A tumor-targeted composite nanozyme material, its preparation method and application

Abstract

The present disclosure provides a tumor-targeting composite nanozyme material, a preparation method thereof, and an application thereof. A copper ion-doped metal-organic framework material UiO66-NH2(Cu) is in-situ grown on the surface of magnetic nanoparticles to form a core-shell structure, and then oxidized hyaluronic acid is bonded to the surface of the core-shell structure, and then a chemotherapeutic drug is loaded to obtain the tumor-targeting composite nanozyme material. The present invention utilizes the magnetism of the magnetic nanoparticles to exert targeting through physical action and enrich the nanoparticles at the tumor site. Further, by modifying the surface of the nanoparticles with oxidized hyaluronic acid, the nanoparticles conjugated with oxidized hyaluronic acid are transported into tumor cells under the mediation of CD44 molecules, realizing precise drug release with dual targeting. In addition, the composite nanozyme catalyzes H2O2 into toxic ·OH to cause apoptosis of tumor cells, consumes GSH, inhibits the in vivo antioxidant system, reduces the consumption of ROS, and generates a cascade amplification enzymatic reaction through the cycle of multivalent metal ions, achieving the maximum killing effect on tumors.

Description

Technical Field

[0001] The present disclosure relates to the technical field of pharmaceutical products, and particularly to a tumor-targeted composite nanozyme material, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, cancer is the biggest killer threatening human health. Conventional methods for cancer treatment clinically include surgical resection, radiotherapy, chemotherapy, or a combination therapy of the above regimens. Among them, chemotherapy is one of the preferred clinical treatment regimens. However, traditional chemotherapy drugs have poor targeting and are toxic and side effects to normal cells.

[0003] During the occurrence and metastasis of tumors, the relationship between the internal and external environments of tumor cells is the tumor microenvironment (TME). TME is relatively hypoxic, and the contents of hydrogen peroxide (H2O2) and glutathione (GSH) are relatively high. These factors hinder the treatment effects of conventional methods to a certain extent.

[0004] In recent years, ferroptosis has attracted extensive attention as a new cancer treatment approach. Ferroptosis is an iron-dependent regulated cell death form, mainly depending on iron-mediated oxidative damage and subsequent cell membrane damage, and finally caused by excessive lipid peroxidation. By increasing intracellular iron ions (Fe 3+ ) or other oxidizing metal ions (such as Cu 2+ ), the generation of hydroxyl radicals (·OH) is increased, or the inactivation of GPX4 is caused by reducing GSH to induce lipid peroxidation, ultimately leading to ferroptosis of tumor cells. Therefore, developing a treatment method based on the combination of multiple therapies and ferroptosis has more obvious advantages in clinical cancer treatment.

[0005] Currently, tumor treatment based on ferroptosis mainly designs nanozyme materials. By increasing the concentration of intracellular ferrous ions, the rate and quantity of ·OH generation are increased, thereby triggering ferroptosis. However, the nanozyme materials reported currently are difficult to accumulate at the tumor site, and the catalytic activity of the nanozymes is insufficient. Summary of the Invention

[0006] The present disclosure provides a tumor-targeted composite nanozyme material, a preparation method thereof, and an application thereof, so as to at least solve the above technical problems existing in the prior art.

[0007] According to the first aspect of the present disclosure, a tumor-targeted composite nanozyme material is provided. A copper ion-doped metal-organic framework material UiO66-NH2(Cu) is in-situ grown on the surface of magnetic nanoparticles to form a core-shell structure, and then oxidized hyaluronic acid is bonded to the surface of the core-shell structure, and then a chemotherapeutic drug is loaded to obtain the tumor-targeted composite nanozyme material, and the magnetic nanoparticles are magnetic nano materials composed of multiple metals.

[0008] In an implementable embodiment, the magnetic nanoparticles are zinc ferrite nanozyme or copper ferrite nanozyme.

