Bionic integrated nano enzyme capable of effectively relieving gouty arthritis and inhibiting recurrence

Abstract

The invention belongs to the technical field of biological medicines, and relates to a bionic integrated nano enzyme for effectively relieving gouty arthritis and inhibiting recurrence. The biomimetic integrated nano-enzyme is composed of a platinum nano-enzyme, hollow Prussian blue and a macrophage cell membrane. The bionic integrated nano enzyme can quickly and effectively relieve joint swelling caused by gouty arthritis and inhibit repeated recurrence of the gouty arthritis. The hollow Prussian blue loaded with the platinum nano-enzyme and a bionic preparation coated with a macrophage membrane are further prepared, the capabilities of catalyzing uric acid decomposition and removing active oxygen of the platinum nano-enzyme and the effects of targeting in vivo inflammation and rapidly relieving gout can be remarkably improved, and recurrence of the gout is inhibited. According to the invention, multi-strategy and multi-means treatment of gouty arthritis is facilitated, and the problems of rapid onset and easy recurrence of gouty arthritis are solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a biomimetic integrated nanozyme, and specifically relates to a biomimetic integrated nanozyme that can quickly and effectively relieve gouty arthritis and inhibit its recurrence, as well as a construction method and application thereof. Technical Background

[0002] According to literature, gouty arthritis (GA) is one of the most common inflammatory arthritis in the world, with a prevalence of 0.68%-3.9% among adults in different countries, and with the improvement of living standards, it shows a significant upward and younger trend. Studies have shown that due to purine metabolism disorders, high-purine diets and other reasons, the patient's blood uric acid level always exceeds the saturated solubility, and then forms monosodium urate crystals (MSU) and deposits in the joints, inducing local inflammatory reactions and tissue destruction. If not effectively controlled in time, it can cause serious complications such as nephritis, diabetes and arteriosclerosis. Painfully, the onset of GA is cyclical, usually sudden in the early morning or midnight, with unbearable pain and easy recurrence. At present, there is no effective drug for the treatment of GA. Its clinical treatment is mainly acute phase relief of inflammation (such as non-steroidal anti-inflammatory drugs, glucocorticoids and colchicine) and long-term uric acid-lowering treatment (such as xanthine oxidase inhibitors, uric acid excretion drugs and recombinant uricase preparations). However, the side effects of these drugs cannot be ignored, and there is an urgent need for drugs that can simultaneously reduce inflammation and reduce uric acid in clinical practice.

[0003] Reactive oxygen species (ROS), including superoxide anion radicals (O2·-), hydrogen peroxide (H2O2) and hydroxyl radicals (·OH), are partially reduced oxygen metabolites with strong oxidative capacity. Excessive production can damage nucleic acids, proteins and lipid components. In addition, the energy required for cell metabolism increases sharply at the site of inflammation, consuming a large amount of O2, ultimately leading to microenvironment hypoxia, which further induces more ROS. When gouty arthritis occurs, MSU activates local macrophages, which then produce a large amount of ROS and form a hypoxic environment, both of which lead to the activation of the NLRP3 inflammasome and NF-KB pathway. Subsequently, the activated local macrophages and endothelial cells release a large number of inflammatory factors, such as IL-1β, TNF-α, CCL2, etc., which in turn recruit a large number of macrophages and neutrophils in the blood circulation, causing immune cell infiltration and inflammatory cascade reactions. Therefore, in the treatment of GA, it is necessary to eliminate excessive ROS and provide sufficient O2 to quickly relieve the hypoxic environment.

