Au-Mn3O4 heterogeneous double-nano enzyme as well as preparation method and application thereof

Abstract

The invention discloses an Au-Mn3O4 heterogeneous double nano enzyme as well as a preparation method and application thereof. According to the Au-Mn3O4 double nano-enzyme with the heterodimer structure, glutathione is deficient in an acute liver injury environment, and a double-enzyme system drug is used for synergistically treating acute liver injury by relieving oxidative stress and eliminating reactive oxygen species (ROS); meanwhile, Mn3O4 in the double-enzyme system cannot release a large amount of Mn < + > in a low-GSH environment, so that'silence 'of a magnetic resonance contrast imaging signal is kept at an acute liver injury part; along with treatment, the GSH level in the liver with acute liver injury gradually recovers, Mn3O4 can release a large amount of Mn < 2 + > and form a metal chelate with GSH, so that MRI contrast imaging is activated, and the treatment effect is evaluated in real time. The novel nano enzyme provided by the invention can be used for visual diagnosis and treatment of acute liver injury, and can also be applied to treatment of acute liver injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to an Au-Mn 3 O 4 heterogeneous dual nanozyme and its preparation method and application. Background Art

[0002] Acute liver injury (ALI) refers to a state in which the liver is damaged within a short period of time, resulting in a sharp decline in liver function. This condition is usually manifested as acute inflammation and necrosis of hepatocytes, accompanied by abnormal increases in liver function indicators, such as transaminases, bilirubin, etc. Acute liver injury is a serious liver pathological state caused by various factors, with diverse etiologies, commonly including drug toxicity, viral infection, alcohol abuse, ischemic injury, and autoimmune diseases, etc. Due to its rapid onset and severe condition, the existing treatment methods usually have limited effects and it is difficult to achieve early diagnosis and precise treatment for it.

[0003] Nanozyme is a material or structure with enzyme-like catalytic activity but in the nanoscale size. In recent years, due to its unique physical and chemical properties, nanozyme has shown great potential in the medical field, especially in the treatment of inflammation, oxidative stress, etc. Nanozyme has broad application prospects in the diagnosis and treatment of diseases such as cancer, infection, and inflammation due to its high stability and durability, strong customizability, high efficiency, and multifunctionality. In addition, the synthesis and function of nanozyme can be precisely regulated through nanotechnology, enabling it to exhibit optimal performance in different biological environments.

[0004] Based on the above advantages, constructing a new type of nanozyme with good biocompatibility, strong specificity, and high sensitivity is of extremely important significance for realizing the precise diagnosis and efficient treatment of acute liver injury. Summary of the Invention

[0005] The purpose of the present invention is to address the above problems and provide an Au-Mn 3 O 4 heterogeneous dual nanozyme and its preparation method and application.

[0006] In order to achieve its purpose, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a preparation method of an Au-Mn 3 O 4 heterogeneous dual nanozyme, comprising the following steps:

[0008] S1. Prepare gold nanoparticles Au@OAm modified with oleylamine as a ligand

[0009] Dissolve chloroauric acid in organic solvent A, add oleylamine, mix well, add reducing agent borane-tert-butylamine complex TBAB to react fully to obtain Au@OAm;

[0010] S2. Au@OAm surface ligand thiolation

[0011] Take the Au@OAm and octadecyl mercaptan obtained in step S1, add them to toluene, and react them fully to obtain Au@ODT with thiolated Au@OAm surface ligands;

[0012] S3. Synthesis of Nano-Au-Mn 3 O 4 :

[0013] Manganese oleate, oleylamine, oleic acid, and organic solvent B are mixed and heated. Under the protection of inert gas, the Au@ODT prepared in step S2 is added, and the mixed solution is heated to reflux state and fully reacted at this reflux temperature to obtain a reaction solution; oleylamine, oleic acid, and organic solvent B are used as catalysts for Mn 3 O 4 and Au double nanoparticles combined and synthesized reaction solvent system;

[0014] After the reaction is completed, the reaction solution is cooled, and a precipitant is added to the reaction solution, mixed, centrifuged, and the supernatant is discarded. The lower layer of the precipitate attached to the wall is the product nano-Au-Mn 3 O 4 ;

[0015] S4, Caffeic acid modified Au-Mn 3 O 4 Preparation of nanozymes

[0016] The nano Au-Mn prepared in step S3 3 O 4 Add caffeic acid dissolved in organic solvent C, heat to 40-60°C for full reaction, centrifuge, collect the precipitate, and obtain caffeic acid-modified Au-Mn 3 O 4 Nanozyme, that is, water-phase Au-Mn 3 O 4 Heterogeneous dual nanozymes. In this step, caffeic acid can provide functional groups to convert the oil-phase material that cannot be used as a drug into an aqueous phase material that can be used as a drug, thereby obtaining a nanoparticle containing two nanoparticles (Mn 3 O 4 and Au) in aqueous phase heterogeneous dual nanozymes.