[0009] According to the second aspect of the present disclosure, a method for preparing a tumor-targeting composite nanozyme material is provided, the method comprising:

[0010] Preparing magnetic nanoparticles;

[0011] In-situ growing a copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of the magnetic nanoparticles to obtain ZnFe2O4@UiO66-NH2(Cu), then dropwise adding ZnFe2O4@UiO66-NH2(Cu) into an aqueous sodium citrate solution, reacting at 60 - 100 °C for 5 - 60 min, after cooling to room temperature, continuing to stir; centrifuging and separating to obtain ZnFe2O4@UiO66-NH2(Cu)-Cit;

[0012] Preparing oxidized hyaluronic acid and dissolving the oxidized hyaluronic acid in a buffer solution;

[0013] Dispersing ZnFe2O4@UiO66-NH2(Cu)-Cit in a buffer solution, and then dropwise adding it into the oxidized hyaluronic acid solution; adjusting the pH of the mixed solution to 11 - 13, reacting for 1 - 3 h, then adjusting the pH to 7.4, stirring in the dark, centrifuging and collecting to obtain the tumor-targeting composite nanozyme material.

[0014] In an implementable embodiment, the preparing of the magnetic nanoparticles includes:

[0015] Mixing a soluble metal salt of iron and another metal, a surfactant, a reducing agent and an alcohol solution uniformly and placing them in a reaction kettle, reacting at 180 - 220 °C for 10 - 15 h, wherein the other metal is zinc or copper;

[0016] Washing the product obtained from the reaction successively with pure water and absolute ethanol, then centrifuging and vacuum drying to obtain the magnetic nanoparticles.

[0017] In an implementable embodiment, the molar ratio of the iron to the other metal is 1:1 - 4.

[0018] In an implementable embodiment, the surfactant is sodium citrate dihydrate, and the reducing agent is sodium acetate.

[0019] In an implementable embodiment, the alcohol solution is diethylene glycol.

[0020] In an implementable embodiment, the in-situ growing of the copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of the magnetic nanoparticles to obtain ZnFe2O4@UiO66-NH2(Cu) includes:

[0021] Disperse magnetic nanoparticles in 3-mercaptopropionic acid solution, and then wash with ultrapure water and ethanol;

[0022] Add magnetic nanoparticles to N,N-dimethylformamide (DMF) and disperse them by ultrasonic treatment, and then add zirconium salt, copper acetate, and 2-aminoterephthalic acid and stir evenly;

[0023] Pour the evenly stirred mixture into a polytetrafluoroethylene autoclave and react at 80 - 120 °C for 12 - 36 h

[0024] Centrifuge and collect the product after the reaction, wash it with N,N-dimethylformamide (DMF) and absolute ethanol, and dry it under vacuum to obtain ZnFe2O4@UiO66-NH2(Cu).

[0025] In one embodiment, in the copper ion-doped metal-organic framework material UiO66-NH2(Cu), the molar ratio of zirconium ions to copper ions is 1:1 - 4.

[0026] According to the third aspect of the present disclosure, there is provided an application of a tumor-targeted composite nanozyme material in tumor-targeted drugs or in MIR contrast agents.

[0027] A tumor-targeted composite nanozyme material, its preparation method and application disclosed by the present invention form a core-shell structure by in-situ growth of a copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of magnetic nanoparticles, then adsorb oxidized hyaluronic acid onto the surface of the core-shell structure, and then load chemotherapy drugs to prepare the tumor-targeted composite nanozyme material. The present invention utilizes the magnetism of magnetic nanoparticles to exert targeting through physical action, and uses the in vitro magnetic field effect to guide the directional movement and localization concentration of chemotherapy drugs in the body, and enrich the nanoparticles to the tumor site. And by modifying the surface of the nanoparticles with oxidized hyaluronic acid, the nanoparticles conjugated with oxidized hyaluronic acid are transported into tumor cells under the mediation of CD44 molecules. Due to the characteristics of the weak acidic microenvironment of tumors, the acid-sensitive Schiff base bond formed between oxidized hyaluronic acid and nanoparticles is specifically cleaved to achieve precise drug release with dual targeting. In addition, the composite nanozyme catalyzes H2O2 into toxic ·OH to cause apoptosis of tumor cells, consumes glutathione (GSH), inhibits the in vivo antioxidant system, reduces the consumption of reactive oxygen species (ROS), and produces a cascade amplification enzymatic reaction through the cycle of multivalent metal ions to achieve the maximum killing effect on tumors.