[0004] Nanozymes are nanomaterials with enzyme activity. In recent years, they have been widely used in the biomedical field due to their high activity, low cost, and storage resistance. For example, anti-oxidative stress treatment based on catalase (CAT) activity and superoxide dismutase (SOD) activity, biosensor detection based on peroxidase (POD) activity and glucose oxidase (GOx) activity, and the application of cancer photodynamics and chemokinetics based on CAT activity and POD activity. Platinum (Pt) nanozymes are metal materials with multiple catalytic activities, such as CAT, SOD, POD, ascorbic acid oxidase, polyphenol oxidase (PPO), etc. In recent years, researchers have found that Pt nanozymes also have superior urate oxidase (UOD) activity, which can be used for the treatment of GA and hyperuricemia, and are expected to solve the shortcomings of poor stability and high immunogenicity of recombinant UOD. In addition, the metabolism of uric acid consumes O2 and produces byproducts such as H2O2, which promotes the process of inflammation. Although the CAT and SOD activities of Pt nanozymes can mitigate the harm of H2O2 and produce O2, the particle size of Pt nanozymes is too small, which limits its circulation time and efficacy in the body.

[0005] Based on the research and current status of the prior art, the inventors of this application intend to provide a biomimetic integrated nanozyme that effectively relieves gouty arthritis and inhibits recurrence. The biomimetic integrated nanozyme can catalyze the decomposition of uric acid, the source of gout, while efficiently removing ROS and supplying oxygen, improving the inflammatory microenvironment, quickly and effectively relieving the inflammatory level of gouty arthritis and effectively inhibiting its recurrence. Summary of the invention

[0006] The purpose of the present invention is to provide a bionic integrated nanozyme based on the research and current status of the prior art, specifically to provide a bionic integrated nanozyme that can quickly and effectively relieve gouty arthritis and inhibit its recurrence, as well as a construction method and application thereof.

[0007] The present invention aims at the two major characteristics of gouty arthritis: rapid onset and easy recurrence, and utilizes the specific catalytic uric acid decomposition, efficient ROS removal and spontaneous O2 generation activity of Pt nanozymes, the particle size regulation and cavity loading capacity of hollow Prussian blue, and the inflammation targeting function of macrophage membrane to design and prepare a biomimetic integrated nanozyme. The biomimetic integrated nanozyme can catalyze the decomposition of uric acid, the source of gout, while efficiently removing ROS and supplying oxygen, improving the inflammatory microenvironment, quickly and effectively alleviating the inflammation level of gouty arthritis and effectively inhibiting its recurrence.

[0008] Specifically, the biomimetic integrated nanozyme of the present invention is composed of Pt nanozyme, hollow Prussian blue and macrophage membrane, wherein the platinum nanozyme is prepared by chemical reduction of platinum tetrachloride, and the particle size and zeta potential are adjusted by adjusting the reaction temperature and time, the reaction time is 2-5 hours, and the reaction temperature is 140-180°C;

[0009] The hollow Prussian blue is prepared by a hydrothermal method, and the particle size can be controlled by the reaction time, reaction temperature and hydrochloric acid concentration. The reaction time is 3-12 hours, the reaction temperature is 37-160°C, and the hydrochloric acid concentration is 0.1-0.01M.

[0010] The cell membrane is derived from RAW264.7 macrophage cell line, J774A.1 macrophage cell line, bone marrow-derived macrophages or peritoneal macrophages.

[0011] In the present invention, the reducing agent in the platinum nanozyme is sodium citrate, ascorbic acid and polyvinyl pyrrolidone.

[0012] In the present invention, the Prussian blue is obtained by thermal aging reaction of potassium ferrocyanide and polyvinyl pyrrolidone at 80°C. The particle size and shape can be adjusted by adjusting the ratio of the raw materials. The mass ratio of polyvinyl pyrrolidone:potassium ferrocyanide can be selected in the range of (11-23):1.

[0013] In the present invention, the mass ratio of the hollow Prussian blue to the platinum nanozyme is 5:1-1:5.

[0014] In the present invention, the mass ratio of the macrophage membrane to the platinum nanozyme is 1:(1-5).

[0015] In the present invention, the biomimetic integrated nanozyme is prepared by loading platinum nanozyme onto the surface and pores of hollow Prussian blue through stirring.