[0017] The above-mentioned preparation method specifically comprises the following steps:

[0018] S1. Preparation of Au@OAm nanoparticles modified with oleylamine as a ligand

[0019] At a reaction temperature of 6 - 12 °C, dissolve chloroauric acid in organic solvent A, add oleylamine, mix well, add borane - tert - butylamine complex TBAB and react fully. After the reaction, add a precipitant to the reaction solution, centrifuge, and disperse the precipitate in organic solvent A to obtain the Au@OAm dispersion;

[0020] S2. Thiolation of the ligand on the surface of Au@OAm

[0021] Add a precipitant to the Au@OAm dispersion obtained in step S1, mix well, centrifuge, dissolve the precipitate in toluene, add octadecanethiol, stir and react fully; after the reaction, add a precipitant, mix well and centrifuge, dissolve the precipitate in organic solvent A to obtain the Au@ODT dispersion with thiolated ligand on the surface of Au@OAm;

[0022] S3. Synthesis of nano Au - Mn 3 O 4 :

[0023] Take manganese oleate, oleylamine, oleic acid, and organic solvent B, heat up to 70 - 90 °C, under the protection of inert gas, add the Au@ODT dispersion prepared in step S2, heat the mixed solution to the reflux state and react fully at this reflux temperature to obtain a reaction solution;

[0024] After the reaction, cool the reaction solution to 50 - 70 °C, add a precipitant to the reaction solution, mix well and centrifuge, discard the supernatant, and the lower adherent precipitate is the product. Redisperse the product in organic solvent A to obtain the nano Au - Mn 3 O 4 dispersion with a heterodimer structure;

[0025] S4. Preparation of caffeic acid - modified Au - Mn 3 O 4 Preparation of nanozyme

[0026] Add the nano Au - Mn 3 O 4 dispersion prepared in step S3 to caffeic acid dissolved in organic solvent C, heat to 40 - 60 °C and react fully. After the reaction, add a base to neutralize the excess caffeic acid, mix well, centrifuge, and collect the precipitate to obtain caffeic acid - modified Au - Mn 3 O 4 nanozyme, which is the aqueous - phase Au - Mn 3 O 4 heterogeneous dual - nanozyme; the base is NaOH or KOH;

[0027] The organic solvent A is selected from n-hexane, the organic solvent B is selected from 1-octadecene, the organic solvent C is selected from tetrahydrofuran, and the precipitant is selected from absolute ethanol and isopropanol.

[0028] In step S1, the ratio of chloroauric acid, the organic solvent A for dissolving chloroauric acid, oleylamine, TBAB, and the organic solvent A for dispersing the precipitate is (10 - 200) mg : (1 - 20) mL : (1 - 20) mL : (3 - 60) mg : (1 - 20) mL; the volume ratio of the added amount of the precipitant to the volume of the reaction solution is (10 - 500) mL : (1 - 100) mL; the reaction time after adding TBAB is 50 - 70 min;

[0029] Preferably, the ratio of chloroauric acid, the organic solvent A for dissolving chloroauric acid, oleylamine, TBAB, and the organic solvent A for dispersing the precipitate is (100 - 150) mg : (6 - 15) mL : (6 - 15) mL : (40 - 50) mg : (6 - 14) mL.

[0030] In step S2, the added amount of the precipitant added to Au@OAm is such that the volume ratio of the precipitant to the Au@OAm dispersion is (5 - 10) mL : (1 - 2) mL; the ratio of the Au@OAm dispersion, toluene, octadecanethiol, and the organic solvent A is (1 - 2) mL : (8 - 20) mL : (1 - 2) mL : (5 - 10) mL.

[0031] In step S3, the ratio of manganese oleate, the Au@ODT dispersion, oleic acid, oleylamine, and the organic solvent B is (10 - 250) mg : (1 - 2) mL : (10 - 2000) μL : (10 - 2000) μL : (1 - 20) mL; the reaction is to first mix manganese oleate, oleic acid, oleylamine, and the organic solvent B and heat to 70 - 90 °C, then add the Au@ODT dispersion, and then raise the temperature to 95 - 105 °C and react for 50 - 70 min, and finally raise the temperature to 300 - 330 °C and react for 1.5 - 2.5 h; preferably, the ratio of manganese oleate, the Au@ODT dispersion, oleic acid, oleylamine, and the organic solvent B is (200 - 250) mg : (1 - 2) mL : (1000 - 2000) μL : (1000 - 2000) μL : (10 - 20) mL;

[0032] After the reaction, the added amount of the precipitant in the reaction solution is such that the volume ratio of the precipitant to the reaction solution is (10 - 500) mL : (1 - 100) mL.