[0028] It should be understood that the content described in this part is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it used to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] By reading the following detailed description with reference to the accompanying drawings, the above and other objects, features, and advantages of the exemplary embodiments of the present disclosure will become readily understood. In the drawings, several embodiments of the present disclosure are shown by way of illustration and not limitation, wherein:

[0030] In the drawings, the same or corresponding reference numerals denote the same or corresponding parts.

[0031] Figure 1 A schematic flowchart showing the implementation process of the preparation method of the tumor-targeted composite nanozyme material in the embodiment of the present disclosure is shown;

[0032] Figure 2 A scanning electron microscope (SEM) image of the zinc ferrite nano material (ZnFe2O4) prepared in the embodiment of the present disclosure is shown;

[0033] Figure 3 An XRD pattern of the zinc ferrite nano material (ZnFe2O4) prepared in the embodiment of the present disclosure is shown;

[0034] Figure 4 A scanning electron microscope (SEM) image of UiO66-NH2(Cu) prepared in the embodiment of the present disclosure is shown;

[0035] Figure 5 A diagram showing the Zr 4+ and Cu 2+ XRD patterns of UiO66-NH2(Cu) with different ratios;

[0036] Figure 6 A diagram showing the Zr 4+ and Cu 2+ CAT activities of UiO66-NH2(Cu) with different ratios;

[0037] Figure 7 A Fourier transform infrared spectrum (FTIR) of OHA prepared in the embodiment of the present disclosure is shown;

[0038] Figure 8 A scanning electron microscope (SEM) image of the tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO) prepared in the embodiment of the present disclosure is shown;

[0039] Figure 9 An XRD pattern of the tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO) prepared in the embodiment of the present disclosure is shown;

[0040] Figure 10The Fourier transform infrared spectroscopy (FTIR) of the tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO) prepared in the embodiments of the present disclosure is shown;

[0041] Figure 11 The ultraviolet absorption spectrum of the tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO) prepared in the embodiments of the present disclosure is shown;

[0042] Figure 12 The enzymatic kinetic curve of the tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO) prepared in the embodiments of the present disclosure with H2O2 as the substrate is shown;

[0043] Figure 13 The consumption diagram of H2O2 by the tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO) prepared in the embodiments of the present disclosure is shown;

[0044] Figure 14 The dissolved oxygen curve generated by catalyzing H2O2 by each material prepared in the embodiments of the present disclosure under different pH conditions is shown;

[0045] Figure 15 The consumption of GSH by different D / Z@UCO concentrations prepared in the embodiments of the present disclosure is shown;

[0046] Figure 16 The T2-weighted images and signal intensities at different D / Z@UCO concentrations prepared in the embodiments of the present disclosure are shown;

[0047] Figure 17 The magnetic pictures of D / Z@UCO prepared in the embodiments of the present disclosure are shown (left: without magnet, right: with magnet). Detailed implementation manners

[0048] To make the objectives, features, and advantages of the present disclosure more obvious and understandable, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present disclosure.

[0049] During tumorigenesis and metastasis, the tumor microenvironment (TME) is relatively hypoxic, and the contents of hydrogen peroxide (H2O2) and glutathione (GSH) are relatively high. These factors hinder the therapeutic effects of conventional methods to a certain extent. Therefore, it is crucial to develop novel nanomaterials targeting the TME for cancer treatment research. Among them, nanozymes, as a class of nanomaterials with enzyme-mimicking properties, possess controllable catalytic activity and high stability. Developing nanozymes with multi-enzyme activities will inhibit tumor development through a "one stone, multiple birds" strategy. Therefore, constructing a nanozyme composite system with high enzymatic reaction efficiency for cancer treatment targeting the tumor microenvironment has great research significance. However, current nanozymes have the following technical problems: they are difficult to accumulate at the tumor site and cannot be specifically released in the tumor microenvironment, resulting in the inability of nanozymes to meet actual requirements. In addition, the low catalytic activity of nanozymes limits their further application. Based on the above technical problems, the present invention proposes a tumor-targeting composite nanozyme material.

