fluorescence spectrophotometer.

[0123] The results are shown in Figure 8. Figure a shows the change in fluorescence intensity with the mixing time of CyP and ALP; Figure b shows the change in fluorescence intensity with the increase of ALP concentration.

[0124] As shown in Figure 8, the fluorescence intensity gradually increased with increasing mixing time of CyP and ALP and increasing ALP concentration. The HPLC monitoring results are shown in Figure 9, indicating that the addition of ALP can convert the CyP probe to Cy.

[0125] III. Preparation of CyP-CuGA-UDCA NSs

[0126] S1. Disperse the pre-prepared CuGA-UDCA NSs in water to obtain an aqueous dispersion of CuGA-UDCA NSs;

[0127] S2. Add excess EDCI and NHS to the aqueous dispersion of CuGA-UDCA NSs under ice bath conditions to activate the carboxyl groups on the particle surface for 1 hour.

[0128] S3, then CyP was added and stirred at room temperature for 24 hours to carry out the coupling reaction;

[0129] S4. After the reaction is complete, centrifuge at 10,000 rpm for ten minutes, wash with water three times to remove free CyP, and finally sonicate to disperse CyP-CuGA-UDCA NSs.

[0130] Example 2 Preparation of CyP-CuGA-UDCA NSs

[0131] The difference between this embodiment and Embodiment 1 is that the synthesis of CuGA-UDCA NSs and its coupling parameters with CyP were changed.

[0132] I. Preparation of CuGA-UDCA NSs

[0133] Specifically, in step S11, the mass ratio of gallic acid to ursodeoxycholic acid is controlled at 8.0:1. In step S13, the temperature of the solvothermal reaction is controlled at 105°C, and the reaction time is controlled at 19 hours. The remaining steps and parameters are the same as in Example 1.

[0134] II. Preparation of CyP-CuGA-UDCA NSs

[0135] Specifically, in step S1, the concentration of the nanozyme carrier aqueous dispersion is controlled at 0.5 mg / mL. In step S2, the activation temperature is controlled at 0°C, and the activation time is controlled at 1.5 hours; in the EDCI / NHS mixture, the amount of EDCI added is 1.5 equivalents of the carboxyl groups on the surface of the nanozyme carrier, and the molar ratio of EDCI to NHS is 1:1. In step S3, the amount of probe molecule CyP added is controlled at 0.5 equivalents of the carboxyl groups, and the coupling reaction time is 36 hours. The remaining steps and parameters are the same as in Example 1.

[0136] Example 3 Preparation of CyP-CuGA-UDCA NSs

[0137] The difference between this embodiment and Embodiment 1 is that the synthesis of CuGA-UDCA NSs and its coupling parameters with CyP were changed.

[0138] I. Preparation of CuGA-UDCA NSs

[0139] Specifically, in step S11, the mass ratio of gallic acid to ursodeoxycholic acid is controlled at 8.8:1. In step S13, the temperature of the solvothermal reaction is controlled at 115°C, and the reaction time is controlled at 17 hours. The remaining steps and parameters are the same as in Example 1.

[0140] II. Preparation of CyP-CuGA-UDCA NSs

[0141] Specifically, in step S1, the concentration of the nanozyme carrier aqueous dispersion is controlled at 2.0 mg / mL. In step S2, the activation temperature is controlled at 5°C, and the activation time is controlled at 0.5 hours; in the EDCI / NHS mixture, the amount of EDCI added is 5.0 equivalents of the carboxyl groups on the surface of the nanozyme carrier, and the molar ratio of EDCI to NHS is 1:1.5. In step S3, the amount of probe molecule CyP added is controlled at 2.0 equivalents of the carboxyl groups, and the coupling reaction time is 12 hours. The remaining steps and parameters are the same as in Example 1.

[0142] Example 4 Preparation of CyP-CuGA-UDCA NSs

[0143] The difference between this embodiment and Embodiment 1 is that the key parameters in the CyP probe synthesis step have been changed.

[0144] In step S31, the molar ratio of 2,3,3-trimethylindole to 3-bromopropylamine hydrobromide is controlled to be 1:1.1, and the reaction temperature is 95°C.

[0145] In step S32, the molar ratio of compound 2 to 2-chloro-3-(hydroxymethyl)-cyclohexenecarboxaldehyde is controlled to be 2.0:1; the dehydration condensation reaction is first refluxed at 130°C to remove water, and then the temperature is lowered to 70°C and stirred for 7 hours.