[0016] In the present invention, the biomimetic integrated nanozyme is coated with a macrophage membrane on the surface of the integrated nanozyme by a polycarbonate membrane co-extrusion method, and the process includes: extruding a macrophage membrane solution and an integrated nanozyme solution through a polycarbonate membrane to obtain a biomimetic integrated nanozyme.

[0017] The biomimetic integrated nanozyme prepared by the present invention can be used to prepare preparations for intravenous administration; it can quickly and effectively relieve gouty arthritis and inhibit its recurrence.

[0018] The present invention utilizes the specific catalytic uric acid decomposition function of Pt nanozyme to treat uric acid reduction from the source; utilizes the efficient ROS scavenging ability of Pt nanozyme to reduce immune cell recruitment and reduce inflammation level; utilizes the ability of Pt nanozyme to spontaneously produce O2 to relieve the local hypoxic environment of inflammation; utilizes the particle size regulation and cavity loading capacity of hollow Prussian blue to adjust the particle size of integrated nanozyme and improve the in vivo circulation time; utilizes the inflammation targeting function of macrophage membrane to quickly and effectively accumulate in the inflammation site. The biomimetic integrated nanozyme of the present invention combines the biological functions of Pt nanozyme, hollow Prussian blue and macrophage membrane, and effectively combines them to quickly and effectively relieve gouty arthritis and inhibit its recurrence.

[0019] More specifically,

[0020] The present invention discloses a biomimetic integrated nanozyme and a preparation method and physicochemical characterization thereof, which comprises:

[0021] (1) Platinum nanozymes were prepared by chemical reduction method. 100 ml of PtCl4 aqueous solution (0.05 mg / ml) was heated to 140-180°C, and then 10 ml of sodium citrate or ascorbic acid aqueous solution (2-4 mg / ml) was added. After reflux heating for 2-5 hours, the solution was cooled under stirring at room temperature to obtain platinum nanozymes of different particle sizes.

[0022] (2) Prussian blue nanoparticles were prepared by thermal aging reaction. 132-264 mg potassium ferrocyanide and 3 g polyvinyl pyrrolidone were weighed and added to 40 ml HCl (0.01 M). The mixture was uniformly mixed and reacted at 80° C. for 20 hours to obtain Prussian blue nanoparticles.

[0023] (3) The hollow Prussian blue was prepared by a hydrothermal method. 20 mg of the Prussian blue nanoparticles obtained in (2) and 100 mg of polyvinyl pyrrolidone were weighed and added to 20 ml of HCl (0.1-0.01 M), mixed evenly, and reacted at 37-160° C. for 3-12 hours. Hollow Prussian blue nanoparticles with different particle sizes and pore sizes were obtained.

[0024] (4) Preparation of integrated nanozymes using electrostatic effects: Platinum nanozymes and hollow Prussian blue were mixed under ultrasonic conditions at a mass ratio of 5:1-1:5 and stirred for 12 hours.

[0025] (5) Extraction of macrophage membranes: RAW264.7 cells were washed with cold PBS (pH 7.4) and collected in cell membrane protein extraction reagent A containing protease inhibitors. After lysis on ice for 15 min, the membrane suspension was repeatedly extruded through a 400 nm carbonate membrane and then centrifuged at 2000 g for 10 min at 4°C to collect the supernatant. Next, the supernatant was centrifuged at 14,000 g for 30 min at 4°C to collect the macrophage membranes. The concentration of macrophage membranes can be quantified using a BCA protein concentration assay kit.

[0026] (6) Preparation of biomimetic integrated nanozyme: Macrophage membrane and integrated nanozyme were mixed in a mass ratio of macrophage membrane: platinum nanozyme = 1: (1-5), and co-extruded through polycarbonate membranes of different particle sizes.