[0050] A tumor-targeting composite nanomaterial is prepared by in-situ growing copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of magnetic nanoparticles to form a core-shell structure, then bonding oxidized hyaluronic acid to the surface of the core-shell structure, and finally loading chemotherapy drugs, thereby obtaining the tumor-targeting composite nanozyme material, wherein the magnetic nanoparticles are magnetic nanomaterials composed of multiple metals.

[0051] The preparation method of the tumor-targeting composite nanozyme material includes the following steps:

[0052] S1. Prepare magnetic nanoparticles;

[0053] In one example, preparing magnetic nanoparticles includes:

[0054] S11. Mix soluble metal salts of iron and another metal, a surfactant, a reducing agent, and an alcohol solution evenly and place them in a reaction kettle, and react at 180 - 220 °C for 10 - 15 h, where the other metal is zinc or copper;

[0055] The surfactant and the reducing agent can be sodium citrate dihydrate and sodium acetate. Sodium citrate dihydrate can act as both a surfactant and a reducing agent, and sodium acetate mainly acts as a reducing agent. It should be noted that other reducing substances can also be used as reducing agents, and the present invention makes no limitation thereto.

[0056] S12. Wash the reaction product successively with pure water and absolute ethanol, then centrifuge and vacuum dry to obtain magnetic nanoparticles.

[0057] S2. In-situ grow copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of magnetic nanoparticles to obtain ZnFe2O4@UiO66-NH2(Cu), and then dropwise add ZnFe2O4@UiO66-NH2(Cu) into an aqueous sodium citrate solution, react at 60 - 100 °C for 5 - 60 min, after cooling to room temperature, continue stirring; centrifuge to obtain ZnFe2O4@UiO66-NH2(Cu)-Cit;

[0058] Specifically, in-situ grow copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of magnetic nanoparticles to obtain ZnFe2O4@UiO66-NH2(Cu), including:

[0059] S21. Disperse magnetic nanoparticles in 3-mercaptopropionic acid solution, and then wash with ultrapure water and ethanol;

[0060] S22. Add magnetic nanoparticles into N,N-dimethylformamide for ultrasonic dispersion, and then add zirconium salt, copper acetate, and 2-aminoterephthalic acid and stir evenly;

[0061] S23. Pour the evenly stirred mixture into a polytetrafluoroethylene autoclave and react at 80 - 120 °C for 12 - 36 h;

[0062] S24. Centrifuge and collect the reaction product, wash with N,N-dimethylformamide and absolute ethanol and dry in vacuum to obtain ZnFe2O4@UiO66-NH2(Cu).

[0063] S3. Prepare oxidized hyaluronic acid (OHA) and dissolve oxidized hyaluronic acid in a buffer solution;

[0064] Dissolve hyaluronic acid in ultrapure water, and then add an aqueous NaIO4 solution to make the molar ratio of HA to NaIO4 1:1, stir in the dark at room temperature for 12 h, dialyze for 3 days and freeze-dry to obtain oxidized hyaluronic acid (OHA).

[0065] S4. Disperse ZnFe2O4@UiO66-NH2(Cu)-Cit in a buffer solution, and then dropwise add it into an oxidized hyaluronic acid solution; adjust the pH of the mixed solution to 11 - 13, react for 1 - 3 h, and then adjust the pH to 7.4, stir in the dark, centrifuge and collect to obtain a tumor-targeted composite nanozyme material.

[0066] In the present invention, copper ion-doped metal-organic framework material UiO66-NH2(Cu) is in-situ grown on the surface of magnetic nanoparticles to form a core-shell structure. Then, oxidized hyaluronic acid is adsorbed onto the surface of the core-shell structure, and a chemotherapeutic drug is loaded to prepare a tumor-targeting composite nanozyme material. The present invention utilizes the magnetism of magnetic nanoparticles to exert targeting through physical action, and uses the in vitro magnetic field effect to guide the directional movement and localization concentration of chemotherapeutic drugs in the body, enriching the nanoparticles at the tumor site. By modifying the surface of the nanoparticles with oxidized hyaluronic acid, the nanoparticles conjugated with oxidized hyaluronic acid are transported into tumor cells under the mediation of CD44 molecules. Due to the characteristics of the weak acidic microenvironment of tumors, the acid-sensitive Schiff base bonds formed between oxidized hyaluronic acid and the nanoparticles are specifically cleaved to achieve precise drug release with dual targeting. In addition, the composite nanozyme catalyzes H2O2 into toxic ·OH to induce apoptosis of tumor cells, consumes glutathione (GSH), inhibits the in vivo antioxidant system, reduces the consumption of reactive oxygen species (ROS), and generates a cascading enzymatic reaction through the cycle of multivalent metal ions to achieve the maximum killing effect on tumors.