[0033] In step S4, the Au-Mn 3 O 4The ratio of the dispersion liquid, caffeic acid, and organic solvent C is (0.5 - 10) mL : (20 - 100) mg : (6 - 60) mL, and the preferred ratio is (0.5 - 1.5) mL : (25 - 35) mg : (6 - 15) mL;

[0034] After precipitating the Au-Mn 3 O 4 dispersion liquid prepared in step S3 with a precipitant, dissolving it with organic solvent C, then adding caffeic acid dissolved in organic solvent C, heating to 40 - 60 °C and reacting for 2.5 - 4 h. After the reaction, add an alkali solution, mix well, centrifuge, and collect the precipitate.

[0035] Preferably, the preparation method of manganese oleate is as follows: dissolve the metal salt of manganese element in methanol, add oleic acid and stir evenly, then add the methanol solution of NaOH, react and cool to room temperature, discard the supernatant, dissolve the precipitate in organic solvent A, and add deionized water for centrifugal washing. After centrifugation, take the supernatant in reverse and let it evaporate naturally to obtain manganese oleate; the organic solvent A is selected from n-hexane.

[0036] In the preparation method of manganese oleate, the ratio of the metal salt of manganese element, methanol, NaOH, and oleic acid is (500 - 800) mg : (5 - 60) mL : (200 - 400) mg : (500 - 3000) μL, and the preferred ratio is (600 - 800) mg : (15 - 30) mL : (250 - 350) mg : (2000 - 3000) μL; the metal salt of manganese element is selected from MnCl 2 、manganese acetylacetonate; the reaction temperature is 65 - 75 °C; the reaction time can be 1.5 - 3 h.

[0037] The second aspect of the present invention provides an Au-Mn 3 O 4 heterogeneous dual nanozyme prepared by the method described in any one of the above.

[0038] The third aspect of the present invention provides the application of the above Au-Mn 3 O 4 heterogeneous dual nanozyme in the preparation of an acute liver injury diagnostic kit or in the preparation of a drug for treating acute liver injury; preferably, the diagnosis is a visual diagnosis.

[0039] The beneficial effects of the present invention are:

[0040] Based on the environmental differences between acute liver injury and normal liver, the present invention provides an Au-Mn with a heterodimer structure 3 O 4A dual nanozyme was developed and its visual diagnosis and treatment effects in an acute liver injury model were verified. In the acute liver injury environment, glutathione (GSH) is deficient. This dual-enzyme system drug synergistically treats acute liver injury by reducing oxidative stress and eliminating reactive oxygen species (ROS). At the same time, Mn in this dual-enzyme system 3 O 4 cannot be released in large amounts in a low GSH environment, thus keeping the magnetic resonance imaging signal "silent" at the acute liver injury site. As the treatment progresses, the GSH level in the liver with acute liver injury gradually recovers, and Mn + O 3 O 4 can release Mn in large amounts 2+ and form metal chelates with GSH, thereby activating MRI imaging to evaluate the treatment effect in real time. More importantly, the unchanged Au can eliminate the potential free radicals generated by the Mn 2+ through the Fenton-like reaction, reducing the impact on the treatment effect. This real-time imaging and evaluation method not only improves the accuracy of diagnosis and the pertinence of treatment, but also reduces the side effects on normal tissues. Through this innovative treatment strategy, it is expected to significantly improve the prognosis of patients with acute liver injury, shorten the course of the disease and reduce the occurrence of complications.

[0041] The novel nanozyme of the present invention can be used for the visual diagnosis and treatment of acute liver injury and can also be used to treat acute liver injury. It not only provides new treatment options and new drugs for patients with acute liver injury, but also lays a foundation for the application of nano-drugs in other diseases. By deeply studying the environmental response mechanism of nano-drugs and optimizing their preparation processes, new ideas and methods can be provided for the diagnosis and treatment of other diseases, with broad application prospects. Brief Description of the Drawings

[0042] Figure 1 Schematic diagram of the preparation process and mechanism of action of the Au-Mn 3 O 4 heterogeneous dual nanozyme system.