[0146] In step S34, the molar ratio of compound 4 to 4-chloro-1,3-dihydroxybenzene is controlled to be 1:2.8, and the reaction temperature is 70°C.

[0147] In step S35, the phosphorylation reaction is controlled to be carried out at 0°C.

[0148] Example 5 Preparation of CyP-CuGA-UDCA NSs

[0149] The difference between this embodiment and Embodiment 1 is that the key parameters in the CyP probe synthesis step have been changed.

[0150] In step S31, the molar ratio of 2,3,3-trimethylindole to 3-bromopropylamine hydrobromide is controlled to be 1:1.3, and the reaction temperature is 105℃.

[0151] In step S32, the molar ratio of compound 2 to 2-chloro-3-(hydroxymethyl)-cyclohexene formaldehyde is controlled to be 2.2:1; the dehydration condensation reaction is first refluxed at 140°C to remove water, and then the temperature is lowered to 80°C and stirred for 5 hours.

[0152] In step S34, the molar ratio of compound 4 to 4-chloro-1,3-dihydroxybenzene is controlled to be 1:3.2, and the reaction temperature is 80°C.

[0153] In step S35, the phosphorylation reaction is controlled to be carried out at 5°C.

[0154] Comparative Example 1 Preparation and characterization of CuGA NSs

[0155] Gallic acid (170 mg) was dissolved in 15 mL of LDM, and 20 μL of triethylamine was added. The mixture was then sonicated in a water bath (300 W, 10 min). Copper sulfate pentahydrate (250 mg) was dissolved in 10 mL of ddH2O. The above solutions were slowly mixed into a 100 mL round-bottom flask and stirred at room temperature for half an hour. The mixture was then transferred to a high-pressure reactor and placed in an oven to be slowly heated to 110 °C for 18 h. After the reaction was completed, the crude product was transferred to a centrifuge tube, centrifuged, and the supernatant was discarded. The product was washed three times with water and three times with anhydrous ethanol, and then freeze-dried under vacuum to obtain CuGA NSs. It was stored at 4 °C for later use.

[0156] The results of hydration particle size and potential measurements are shown in Figure 10, where Figure a shows the hydration particle size of CuGA NSs, and Figure b shows the potential of CuGA NSs. Figure 10 shows that its size is around 100 nm, and the surface is negatively charged (Figure 10, Figure b). As shown in Figure 11, CuGA NSs was observed to have a plate-like structure under a transmission electron microscope. The EDS spectrum results shown in Figure 12 show that CuGA NSs mainly contains three elements: C, O, and Cu. As shown in the infrared spectral analysis results in Figure 13, gallic acid (GA) is at 3268 cm⁻¹. -1 A broad absorption peak appeared nearby, which was caused by the stretching vibration of the phenolic hydroxyl group in gallic acid, while this absorption peak almost disappeared in CuGA NSs; 1016 cm⁻¹ in GA -1 The CO tensile vibration peak at that location shifts to 1050 cm⁻¹ in CuGA NSs. -1 The presence of gallic acid at this location confirms the successful coordination of gallic acid with copper ions. The XPS results are shown in Figure 14, where figure a is the total XPS elemental spectrum; figure b is the XPS analysis spectrum of Cu2p. Figure 14 indicates the presence of Cu in CuGA NSs. 2+ and Cu + Two valence states.

[0157] Comparative Example 2 Preparation of CuGA-UDCA NSs

[0158] Same as Example 1.

[0159] Comparative Example 3 Preparation of CyP-CuGA NSs

[0160] I. Preparation of CuGA NSs

[0161] Same as Comparative Example 1.

[0162] II. Synthesis of the corresponding CyP probe for alkaline phosphatase

[0163] Same as Example 1.

[0164] III. Preparation of CyP-CuGA NSs

[0165] The steps are exactly the same as in Example 1, except that CuGA-UDCA NSs in step S1 is replaced with CuGA NSs.

[0166] Experimental Example 1 Test the ability of CyP-CuGA-UDCA NSs to respond to ALP and light up fluorescence.