[0067] The following specifically describes in detail the preparation and characterization of the tumor-targeting composite nanozyme material prepared by the present invention in conjunction with specific embodiments.

[0068] Example 1

[0069] A preparation method of a tumor-targeting composite nanozyme material, as Figure 1 shown in the schematic diagram of the preparation method, the preparation method includes the following steps:

[0070] 1. Preparation of zinc ferrite nanozyme (ZnFe2O4):

[0071] 0.164 g of zinc chloride, 0.649 g of ferric chloride hexahydrate, 0.240 g of sodium citrate dihydrate and 1.200 g of sodium acetate are dissolved in a diethylene glycol solution. The above mixture is ultrasonically stirred at room temperature, and then the obtained suspension is transferred into a reaction kettle and heated at 200 °C for 12 h; the obtained product is washed successively with pure water and absolute ethanol, centrifuged at 11000 r / min for 20 min, and vacuum dried at 60 °C for 12 h to obtain ZnFe2O4 (Z) nanomaterial.

[0072] 1.1 Characterization of ZnFe2O4

[0073] The morphology of the zinc ferrite nanomaterial is obtained by scanning electron microscopy (SEM). As Figure 2 shown, the obtained ZnFe2O4 has a spherical structure with a particle size of about 30 nm. The crystal structure is analyzed and verified by an X-ray powder diffractometer (XRD), and the results are as Figure 3As shown, the obtained ZnFe2O4 has an XRD pattern corresponding to the ZnFe2O4 standard card (JCPDS 22-1012), indicating the successful preparation of ZnFe2O4.

[0074] 2. Prepare ZnFe2O4@UiO66-NH2(Cu) (Z@UC) powder, that is, in-situ grow the copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of the zinc ferrite nanomaterial;

[0075] 2.1. First, prepare UiO66-NH2(Cu) separately in the present invention, and the preparation process is as follows:

[0076] Dissolve 0.0814 g of zirconium chloride and 0.1187 g of copper chloride in 10 mL of N,N-dimethylformamide (DMF) to obtain solution A; dissolve 2-aminoterephthalic acid in 10 mL of DMF to obtain solution B; place solution A, solution B, and 0.6 mL of acetic acid in a beaker and mix evenly, ultrasonic for 5 min, pour into a polytetrafluoroethylene reaction kettle, place it in an oven at a temperature of 100 °C for 24 h, after cooling to room temperature, obtain suspension C; centrifuge suspension C at a speed of 11000 r / min for 5 min, discard the supernatant, obtain precipitate D, wash precipitate D with DMF three times, then wash precipitate D with absolute ethanol three times, and finally place it in a vacuum oven at a temperature of 60 °C to dry overnight to obtain UiO66-NH2(Cu) (UC) powder.

[0077] 2.2. Characterization of UiO66-NH2(Cu)

[0078] The SEM image of UiO66-NH2(Cu) nanoparticles is as Figure 3 shown. The UiO66-NH2(Cu) nanoparticles are octahedrons, and the crystal size is about 100 nm. The XRD pattern is as Figure 4 shown. The XRD characterization peaks are consistent with the crystal structure of the nanoparticles reported in the literature, confirming the successful synthesis of UiO66-NH2(Cu) nanoparticles. In addition, the effects of the ratios of Zr 4+ and Cu 2+ on the structure and enzyme activity were investigated. Since the lattice of Cu 2+ is smaller than that of Zr 4+ , the XRD peaks will shift slightly to the right, as Figure 3 shown. As the proportion of Cu 2+ increases, its characteristic peaks shift more to the right. In addition, due to the doping of Cu 2+ affecting its enzyme activity, considering the importance of the factors of structural stability and enzyme activity, the final determination of Zr 4+ : Cu 2+UiO66-NH2(Cu) prepared in a 1:2 ratio was used for subsequent experiments. Figure 5 As shown, the CAT activity was investigated using the titanium sulfate colorimetric method. After adding 50 μL of H2O2 (150 mM) and 200 μL of UiO66-NH2(Cu) (1 mg / mL) to the buffer and mixing for 30 min, it was added to the titanium sulfate solution. After 10 min, the concentration of H2O2 was obtained by measuring the absorbance at 410 nm. The stronger the CAT activity, the less H2O2 remained, and the lighter the color. As Figure 6 shown, as the Cu 2+ proportion increased, the color gradually became lighter and the CAT activity was higher.