[0043] Figure 2 For the Au-Mn 3 O 4 nano-drug of Example 1, (a) transmission electron microscopy (TEM) image, (b) elemental mapping spectra of Au-Mn3O4.

[0044] Figure 3 For the Au-Mn 3 O 4 nano-drug of Example 1, (a) X-ray diffraction pattern (XRD), (b) dynamic light scattering (DLS).

[0045] Figure 4For Au-Mn of Example 1 3 O 4 (a) The hysteresis loop test (VSM) and (Figs. b, c, d, and e) of the Au-Mn3O4 nanomedicine were X-ray photoelectron spectroscopy (XPS spectra), which determined the main elemental composition of Au-Mn3O4 and confirmed the presence of Au, Mn, and O (Figs. b, c, d, and e).

[0046] Figure 5 For Au-Mn of Example 1 3 O 4 Ultraviolet absorption spectrum of the Au-Mn3O4 nanomedicine for scavenging hydroxyl radicals.

[0047] Figure 6 For Au-Mn of Example 1 3 O 4 Superoxide anion (O 2 .- ) scavenging rate of the Au-Mn3O4 nanomedicine.

[0048] Figure 7 For Au-Mn of Example 1 3 O 4 Scavenging activity results of singlet oxygen ( 1 O 2 ) of the Au-Mn3O4 nanomedicine.

[0049] Figure 8 For Au-Mn of Example 1 3 O 4 Longitudinal relaxation rate of the Au-Mn3O4 nanomedicine at a magnetic field strength of 1T 1

[0050] Figure 9 For Au-Mn of Example 1 3 O 4 Drug toxicity results of the Au-Mn3O4 nanomedicine at the LO2 cell level.

[0051] Figure 10 For Au-Mn of Example 1 3 O 4 Effect of the Au-Mn3O4 nanomedicine on scavenging reactive oxygen species at the LO2 cell level.

[0052] Figure 11 For Au-Mn of Example 1 3 O 4 H&E staining map of the Au-Mn3O4 nanomedicine for treating acute liver injury mice. Detailed implementation manners

[0053] The present invention will be further described below in conjunction with examples, but the present invention is not limited thereby.

[0054] The experimental methods in the following examples are all conventional methods unless otherwise specified.​

[0055] Example 1: Preparation of Au-Mn 3 O 4 nanozyme

[0056] I. Au-Mn 3 O 4 nanozyme

[0057] The preparation process and mechanism of action of Au-Mn 3 O 4 nanozyme are as Figure 1 shown. The detailed preparation steps are as follows:

[0058] ① Synthesis of Au@OAm: Put 123 mg of chloroauric acid HAuCl4·4H 2 O, 10 mL of oleylamine (OAm, CAS No. 112-90-3), and 10 mL of n-hexane into a reaction flask, stir evenly at 10 °C (ice bath), the solution color is orange-yellow. Weigh 43.5 mg of TBAB (borane-tert-butylamine complex) and dissolve it in 1 mL of oleylamine and 1 mL of n-hexane to obtain a TBAB solution. Add the TBAB solution to the aforementioned orange-yellow reaction solution and react for 1 h. After adding TBAB, the solution immediately turns darker, showing dark brown. After reacting for 10 min, the color turns dark red until the reaction ends. After the reaction ends, add absolute ethanol to the reaction solution, mix well, centrifuge at 8000 rpm, and disperse the obtained precipitate in 8 - 10 mL of n-hexane to obtain gold nanoparticles Au@OAm modified with oleylamine as a ligand.

[0059] ② Thiolate the surface ligand of Au@OAm: Take 1 - 2 mL of the Au@OAm dispersion and place it in a centrifuge tube. Add absolute ethanol to precipitate Au@OAm, centrifuge at high speed, dissolve the precipitate in 10 mL of toluene, add 1015 μL of octadecanethiol (ODT), and stir overnight at room temperature; the next day the solution turns red, add absolute ethanol, mix well and centrifuge at 8000 rpm, and disperse the obtained precipitate in 5 - 8 mL of n-hexane, which is a bit purple, to obtain the Au@ODT dispersion.

[0060] ③ Synthesis of manganese oleate: Weigh 800 mg of MnCl 2 ·4H 2 O and dissolve it in 20 mL of methanol. Add 2530 μL of oleic acid (CAS No. 112-80-1) and place it on an electric heating mantle, set the external temperature to 70 °C; take 328.5 g of NaOH and dissolve it in 20 mL of methanol, and then slowly drip all the NaOH solution into the MnCl 2In the reaction flask with oleic acid, as NaOH was added, the color changed from colorless to brick red, with a relatively deep color. The reaction was timed for 2 h. After 2 h, heating was stopped and the mixture was cooled to room temperature. The oily substance adhered to the bottom of the flask. The supernatant was poured off, and the precipitate was redissolved in 20 mL of n - hexane and placed in a centrifuge tube. 5 mL of H 2 O was added and shaken (to wash away surface impurities), and centrifuged three times at 8000 rpm. The supernatant of the centrifuged sample was taken, placed in a glass petri dish, and naturally dried, obtaining brick - red manganese oleate. Manganese oleate can also be obtained directly through commercial purchase.