[0167] 100 U / L ALP was added to CyP-CuGA-UDCA NSs (prepared in Example 1) with a concentration of 1 mg / mL. After reacting for half an hour, the fluorescence intensity of CyP-CuGA-UDCA NSs with and without ALP was measured using a fluorescence spectrophotometer. The results are shown in Figure 15. The fluorescence intensity of the mixed solution was significantly enhanced with the addition of ALP.

[0168] The CyP-CuGA-UDCA NSs prepared in other examples were also subjected to the above tests, and the results were similar, with a significant increase in fluorescence intensity after ALP treatment.

[0169] Experimental Example 2 Testing the ability of CyP-CuGA-UDCA NSs to respond to ALP in PSC mice for in vivo imaging.

[0170] Male C57BL / 6 mice (purchased from Guangdong Yaokang, weighing approximately 25g) were used in the experiment. Mice were randomly divided into three groups: a PBS group, a CyP-CuGA NSs treatment group, and a CyP-CuGA-UDCA NSs treatment group, with three mice in each group. Except for the PBS group, the other two groups were fed a diet containing 0.1% DDC for 10 days beforehand to establish the PSC mouse model. After modeling, the mice were treated with the corresponding drugs. Specifically: PBS group: intravenous injection of PBS; CyP-CuGA NSs treatment group: CyP-CuGA NSs (prepared in Comparative Example 3) was injected into the mice via the tail vein, once every two days at a dose of 10mg / kg; CyP-CuGA-UDCA NSs treatment group: CyP-CuGA-UDCA NSs (prepared in Example 1) was injected into the mice via the tail vein, once every two days at a dose of 10mg / kg. One hour after tail vein administration on days 0, 5, and 10 of treatment, the fluorescence intensity in mice was detected using a small animal in vivo imaging system to assess the severity of PSC disease and treatment progress.

[0171] As shown in Figure 16, the liver fluorescence was strongest on day 0 of treatment, corresponding to the peak ALP level. The signal significantly weakened on day 5, indicating disease remission. By day 10, the fluorescence was no longer significant, indicating a significant treatment effect. Notably, the fluorescence decay in the CyP-CuGA-UDCA NSs group was faster than that in the CyP-CuGA NSs group at all time points, confirming its superior treatment efficacy.

[0172] The imaging of the ex vivo organs is shown in Figure 17. Figure a shows the fluorescence distribution of each organ in each treatment group of mice; Figure b shows the quantification of fluorescence signals of each organ in each group of mice.

[0173] As shown in Figure 17, the livers of the two groups of treated mice exhibited the strongest fluorescence, and the fluorescence signal of the CyP-CuGA-UDCA NSs group was significantly lower than that of the CyP-CuGA NSs group, consistent with the results of in vivo imaging.

[0174] The above results demonstrate that CyP-CuGA-UDCA NSs can improve cholestatic hepatitis and monitor the progression of cholestatic hepatitis.

[0175] Experimental Example 3 Detection of the effect of CuGA-UDCA NSs on improving liver edema in PSC mice

[0176] Male C57BL / 6 mice (purchased from Guangdong Yaokang, weighing approximately 25g) were used in the experiment. Mice were randomly divided into 5 groups: PBS group, DDC model group, CuGA NSs treatment group, UDCA treatment group, and CuGA-UDCA NSs treatment group, with 6 mice in each group. Except for the PBS group, the other four groups were fed a diet containing 0.1% DDC for 10 days to establish the PSC mouse model. After modeling, the mice were treated with the corresponding drugs. Specifically, the PBS and DDC groups received intravenous PBS; the CuGA NSs treatment group received intravenous 10mg / kg CuGANSs (prepared in Comparative Example 1); the UDCA treatment group received intravenous 10mg / kg UDCA; and the CuGA-UDCA NSs treatment group received intravenous 10mg / kg CuGA-UDCA NSs (prepared in Comparative Example 2), administered every 2 days for approximately 10 days. After treatment, the mice were sacrificed, and their livers were harvested for observation of size, appearance, and weight.

[0177] The results are shown in Figure 18, where Figure a shows the livers of mice in each group; and Figure b shows the liver weights of mice in each group.

[0178] As shown in Figure 18, the liver size, appearance and weight of mice treated with CuGA-UDCA NSs were closest to those of mice in the normal group, indicating that CuGA-UDCA NSs significantly improved liver edema in PSC mice.

[0179] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself has a significant effect on improving liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it is reasonable to infer that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain a similar effect on improving liver edema in PSC mice in vivo.