[0027] (7) The particle size and zeta potential of the nanozyme were measured by a Malvern particle size analyzer, and its morphology was observed by transmission electron microscopy. The successful combination of platinum nanozyme and hollow Prussian blue was verified by energy dispersive X-ray spectroscopy. The macrophage membrane on the surface of the biomimetic integrated nanozyme was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and protein blotting.

[0028] The present invention successfully prepared biomimetic integrated nanozymes, such as Figure 1 As shown, the particle sizes of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM are approximately 2.49, 164.40, 176.20, and 181.77 nm, respectively. The size of each preparation is uniform, the surface is uniform, and the corresponding particle size is basically consistent with the results measured by the aforementioned dynamic light scattering method. HMPB-Pt was analyzed by energy dispersive X-ray spectroscopy (EDS), and the presence of Pt and Fe elements was detected, proving the successful binding of Pt nanozyme to HMPB. The protein integrity of the macrophage membrane on the surface of HMPB-Pt@MM was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the presence of macrophage membrane-specific receptors CCR2, IL-1R1 and LFA-1 on the surface of HMPB-Pt@MM was verified by protein blotting.

[0029] The biomimetic integrated nanozyme of the present invention has shown its anti-inflammatory activity in vivo through animal experiments, including in vivo targeting analysis of HMPB-Pt and HMPB-Pt@MM by in vivo imaging and the swelling elimination rate of the right soles of each group of mice 4 hours after administration, which evaluates the effect of the drug in rapidly relieving swelling caused by gouty arthritis, the swelling elimination rate of the right soles of each group of mice 12 hours after administration, which evaluates the therapeutic effect of the drug on gouty arthritis, the inflammation score of the right ankle joint of each group of mice 12 hours after administration, which evaluates the therapeutic effect of the drug on gouty arthritis, and the Photos of the right ankle joints of mice in each group after 12 hours, HE staining sections of the right ankle joints of mice in each group after 12 hours of administration, CD86 immunohistochemical staining sections of the right ankle joints of mice in each group after 12 hours of administration to characterize the infiltration of M1 macrophages, swelling elimination rate of the right soles of mice in each group at different time points, characterization of the rapid treatment of gouty arthritis and the effect of inhibiting recurrence, and inductively coupled plasma mass spectrometry (ICP-MS) analysis of the Pt content of the right ankle joints of mice in the HMPB-Pt and HMPB-Pt@MM groups at different time points. The results show that the combined use of Pt nanozymes, hollow Prussian blue and macrophage membranes in the present invention can target uric acid reduction while inhibiting inflammation, and the prepared biomimetic integrated nanozymes can quickly and effectively relieve gouty arthritis and inhibit its recurrence. It has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Preparation and characterization of biomimetic integrated nanozymes, including:

[0031] (a) Preparation process of biomimetic integrated nanozyme,

[0032] (b) Platinum nanozyme (Pt nanozyme), hollow Prussian blue (HMPB), integrated nanozyme (HMPB-Pt)

[0033] And transmission electron microscopy photos of biomimetic integrated nanozyme (HMPB-Pt@MM). Scale bar: 50nm, (c) Energy dispersive X-ray spectroscopy analysis of HMPB-Pt,

[0034] (d) Particle sizes of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0035] (e) Zeta potential of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0036] (f) Sodium dodecyl sulfate polyacrylamide gel electrophoresis results characterize the protein integrity of the cell membrane on the surface of HMPB-Pt@MM.

[0037] (g) Western blotting verified the characteristic proteins CCR2, IL-1R1 and LFA-1 on the macrophage membrane surface. Figure 2 Characterization of multiple enzyme activities of biomimetic integrated nanozymes, including:

[0038] (a) The bubbles in the solutions of Pt nanozyme, HMPB, HMPB-Pt, and HMPB-Pt@MM indicate the generation of O2.