[0061] ④ Synthesis of nano - Au - Mn 3 O 4 : Add 247 mg of manganese oleate, 1.97 mL of oleylamine, 1.9 mL of oleic acid, and 20 mL of 1 - octadecene (ODE) into a 100 - mL three - necked flask. Heat up to 80 °C. The mixed solution was evacuated at room temperature to remove internal oxygen, and then backfilled with nitrogen protectively. The solution was transparent yellow. Add 1 mL of the Au@ODT dispersion obtained in step ②, and the solution immediately turned dark red. Continue to evacuate at 80 °C for 20 min. Heat up to 100 °C, evacuate and refill nitrogen 2 times, and continue to react for 1 h. The color of the solution did not change significantly and remained a dark - red transparent liquid. Finally, heat up to 320 °C and react for 2 h. During the reaction, liquid boiling was visible at about 290 °C, and the color gradually deepened with the increase of reaction time. After the reaction ended, the solution was dark gray - purple. After the reaction ended, when the solution was cooled to about 50 - 70 °C, add isopropanol with a volume twice that of the reaction solution (about 40 mL), mix well, centrifuge at 9000 rpm for 10 min, centrifuge twice repeatedly, and finally disperse the precipitate in 5 - 8 mL of n - hexane, obtaining a nano - Au - Mn 3 O 4 dispersion with a particle size of about 20 - 30 nm.

[0062] ⑤ Preparation of aqueous - phase caffeic - acid - modified Au - Mn 3 O 4 nanozyme

[0063] Add 500 μL of the previously prepared Au - Mn 3 O 4 dispersion into a 2 - mL EP tube, and add 1 mL of absolute ethanol to precipitate the particles. Discard the supernatant. The precipitate was redissolved in 1 mL of tetrahydrofuran. Weigh 30 mg of caffeic acid (CAS No. 331 - 39 - 5) and dissolve it in 6 mL of tetrahydrofuran, pour it into a 50 - mL reaction flask, and add 1 mL of Au - Mn 3 O 4The solution was heated to 50 °C and stirred for reaction for 3 h. After the reaction was completed, it was cooled to room temperature. 50 μL of 0.5 mM sodium hydroxide aqueous solution was added, and the mixture was mixed well. Then it was centrifuged at 9000 rpm for 5 min, and the supernatant was discarded. The obtained precipitate was added with deionized water and ultrasonically dispersed evenly to obtain the hydrophilic Au-Mn modified with caffeic acid 3 O 4 nanozyme, Figure 2 The transmission electron microscopy (TEM) image and the elemental mapping spectra of each element in Au-Mn 3 O 4 are shown.

[0064] II. Characterization of Au-Mn 3 O 4 nanozyme

[0065] The structure of Au-Mn Figure 2 nanozyme was observed by transmission electron microscopy ( 3 O 4 ). It can be seen that the nanoparticles are evenly dispersed and the particle size is about 20 - 30 nm. The X-ray diffraction pattern (XRD) ( Figure 3 a) confirmed that the nanoparticles are Au-Mn 3 O 4 , the synthesis was successful and the crystal form was stable; dynamic light scattering (DLS) ( Figure 3 b) showed that the hydrodynamic diameter of Au-Mn 3 O 4 nanozyme was about 33.5 nm, which is suitable for subsequent diagnostic and therapeutic research. The hysteresis loop test (VSM) ( Figure 4 a), X-ray photoelectron spectroscopy (XPS spectrum) determined the main elemental composition of Au-Mn 3 O 4 and confirmed the existence of Au, Mn and O elements ( Figure 4 b, c, d and e).

[0066] The prepared caffeic acid-modified Au-Mn 3 O 4 nanozyme was dissolved in deionized water and used for further research experiments in Examples 2 - 6, and was formulated into different concentrations according to the experimental design.