[0079] 2.3 Preparation of ZnFe2O4@UiO66-NH2(Cu) (Z@UC) powder

[0080] 80 mg of ZnFe2O4 was dispersed in a 0.6 mM 3-mercaptopropionic acid (3-MPA) solution (20 mL), magnetically stirred for 24 h, and then washed with ultrapure water and ethanol. 0.414 mmol, 80 mg of ZnFe2O4 was added to 10 mL of DMF and sonicated for 20 min. Zirconium chloride (0.3493 mmol, 0.0814 g) was added and stirred for 30 min, then copper chloride (0.0874 mmol, 0.0140 g) was added and stirred for 30 min, then acetic acid (0.6 mL) was added and stirred for 3 min. 2-Aminoterephthalic acid (0.3493 mmol, 0.0621 g) was continuously added and stirred for 1 h. Finally, the reaction mixture was poured into a polytetrafluoroethylene autoclave, sealed and reacted at 100 °C for 12 h. The final product was collected by centrifugation, rinsed with DMF / absolute ethanol and vacuum dried at 70 °C to obtain ZnFe2O4@UiO66-NH2(Cu) (Z@UC) powder.

[0081] To further improve the dispersion effect of ZnFe2O4@UiO66-NH2(Cu) (Z@UC) powder, 10 mg of Z@UC powder was further added to 5 mL of ultrapure water and sonicated for 1 h. 100 mg of sodium citrate (Na3Cit·2H2O) was weighed and dissolved in 15 mL of ultrapure water. The ZnFe2O4@UiO66-NH2(Cu) dispersion was added dropwise to the Na3Cit·2H2O aqueous solution, reacted at 80 °C for 30 min, cooled to room temperature, and stirred for 12 h. It was collected by centrifugation to obtain ZnFe2O4@UiO66-NH2(Cu)-Cit.

[0082] 3 Preparation of oxidized hyaluronic acid (OHA)

[0083] Dissolve 0.5 g of hyaluronic acid (HA) in 50 mL of ultrapure water, add 8 mL of an aqueous solution of NaIO4 with a mass fraction of 3.3%, so that the molar ratio of HA to NaIO4 is 1:1, stir in the dark at room temperature for 12 h, dialyze for 3 days and freeze-dry to obtain OHA. The reaction formula is as follows:

[0084]

[0085] 3.1 Characterization of OHA

[0086] Figure 7 The Fourier transform infrared spectroscopy (FTIR) of the OHA sample prepared in this example is shown below. According to Figure 7 it can be seen that all HA and OHA have polysaccharide characteristics. The presence of aldehyde groups in OHA can be determined by the sharp peak at 1730 cm -1 (-C=O stretching vibration), proving that OHA was successfully synthesized.

[0087] 4. Preparation of tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO)

[0088] In this example, the chemotherapeutic drug is doxorubicin (DOX) as an example. Dissolve 10 mg of OHA in 20 mL of PBS solution. Disperse 10 mg of ZnFe2O4@UiO66-NH2(Cu)-Cit and 10 mg of doxorubicin (DOX) in PBS (pH = 7.4) solution and ultrasonically mix for 1 h, then gradually add it to the OHA solution. Adjust the pH of the mixed solution to 12, react for 2 h, then adjust the pH to 7.4, stir in the dark for 12 h, centrifuge and collect to obtain the final tumor-targeted composite nanozyme material DOX / ZnFe2O4@UiO66-NH2(Cu)-OHA (D / Z@UCO).