[0039] (b) CAT-like activity of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0040] (c) SOD-like activity of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0041] (d) OH- scavenging activity of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0042] (e) Dose-dependent UOD-like activity of Pt nanozyme,

[0043] (f) UOD-like activity of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0044] (g) Effect of O2 content on the UOD-like activity of Pt nanozyme.

[0045] (h) Effect of pH on the UOD-like activity of Pt nanozyme.

[0046] Figure 3 In vitro anti-inflammatory activity of biomimetic integrated nanozymes,

[0047] (a) Cell safety of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0048] (b) Confocal microscopy images characterizing the uptake of HMPB-Pt and HMPB-Pt@MM in M0 and M1 macrophages, scale bar = 20 μm,

[0049] (c) Confocal microscopy images showing the cell adhesion of HMPB-Pt and HMPB-Pt@MM to HUVEC and inflammatory HUVEC, scale bar = 20 μm,

[0050] (d) Flow cytometry quantification of HMPB-Pt and HMPB-Pt@MM uptake in M0 and M1 macrophages, (e) Flow cytometry quantification of HMPB-Pt and HMPB-Pt@MM cell adhesion to HUVEC and inflammatory HUVEC,

[0051] (f) Confocal microscopy images showing that Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM reduce ROS at the cellular level, scale bar = 20 μm,

[0052] (g) Flow cytometry quantification of the reduction of ROS at the cellular level by Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0053] (h) Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM reduce the infiltration of M1 macrophages. (i) Western blot analysis of the expression of HIF-1α and CD86 in cells treated with Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0054] (j) Semi-quantitative analysis of HIF-1α in (i),

[0055] (k) Semi-quantitative analysis of CD86 in (i),

[0056] (l) ELISA kit was used to characterize the IL-1β levels in the cell supernatants treated with Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM.

[0057] Figure 4 In vivo anti-inflammatory activity of biomimetic integrated nanozymes,

[0058] (a) Analysis of the in vivo targeting of HMPB-Pt and HMPB-Pt@MM by small animal in vivo imaging.

[0059] (b) Semi-quantification of fluorescence of the right ankle joint in (a).

[0060] (c) Illustration of animal experiment design,

[0061] (d) The swelling elimination rate of the right paw of each group of mice 4 hours after administration was used to evaluate the drug's effect of rapidly relieving gouty arthritis swelling.

[0062] (e) The swelling elimination rate of the right paw of each group of mice 12 hours after administration was used to evaluate the therapeutic effect of the drug on gouty arthritis.

[0063] (f) The right ankle joint inflammation scores of mice in each group 12 hours after administration were used to evaluate the therapeutic effect of the drug on gouty arthritis.

[0064] (g) Photos of the right ankle joints of mice in each group 12 hours after administration.

[0065] (h) HE staining sections of the right ankle joints of mice in each group 12 hours after administration.

[0066] (i) CD86 immunohistochemical staining sections of the right ankle joints of mice in each group 12 hours after administration to characterize the infiltration of M1 macrophages,

[0067] (j) The swelling elimination rate of the right paw of each group of mice at different time points, indicating the effect of the drug on the rapid treatment of gouty arthritis and the inhibition of recurrence,

[0068] (k) Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the Pt content in the right ankle joints of mice in the HMPB-Pt and HMPB-Pt@MM groups at different time points. DETAILED DESCRIPTION

[0069] The present invention is further described below in conjunction with specific examples, which are only intended to explain the present invention and are not intended to limit its contents. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0070] Example 1: Preparation and physicochemical characterization of biomimetic integrated nanozyme

[0071] Method: (1) Platinum nanozymes were prepared by chemical reduction method. 100 ml of PtCl4 aqueous solution (0.05 mg / ml) was heated to 140-180°C, and then 10 ml of sodium citrate or ascorbic acid aqueous solution (2-4 mg / ml) was added. After reflux heating for 2-5 hours, the solution was cooled under stirring at room temperature to obtain platinum nanozymes of different particle sizes.