[0067] Example 2: In vitro antioxidant activity of Au-Mn 3 O 4 nanozyme

[0068] (1) Hydroxyl radical scavenging activity: FeSO 4 (1.8 mM, 350 μL) and H 2 O 2(5 mM, 350 μL) After mixing, incubate at 37 °C for 10 minutes. Then, add 80 μL of 50 μg / mL Au-Mn 3 O 4 and 300 μL of methylene blue (MB) (100 μg / mL), and react for 1 hour. After the reaction, use a UV-Vis spectrometer to measure the absorbance of the solution in the range of 475 - 750 nm. As Figure 5 shown, the control group without the drug generated hydroxyl radicals, while the Au-Mn 3 O 4 group with the drug eliminated hydroxyl radicals (visible by comparison with MB), indicating that Au-Mn 3 O 4 has good performance in eliminating hydroxyl radicals, laying a foundation for subsequent treatment of acute liver injury.

[0069] (2) Superoxide anion (O 2 .- ) scavenging activity: Take 150 μL of methionine (130 mM), 150 μL of riboflavin (200 μM), 150 μL of NBT (750 μM) and 20 μL of Au-Mn 3 O 4 (at concentrations of 0, 2.5, 5, 10, 25, 50 μg / mL) and mix, then make up to 1.5 mL with deionized water. Then, irradiate the mixed solution with a 365 nm ultraviolet lamp for 15 minutes. Next, immediately measure the absorbance at 560 nm using an enzyme-linked immunosorbent assay reader. Calculate the scavenging efficiency using the following formula: Elimination rate (%) = [(A 0 - A) / A 0 × 100%, where A is the absorbance of the sample and A 0 is the absorbance of the control group. As Figure 6 shown, as the drug concentration increases, the superoxide anion scavenging rate is higher.

[0070] (3) Singlet oxygen ( 1 O 2 ) scavenging activity: The singlet oxygen ( 3 O 4 ) scavenging activity of Au-Mn 1 O 2 was evaluated by electron spin resonance (ESR) spectroscopy. To generate 1 O 2 , hydrogen peroxide (H 2 O 2 , 7.5 mM, 200 μL) was mixed with sodium hypochlorite (2.5 mM, 200 μL) and incubated for 5 minutes. Subsequently, Au-Mn 3 O 4(0.5 μg / mL), TEMP( 1 O 2 scavenger) and deionized water were added to a total volume of 1.6 mL, and incubation was continued for 5 minutes. The remaining 1 O2 was captured by TEMP, and the ESR spectrum was recorded by a Bruker A300 - 10 / 12 spectrometer. As Figure 7 shown, Au - Mn 3 O 4 has good singlet oxygen scavenging activity.

[0071] Example 3: T1 magnetic resonance longitudinal relaxation rate (r 3 O 4 of Au - Mn 1 nanodrugs at 1 T magnetic field strength 1 )

[0072] Au - Mn 3 O 4 magnetic nanodrugs were separately formulated with deionized water and glutathione (GSH) solutions (concentrations of 0 mM, 1 mM, 10 mM) to prepare solutions with manganese ion concentrations of 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM, and 0 mM. These solutions were placed in a magnetic resonance imaging instrument with a 71 T magnetic field strength for T 1 scanning and measurement. After completing the above process, we can obtain the T 3 O 4 longitudinal relaxation rates of Au - Mn 1 magnetic nanodrugs before and after dissociation. As Figure 8 shown, for Au - Mn 3 O 4 nanodrugs in aqueous solution and GSH solution, the r 1 value increases with the increase of drug concentration and GSH, and the r 1 value is also higher in a high GSH environment. This shows that under GSH conditions, manganese ions are released, the T 1 contrast enhancement, and the T 1 contrast performance at this time is more efficient than the T 1 contrast agent used clinically.

[0073] Example 4: Cytotoxicity experiment of Au - Mn 3 O 4 nanodrugs in normal human hepatocytes (LO2)

[0074] First, normal human hepatocytes (LO2) were seeded into 96 - well plates (LO2 was cultured in 1640 medium with 10% FBS in an incubator containing 15% CO 2 at 37 °C), and then Au - Mn 3 O 4After 24 h of co-incubation, the CCK8 staining solution was added and incubated for 40 min. The absorbance was measured at 450 nm using a microplate reader, and the cell viability was calculated using the following formula:

[0075] Cell viability = [(experimental well - blank well) / (negative control well - blank well)] × 100%.

[0076] The results are as Figure 9 shown. Au-Mn 3 O 4 showed no obvious toxic effect on human normal liver cells L02 at concentrations below 50 μg / mL, and toxicity began to appear at concentrations above 100 μg / mL, indicating its good biosafety. Concentrations below 100 μg / mL were subsequently used for further studies on acute liver injury at the cellular level.