[0089] 4.1 Characterization of D / Z@UCO

[0090] The SEM image of D / Z@UCO is as Figure 8 shown. Morphological observation shows that D / Z@UCO is spherical and the crystal size is about 40 nm; Figure 9 The XRD crystal structure shown below shows that the characteristic peaks are consistent with the crystal structures of single-component nanoparticles. The research confirms the successful synthesis of D / Z@UCO; FTIR is as Figure 10 shown. Compared with the individual components, the composite material has all the characteristic peaks of the single components, indicating the successful synthesis of D / Z@UCO.

[0091] 4.2 Enzyme activity characterization of D / Z@UCO

[0092] The POD activity of the prepared D / Z@UCO was determined by the color reaction of 3,3',5,5'-tetramethylbenzidine (TMB). That is, composite nanozyme materials with different concentrations were added to 0.1 M acetic acid-sodium acetate buffer solutions with different pH values. The final concentration of H2O2 was 0.5 M, and the final concentration of TMB was 0.4 mM. After mixing, the absorbance of the solution at 652 nm was measured at different time points.

[0093] As Figure 11 shown by the absorbance results, as the concentration of D / Z@UCO increased, the amount of oxidized TMB produced was more, indicating that the peroxidase (POD) activity was higher. As Figure 12 shown by the enzymatic kinetic curve with H2O2 as the substrate, it was measured from the D / Z@UCO nanozyme dispersion in H2O2 solutions with different concentrations. The final concentration of TMB was 0.4 mM. From Figure 12 it can be obtained that the affinity constant Km of the prepared D / Z@UCO was 1.061 μM and the maximum reaction rate Vmax was 139.4 nM s -1 .

[0094] By measuring the consumption of H2O2 and the oxygen production level of D / Z@UCO, it was proved that the prepared D / Z@UCO also had CAT activity. As Figure 13 shown, as the concentration of D / Z@UCO increased, the consumption of H2O2 gradually increased, indicating that the CAT activity was higher. Figure 14 Shown is a schematic diagram of the curve of D / Z@UCO catalyzing H2O2 to produce dissolved oxygen under different pH conditions. The highest amount of O2 was produced by D / Z@UCO in an environment with a pH of 6.5, indicating that D / Z@UCO could effectively produce O2 under the weakly acidic conditions at the tumor site, relieve hypoxia at the tumor site, and thus improve the killing ability against tumors.

[0095] By measuring the consumption of GSH by D / Z@UCO, it was proved that the prepared D / Z@UCO also had GSH-Ox enzyme activity. As Figure 15 shown, as the concentration of D / Z@UCO increased, the consumption of GSH was faster, indicating that the GSH-Ox activity was better.

[0096] On the other hand, the present invention also provides an application of the tumor-targeting composite nanozyme material in tumor-targeting drugs or in MIR contrast agents.

[0097] As a tumor-targeted drug, this tumor-targeted composite nanozyme material exerts its targeting effect through physical action due to the magnetism of magnetic nanoparticles. By utilizing the in vitro magnetic field effect, it guides the chemotherapeutic drug to move directionally and concentrate in vivo, enriching the nanoparticles at the tumor site. By modifying the surface of the nanoparticles with oxidized hyaluronic acid, the nanoparticles conjugated with oxidized hyaluronic acid are transported into tumor cells under the mediation of CD44 molecules. Due to the characteristics of the weak acidic microenvironment of tumors, the acid-sensitive Schiff base bonds formed between oxidized hyaluronic acid and nanoparticles are specifically cleaved, achieving precise drug release with dual targeting. In addition, the composite nanozyme catalyzes H2O2 into toxic ·OH, causing apoptosis of tumor cells, consuming glutathione (GSH), inhibiting the in vivo antioxidant system, reducing the consumption of reactive oxygen species (ROS), and generating a cascading enzymatic reaction through the cycle of multivalent metal ions, achieving the maximum killing effect on tumors. Therefore, as a tumor-targeted drug, this tumor-targeted composite nanozyme material can produce a good anti-tumor effect.