[0072] (2) Prussian blue nanoparticles were prepared by thermal aging reaction. 132-264 mg potassium ferrocyanide and 3 g polyvinyl pyrrolidone were weighed and added to 40 ml HCl (0.01 M). The mixture was uniformly mixed and reacted at 80° C. for 20 hours to obtain Prussian blue nanoparticles.

[0073] (3) The hollow Prussian blue was prepared by a hydrothermal method. 20 mg of the Prussian blue nanoparticles obtained in (2) and 100 mg of polyvinyl pyrrolidone were weighed and added to 20 ml of HCl (0.1-0.01 M), mixed evenly, and reacted at 37-160° C. for 3-12 hours. Hollow Prussian blue nanoparticles with different particle sizes and pore sizes were obtained.

[0074] (4) Preparation of integrated nanozymes using electrostatic effects: Platinum nanozymes and hollow Prussian blue were mixed under ultrasonic conditions at a mass ratio of 5:1-1:5 and stirred for 12 hours.

[0075] (5) Extraction of macrophage membranes: RAW264.7 cells were washed with cold PBS (pH 7.4) and collected in cell membrane protein extraction reagent A containing protease inhibitors. After lysis on ice for 15 min, the membrane suspension was repeatedly extruded through a 400 nm carbonate membrane and then centrifuged at 2000 g for 10 min at 4°C to collect the supernatant. Next, the supernatant was centrifuged at 14,000 g for 30 min at 4°C to collect the macrophage membranes. The concentration of macrophage membranes can be quantified using a BCA protein concentration assay kit.

[0076] (6) Preparation of biomimetic integrated nanozyme: Macrophage membrane and integrated nanozyme were mixed in a mass ratio of macrophage membrane: platinum nanozyme = 1: (1-5), and co-extruded through polycarbonate membranes of different particle sizes.

[0077] (7) The particle size and zeta potential of the nanozyme were measured by a Malvern particle size analyzer, and its morphology was observed by transmission electron microscopy. The successful combination of platinum nanozyme and hollow Prussian blue was verified by energy dispersive X-ray spectroscopy. The macrophage membrane on the surface of the biomimetic integrated nanozyme was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and protein blotting.

[0078] Results: Figure 1 As shown, the particle sizes of Pt nanozyme, HMPB, HMPB-Pt and HMPB-Pt@MM are approximately: 2.49, 164.40, 176.20, 181.77nm, respectively. The size of each preparation is uniform, the surface is uniform, and the corresponding particle size is basically consistent with the results measured by the aforementioned dynamic light scattering method. HMPB-Pt was analyzed by energy dispersive X-ray spectroscopy (EDS), and the presence of Pt and Fe elements was detected, proving the successful binding of Pt nanozyme to HMPB. The protein integrity of the macrophage membrane on the surface of HMPB-Pt@MM was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the presence of macrophage membrane-specific receptors CCR2, IL-1R1 and LFA-1 on the surface of HMPB-Pt@MM was verified by protein blotting. In summary, the successful preparation of biomimetic integrated nanozyme is shown.

[0079] Example 2: Multiple enzyme activities of biomimetic integrated nanozymes

[0080] Methods: (1) CAT-like activity assessment: Different concentrations of each group of preparations were incubated with 50 μM H2O2, and the oxygen concentration was measured using a portable oxygen meter at multiple time points to evaluate the CAT-like activity of each group of preparations.

[0081] (2) Investigation of SOD-like activity: The SOD-like activity of each group of preparations was investigated using a SOD detection kit (Biyuntian).

[0082] (3) Investigation of ·OH- scavenging activity: Salicylic acid reacts with ·OH- to generate 2,3-dihydroxybenzoic acid with characteristic absorption at 510 nm. 50 μl FeSO4·7H2O (1.8 mM), 50 μl of each preparation, 50 μl salicylic acid and 50 μl H2O2 were added to a 96-well plate in sequence. After 30 minutes of reaction, the absorbance at 510 nm was detected using an ELISA reader.