[0180] Test Example 4 Detecting the effect of CuGA-UDCA NSs on improving liver function in PSC mice

[0181] In the PSC mouse model of Experiment Example 3, after the treatment was completed, the eyeballs of the mice were removed and blood was collected before sacrifice. A biochemical analyzer was used to detect various liver function indicators in the serum samples of each group of mice.

[0182] The results are shown in Figure 19. Figure a shows the results of AST, a liver function indicator in the serum samples of mice in each group; Figure b shows the results of ALT, a liver function indicator in the serum samples of mice in each group; Figure c shows the results of ALP, a liver function indicator in the serum samples of mice in each group; Figure d shows the results of TBA, a liver function indicator in the serum samples of mice in each group; and Figure e shows the results of TBIL, a liver function indicator in the serum samples of mice in each group.

[0183] As shown in Figure 19, compared with the DDC model group, CuGA-UDCA NSs treatment significantly reduced the serum levels of ALT, AST, ALP, TBA and TBIL, and the levels of each index were closer to those of the normal group, indicating that CuGA-UDCA NSs can improve liver function indicators in PSC mice.

[0184] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself has the effect of improving liver function in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its function is mainly as a fluorescent reporter, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it can be reasonably inferred that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain similar efficacy in improving liver function in PSC mice in vivo.

[0185] Experimental Example 5 Detecting the effect of CuGA-UDCA NSs on improving liver fibrosis in PSC mice

[0186] In Experiment 3, after the PSC mouse model was treated, the mice were sacrificed and their livers were harvested. The liver tissue pathology analysis was used to further analyze the improvement effect of each treatment group on PSC mice. The MASSON staining results are shown in Figure 20. The expression level of collagen fibers in the DDC model group was significantly increased, and it was significantly decreased after CuGA-UDCANSs treatment, indicating that CuGA-UDCANSs has the effect of improving liver fibrosis in PSC mice.

[0187] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself significantly improves liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it is reasonable to infer that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain similar efficacy in improving liver fibrosis in PSC mice in vivo.

[0188] Experimental Example 6 Detecting the effect of CuGA-UDCA NSs on improving intrahepatic bile duct injury in PSC mice

[0189] In Experiment 3, after the PSC mouse model was treated, the mice were sacrificed and their livers were harvested. The liver tissue pathology analysis was used to further analyze the improvement effect of each treatment group on PSC mice. The K19 / NIK immunofluorescence staining results are shown in Figure 21. In the liver of the PSC mouse model, the bile duct epithelial cell marker K19 and the bile duct injury-related protein NIK showed obvious co-localization, and the NIK expression level was significantly increased. After CuGA-UDCANSs treatment, the NIK level decreased significantly, indicating that CuGA-UDCANSs has the effect of improving intrahepatic bile duct injury in PSC mice.

[0190] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself significantly improves liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it is reasonable to infer that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain similar efficacy in improving intrahepatic bile duct injury in PSC mice in vivo.

[0191] Experimental Example 7 Detecting the effect of CuGA-UDCA NSs on improving intrahepatic cholestasis in PSC mice

[0192] In Experiment 3, after the PSC mouse model was treated, the mice were sacrificed and their livers were harvested. The liver tissue pathology analysis was used to further analyze the improvement effect of each treatment group on PSC mice. The HE staining results are shown in Figure 22. In the DDC model group, obvious bile salt precipitation and stasis were observed. After treatment with CuGA-UDCA NSs, this lesion was significantly reduced, indicating that CuGA-UDCA NSs has the effect of improving intrahepatic cholestasis in PSC mice.

[0193] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself significantly improves liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it can be reasonably inferred that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain a similar efficacy in improving intrahepatic cholestasis in PSC mice in vivo.

[0194] Experimental Example 8 Detecting the effect of CuGA-UDCA NSs on reducing the level of inflammatory factors in PSC mice

[0195] In the PSC mouse model of Experiment Example 3, after the treatment was completed, the eyeballs of the mice were removed before sacrifice to collect blood and extract serum. The levels of IL-1β, TNF-α, IL-18 and IL-6 in the serum samples of each group of mice were detected by ELISA.

[0196] The results are shown in Figure 23. Figure a shows the detection results of IL-1β in the serum samples of mice in each group; Figure b shows the detection results of IL-18 in the serum samples of mice in each group; Figure c shows the detection results of TNF-α in the serum samples of mice in each group; and Figure d shows the detection results of IL-6 in the serum samples of mice in each group.