[0098] This composite nanozyme material can also be used as a MIR contrast agent, and the application method is as follows:

[0099] Disperse D / Z@UCO in 2% agarose gel at a concentration of 0 to 1000 mg / mL. Use a 3.0T nuclear magnetic resonance instrument from GE Company, such as Figure 16 Shown are the T2-weighted images and signal intensities at different D / Z@UCO concentrations; Figure 17 Is the magnetic picture of D / Z@UCO (left: without magnet, right: with magnet). According to Figure 16 And Figure 17 The results show that D / Z@UCO has certain magnetism, and the T2-weighted images gradually darken as the D / Z@UCO concentration increases, indicating that D / Z@UCO can be used as a T2 contrast agent.

[0100] It should be understood that various forms of processes shown above can be used, with steps reordered, added, or deleted. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in a different order, as long as the desired results of the technical solution disclosed in this disclosure can be achieved. This is not limited herein.

[0101] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of this disclosure, "a plurality" means two or more, unless otherwise specifically defined.

[0102] As described above, it is only the specific implementation manner of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present disclosure can easily think of changes or substitutions, which should all be covered within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claimed rights.

Claims

1. A tumor-targeted composite nanozyme material, characterized in that, In-situ growth of copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of magnetic nanoparticles to form a core-shell structure, then bonding oxidized hyaluronic acid to the surface of the core-shell structure, and then loading chemotherapy drugs to prepare a tumor-targeting composite nanozyme material, wherein the magnetic nanoparticles are ZnFe2O4.

2. The preparation method of the tumor-targeting composite nanozyme material according to claim 1, characterized in that, The method includes: Preparing magnetic nanoparticles; In-situ growth of copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of magnetic nanoparticles to obtain ZnFe2O4@UiO66-NH2(Cu), then dropping ZnFe2O4@UiO66-NH2(Cu) into an aqueous sodium citrate solution, reacting at 60-100 °C for 5-60 min, cooling to room temperature, and then continuing to stir; centrifuging to obtain ZnFe2O4@UiO66-NH2(Cu)-Cit; Preparing oxidized hyaluronic acid and dissolving the oxidized hyaluronic acid in a buffer solution; Dispersing ZnFe2O4@UiO66-NH2(Cu)-Cit in a buffer solution, and then dropping it into the oxidized hyaluronic acid solution; adjusting the pH of the mixed solution to 11-13, reacting for 1-3 h, then adjusting the pH to 7.4, stirring in the dark, centrifuging and collecting to obtain the tumor-targeting composite nanozyme material.

3. The method according to claim 2, characterized in that, The preparation of the magnetic nanoparticles includes: Mixing soluble metal salts of iron and zinc, a surfactant, a reducing agent and a diol solution evenly and placing them in a reaction kettle, reacting at 180-220 °C for 10-15 h; Washing the product obtained from the reaction successively with pure water and absolute ethanol, then centrifuging and vacuum drying to obtain magnetic nanoparticles.

4. The method according to claim 3, characterized in that, The molar ratio of iron to zinc is 1:1-4.

5. The method according to claim 3, wherein The surfactant is sodium citrate dihydrate, and the reducing agent is sodium acetate.

6. The method according to claim 3, wherein The diol solution is diethylene glycol.

7. The method according to claim 3, wherein The in-situ growth of copper ion-doped metal-organic framework material UiO66-NH2(Cu) on the surface of magnetic nanoparticles to obtain ZnFe2O4@UiO66-NH2(Cu) includes: Dispersing the magnetic nanoparticles in 3-mercaptopropionic acid solution, and then washing with ultrapure water and ethanol; Adding the magnetic nanoparticles to N,N-dimethylformamide for ultrasonic dispersion, and then adding zirconium salt, copper salt, acetic acid and 2-aminoterephthalic acid and stirring evenly; Pouring the evenly stirred mixture into a polytetrafluoroethylene autoclave and reacting at 80-120 °C for 12-36 h; Centrifuging and collecting the product after the reaction, rinsing with N,N-dimethylformamide and absolute ethanol and vacuum drying to obtain ZnFe2O4@UiO66-NH2(Cu).

8. The method according to claim 7, wherein In the copper ion-doped metal-organic framework material UiO66-NH2(Cu), the molar ratio of zirconium ion to copper ion is 1:1-4.

9. Use of the tumor-targeting composite nanozyme material according to claim 1 in the preparation of tumor-targeting drugs.

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