[0077] Example 5: Verification of the ability of Au-Mn 3 O 4 nanodrugs to scavenge intracellular reactive oxygen species

[0078] Since acute liver injury is accompanied by the production of a large amount of reactive oxygen species (ROS), we used 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as a probe to study the ROS scavenging effect of Au-Mn 3 O 4 in LO2 cells. Compared with cells treated with saline, cells treated with Rosup (a substance widely used to increase intracellular ROS levels) showed obvious green fluorescence, indicating an increase in the ROS level in LO2 cells. As Figure 10 shown, Au-Mn 3 O 4 treatment significantly reduced the fluorescence intensity, indicating the ROS scavenging activity of Au-Mn 3 O 4 in normal liver cells. Au-Mn 3 O 4 nanodrugs can effectively reduce the ROS level and protect normal liver cells when used to treat acute liver injury.

[0079] Example 6: Treatment of acute liver injury in mice with Au-Mn 3 O 4 nanodrugs

[0080] An acute liver injury model was established by intraperitoneal injection of APAP (paracetamol) in BALB / C mice at a concentration of 350 mg / kg. Two hours after modeling, different concentrations of Au-Mn 3 O 4Nano-drugs (1 mg / kg, 3 mg / kg, 6 mg / kg), and the liver was taken after 12 h for pathological H&E detection of mouse liver tissue.

[0081] The results were as Figure 11 shown. The pathological structure of the liver tissue of mice without injection of Au-Mn 3 O 4 nano-drugs was significantly damaged, indicating that an acute liver injury model was successfully established. However, the livers of mice injected with Au-Mn 3 O 4 nano-drugs were protected, and the therapeutic effect was stronger with the increase of drug concentration, confirming the role of Au-Mn3O4 nano-drugs in the treatment of acute liver injury.

Claims

1. A method for preparing Au-Mn3O4 heterogeneous dual nanozyme, characterized in that: The steps include: S1. Preparation of Au@OAm nanoparticles modified with oleylamine as a ligand Dissolve chloroauric acid in organic solvent A, add oleylamine, mix well, add reducing agent borane-tert-butylamine complex TBAB to react fully to obtain Au@OAm; S2. Au@OAm surface ligand thiolation Take the Au@OAm and octadecyl mercaptan obtained in step S1, add them to toluene, and react them fully to obtain Au@ODT with thiolated Au@OAm surface ligands; S3. Synthesis of nanozyme Au-Mn3O4: Manganese oleate, oleylamine, oleic acid, and organic solvent B are mixed and heated, and Au@ODT prepared in step S2 is added under the protection of an inert gas, and the mixed solution is heated to a reflux state and fully reacted at the reflux temperature to obtain a reaction solution; After the reaction is completed, the reaction solution is cooled, and a precipitant is added to the reaction solution, mixed, centrifuged, and the supernatant is discarded. The lower layer of the precipitate attached to the wall is the product nano-Au-Mn3O4; S4. Preparation of caffeic acid modified Au-Mn3O4 nanozyme Add caffeic acid dissolved in organic solvent C to the nano Au-Mn3O4 prepared in step S3, heat to 40-60°C for full reaction, centrifuge, and collect the precipitate to obtain caffeic acid-modified Au-Mn3O4 nanozyme, which is the aqueous phase Au-Mn3O4 heterogeneous dual nanozyme.

2. The preparation method according to claim 1, characterized in that: The steps include: S1. Preparation of Au@OAm nanoparticles modified with oleylamine as a ligand At a reaction temperature of 6-12°C, chloroauric acid is dissolved in an organic solvent A, oleylamine is added, mixed, and borane-tert-butylamine complex TBAB is added to react fully. After the reaction is completed, a precipitant is added to the reaction solution, centrifuged, and the precipitate is dispersed in the organic solvent A to obtain an Au@OAm dispersion. S2. Au@OAm surface ligand thiolation Add a precipitant to the Au@OAm dispersion obtained in step S1, mix well, centrifuge, dissolve the precipitate in toluene, add octadecyl mercaptan, stir, and react fully; after the reaction, add a precipitant, mix well, centrifuge, and dissolve the precipitate in an organic solvent A to obtain an Au@ODT dispersion with Au@OAm surface ligand thiolation; S3. Synthesis of nano Au-Mn3O4: Manganese oleate, oleylamine, oleic acid, and organic solvent B are mixed and heated to 70-90° C. Under the protection of inert gas, the Au@ODT dispersion prepared in step S2 is added, and the mixed solution is heated to reflux state and fully reacted at the reflux temperature to obtain a reaction solution; After the reaction is completed, the reaction liquid is cooled to 50-70°C, a precipitant is added to the reaction liquid, mixed and centrifuged, the supernatant is discarded, and the lower layer of the precipitate adhering to the wall is the product. The product is redispersed in organic solvent A to obtain a nano Au-Mn3O4 dispersion with a heterodimer structure; S4. Preparation of caffeic acid modified Au-Mn3O4 nanozyme Add caffeic acid dissolved in organic solvent C to the nano Au-Mn3O4 dispersion prepared in step S3, heat to 40-60°C for sufficient reaction, add alkali to neutralize excess caffeic acid after the reaction, mix well, centrifuge, collect precipitate, and obtain caffeic acid-modified Au-Mn3O4 nanozyme, which is aqueous Au-Mn3O4 heterogeneous dual nanozyme; the alkali is NaOH or KOH; The organic solvent A is selected from n-hexane, the organic solvent B is selected from 1-octadecene, the organic solvent C is selected from tetrahydrofuran, and the precipitant is selected from anhydrous ethanol and isopropanol.