[0197] As shown in Figure 23, compared with the model group, CuGA-UDCA NSs treatment significantly reduced the serum levels of IL-1β, TNF-α, IL-18 and IL-6, and the levels of each index were closer to those of the normal group, indicating that CuGA-UDCA NSs can reduce the levels of inflammatory factors in PSC mice.

[0198] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself significantly improves liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it is reasonable to infer that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain similar efficacy in improving the levels of inflammatory factors in PSC mice in vivo.

[0199] Experimental Example 9 Detecting the effect of CuGA-UDCA NSs on improving macrophage infiltration in the liver of PSC mice

[0200] In Experiment 3, after the PSC mouse model was treated, the mice were sacrificed and their livers were harvested. The liver histopathological analysis was used to further analyze the improvement effect of each treatment group on PSC mice. The F4 / 80 histochemical results are shown in Figure 24. F4 / 80 expression was significantly increased in the DDC model group. After treatment with CuGA-UDCA NSs, F4 / 80 expression was significantly decreased, indicating that CuGA-UDCA NSs can improve macrophage infiltration in the liver of PSC mice.

[0201] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself significantly improves liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it is reasonable to infer that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain a similar efficacy in improving macrophage infiltration in the liver of PSC mice in vivo.

[0202] Experimental Example 10 Test the ability of CuGA-UDCA NSs to reduce liver ROS levels in PSC mice.

[0203] In Experiment 3, after the PSC mouse model was treated, the mice were sacrificed and their livers were harvested. The ROS levels in the livers of each treatment group were further analyzed by DHE staining. The DHE staining results are shown in Figure 25. The ROS level was significantly increased in the DDC model group. After treatment with CuGA-UDCA NSs, the ROS fluorescence intensity was significantly reduced, indicating that CuGA-UDCA NSs has the ability to reduce the ROS level in the livers of PSC mice.

[0204] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself significantly improves liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it is reasonable to infer that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain a similar ability to reduce ROS levels in the liver of PSC mice in vivo.

[0205] Experimental Example 11 Test the ability of CuGA-UDCA NSs to reduce the level of hepatocyte apoptosis in PSC mice.

[0206] In Experiment 3, after the PSC mouse model was treated, the mice were sacrificed and their livers were harvested. The apoptosis level of liver cells in each treatment group was further analyzed by TUNEL staining. The TUNEL staining results are shown in Figure 26. The apoptosis level of liver cells was significantly increased in the DDC model group. After treatment with CuGA-UDCA NSs, the apoptosis level of liver cells was significantly decreased, indicating that CuGA-UDCA NSs has the ability to reduce the apoptosis level of liver cells in PSC mice.

[0207] The above results indicate that the CuGA-UDCA NSs nanozyme carrier itself significantly improves liver edema in PSC mice. Given that the CyP probe is covalently linked to the carrier surface and its main function is fluorescence reporting, it is not expected to substantially affect the liver targeting and catalytic therapeutic function of the nanozyme carrier. Therefore, it is reasonable to infer that the final product CyP-CuGA-UDCA NSs constructed from this carrier should retain a similar ability to reduce hepatocyte apoptosis levels in PSC mice in vivo.

[0208] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A copper-based nanozyme, characterized in that, The copper-based nanozyme comprises: a nanozyme carrier, which is CuGA-UDCA NSs; and a probe molecule, which is a CyP probe linked to the surface of the nanozyme carrier via an amide bond, and its structural formula is shown in Formula 1: The probe molecule is configured to be specifically activated by alkaline phosphatase as a substrate through its phosphate group.