3. The preparation method according to claim 1, characterized in that: In step S1, the ratio of chloroauric acid, organic solvent A for dissolving chloroauric acid, oleylamine, TBAB, and organic solvent A for dispersing precipitation is (10-200) mg: (1-20) mL: (1-20) mL: (3-60) mg: (1-20) mL; the volume ratio of the added amount of the precipitant to the reaction solution is (10-500) mL: (1-100) mL; the reaction time after adding TBAB is 50-70 min; Preferably, the ratio of chloroauric acid, organic solvent A for dissolving chloroauric acid, oleylamine, TBAB, and organic solvent A for dispersing precipitation is (100-150) mg: (6-15) mL: (6-15) mL: (40-50) mg: (6-14) mL.

4. The preparation method according to claim 3, characterized in that: In step S2, the amount of the precipitant added to Au@OAm is such that the volume ratio of the precipitant to the Au@OAm dispersion is (5-10) mL:(1-2) mL; the ratio of the Au@OAm dispersion, toluene, octadecyl mercaptan, and organic solvent A is (1-2) mL:(8-20) mL:(1-2) mL:(5-10) mL.

5. The preparation method according to claim 4, characterized in that: In step S3, the ratio of manganese oleate, Au@ODT dispersion, oleic acid, oleylamine, and organic solvent B is (10-250) mg: (1-2) mL: (10-2000) μL: (10-2000) μL: (1-20) mL; the reaction is firstly mixing manganese oleate, oleic acid, oleylamine, and organic solvent B and heating them to 70-90° C., then adding Au@ODT dispersion, then heating to 95-105° C. and reacting for 50-70 min, and finally heating to 300-330° C. and reacting for 1.5-2.5 h; After the reaction is completed, the amount of the precipitant added to the reaction solution is such that the volume ratio of the precipitant to the reaction solution is (10-500) mL:(1-100) mL.

6. The preparation method according to claim 5, characterized in that: In step S4, the ratio of the Au-Mn3O4 dispersion, caffeic acid, and organic solvent C is (0.5-10) mL: (20-100) mg: (6-60) mL; The Au-Mn3O4 dispersion prepared in step S3 is precipitated with a precipitant, dissolved with an organic solvent C, and then caffeic acid dissolved in the organic solvent C is added, heated to 40-60°C for reaction for 2.5-4h, and after the reaction is completed, an alkaline solution is added, mixed, centrifuged, and the precipitate is collected.

7. The preparation method according to claim 1, characterized in that: The preparation method of manganese oleate is as follows: dissolving a metal salt of manganese element in methanol, adding oleic acid and stirring evenly, adding a methanol solution of NaOH, cooling the reaction to room temperature, discarding the supernatant, dissolving the precipitate in an organic solvent A, adding deionized water for centrifugal washing, taking the supernatant after centrifugation, and evaporating it naturally to obtain manganese oleate; the organic solvent A is selected from n-hexane.

8. The preparation method according to claim 7, characterized in that: The ratio of the metal salt of manganese, methanol, NaOH and oleic acid is (500-800) mg: (5-60) mL: (200-400) mg: (500-3000) μL; the metal salt of manganese is selected from MnCl2 and manganese acetylacetonate; the reaction temperature is 65-75°C; and the reaction time can be 1.5-3h.

9. An Au-Mn3O4 heterogeneous dual nanozyme, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the Au-Mn3O4 heterogeneous dual nanozyme according to claim 9 in the preparation of an acute liver injury diagnostic kit or in the preparation of a drug for treating acute liver injury; preferably, the diagnosis is a visual diagnosis.

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