2. A method for preparing a copper-based nanozyme, characterized in that, Includes the following steps: S1. Provide an aqueous dispersion of the nanozyme carrier; S2. Activate the carboxyl groups on the surface of the nanozyme carrier in the presence of a coupling agent; S3. Add probe molecules to perform a coupling reaction; S4. After the reaction is complete, separate and purify the obtained product to obtain the copper-based nanozyme; wherein, in step S1, the nanozyme carrier is CuGA-UDCA NSs; in step S3, the probe molecule is a CyP probe, the structural formula of which is shown in Formula 1: 。 3. The preparation method according to claim 2, characterized in that, In step S1, the concentration of the aqueous dispersion of the nanozyme carrier is 0.5-2.0 mg / mL; and / or, in step S2, the coupling agent is an EDCI / NHS mixture; the amount of the EDCI / NHS mixture, calculated as EDCI, is 1.5-5.0 equivalents relative to the carboxyl groups on the surface of the nanozyme carrier; the molar ratio of EDCI to NHS in the EDCI / NHS mixture is 1:(1-1.5); and / or, in step S2, the activation temperature is 0-5℃ and the time is 0.5-1.5 hours; and / or, in step S3, the coupling reaction is carried out at room temperature for 12-36 hours; and / or, in step S3, the amount of the probe molecule added is 0.5-2.0 equivalents relative to the carboxyl groups on the surface of the nanozyme carrier.

4. The preparation method according to claim 2, characterized in that, In step S1, the preparation of CuGA-UDCA NSs includes the following steps: S11, dissolving gallic acid and ursodeoxycholic acid in an organic solvent and adding a base; S12, providing an aqueous solution of copper salt; S13, mixing the solutions from steps S11 and S12 and carrying out a solvothermal reaction; S14, after the reaction is complete, separating, washing and drying the obtained solid to obtain CuGA-UDCA NSs.

5. The preparation method according to claim 4, characterized in that, In step S11, the mass ratio of gallic acid to ursodeoxycholic acid is (8.0-8.8):1; and / or, in step S11, the organic solvent is DMF and the base is triethylamine; and / or, in step S13, the temperature of the solvothermal reaction is 105-115℃ and the reaction time is 17-19 hours.

6. The preparation method according to any one of claims 2-5, characterized in that, In step S3, the preparation of the CyP probe includes the following steps: S31, Synthesis of compound 2: 2,3,3-trimethylindole is reacted with 3-bromopropylamine hydrobromide in methanol to obtain compound 2, as shown in the following reaction formula: S32. Synthesis of Compound 3: Compound 2 is reacted with 2-chloro-3-(hydroxymethylene)-cyclohexenaldehyde in a 1-butanol / benzene mixed solvent by dehydration condensation to generate cyanine dye skeleton compound 3, as shown in the following reaction formula: S33. Synthesis of compound 4: The amino group of compound 3 is protected with Boc2O to generate compound 4, as shown in the following reaction formula: S34. Synthesis of compound 5: Compound 4 is reacted with 4-chloro-1,3-dihydroxybenzene under alkaline conditions to produce compound 5, as shown in the following reaction formula: S35. Synthesis of Compound 6: Compound 5 was phosphorylated using POCl3 to generate phosphorylated intermediate compound 6, as shown in the following reaction formula: S36. Synthesis of CyP probe: Under acidic conditions, the Boc protecting group of compound 6 was removed to obtain the CyP probe, as shown in the following reaction formula: 。 7. The preparation method according to claim 6, characterized in that, In step S31, the molar ratio of 2,3,3-trimethylindole to 3-bromopropylamine hydrobromide is 1:(1.1-1.3), and the reaction temperature is 95-105℃; and / or, in step S32, the molar ratio of compound 2 to 2-chloro-3-(hydroxymethylene)-cyclohexenecarbaldehyde is (2.0-2.2):1; the dehydration condensation reaction is first refluxed at 130-140℃ until the dehydration is completed, and then the temperature is lowered to 70-80℃ and stirring is continued for 5-7 hours; and / or, in step S34, the molar ratio of compound 4 to 4-chloro-1,3-dihydroxybenzene is 1:(2.8-3.2), and the reaction temperature is 70-80℃; and / or, in step S35, the phosphorylation reaction is carried out at 0-5℃.

8. The use of the copper-based nanozyme of claim 1 or the copper-based nanozyme prepared by the preparation method of any one of claims 2-7 in the preparation of products for any of the following uses: (a) a diagnostic reagent for detecting alkaline phosphatase activity in in vitro solutions or isolated organs; (b) a diagnostic reagent for assessing the severity or treatment progress of diseases related to alkaline phosphatase overexpression at the in vivo level by fluorescence imaging; (c) a medicament for treating diseases related to alkaline phosphatase overexpression; wherein, In uses (b) and (c), the disease associated with alkaline phosphatase overexpression is a hepatobiliary disease; the hepatobiliary disease is primary sclerosing cholangitis.

Images