[0083] (4) UOD-like activity investigation: The UOD activity of each group of preparations was evaluated by detecting the decrease in the absorbance of uric acid at 290 nm. (i) Dose-dependent UOD-like activity of Pt nanozyme: Uric acid (50 μM) was mixed with different concentrations of Pt nanozyme in PBS (pH 7.4), and the system was incubated at 37°C, 120 rpm for different times. (ii) UOD-like activity of Ptnanozyme at different pH: Uric acid (50 μM) was mixed with Pt nanozyme (20 μg / ml) in PBS, and then the pH was adjusted to 2.0, 4.0, 6.0, and 8.0, respectively. These systems were then incubated at 37°C, 120 rpm for 12 hours. (iii) UOD-like activity of Pt nanozyme under different O2 contents: Uric acid (50 μM) was mixed with Pt nanozyme (10 μg / ml) in PBS and filled with different gases (anoxic group: N2, control group: air, oxygen-enriched group: O2). These systems were incubated at 37°C, 120 rpm for 12 hours. (iv) UOD-like activity determination of each group of preparations: Uric acid (50 μM) was mixed with different preparations (20 μg / ml) in PBS (pH 7.4), and these systems were incubated at 37°C, 120 rpm for 12 hours.

[0084] Results: (1) Figure 2 As shown in (a, b), each group of preparations had CAT-like activity, produced O2, and HMPB-Pt@MM had the highest enzyme activity.

[0085] (2) Figure 2 As shown in (c), all preparations have SOD-like activity, and HMPB-Pt@MM has the highest enzyme activity.

[0086] (3) Figure 2 As shown in (d), all preparations have the activity of scavenging ·OH-, and HMPB-Pt@MM has the highest enzyme activity.

[0087] (4) Figure 2 As shown in (e, f, g, h), the UOD-like activity of Pt nanozyme increases with increasing dosage, while HMPB does not have UOD-like activity. The UOD-like activity of HMPB-Pt and HMPB-Pt@MM is derived from Pt nanozyme. Moreover, the activity of Pt nanozyme is basically unaffected by pH in the range of pH 4.0-8.0, and the increase of O2 content is conducive to improving the UOD-like activity of Ptnanozyme.

[0088] Example 3: Cellular uptake and inflammatory cell adhesion ability of biomimetic integrated nanozymes

[0089] Methods: (1) RAW264.7 cells were seeded in a 24-well plate and cultured for 12 hours. M0 cells were not treated, and M1 cells were stimulated for 12 hours by adding DMEM high-glucose serum-free medium containing 100μg / ml LPS + 20μg / ml IFN-γ. The cells were incubated with serum-free DMEM high-glucose medium containing FITC-labeled HMPB-Pt and HMPB-Pt@MM, so that the final concentration of FITC in the sample well was 5μM. The cells were placed in an incubator at 37℃ in the dark for 4 hours and then washed. Each group was operated in parallel for 3 times. The cell uptake was qualitatively observed and photographed by confocal microscopy. Another group was treated with the same operation, washed, digested, and collected, and quantitative analysis was performed by flow cytometry.

[0090] (2) HUVEC cells were seeded in a 12-well plate and cultured for 12 hours. One group of cells was not treated, and the other group of cells was stimulated for 12 hours by adding RPMI 1640 serum-free medium containing 100 ng / ml TNF-α. The cells were incubated with serum-free DMEM high-glucose medium containing FITC-labeled HMPB-Pt and HMPB-Pt@MM, so that the final concentration of FITC in the sample wells was 5 μM. The cells were placed in an incubator at 37°C in the dark for 10 minutes and then washed. Each group was operated in parallel for 3 times. The cell adhesion was qualitatively observed and photographed by confocal microscopy. Another group was treated with the same operation, washed, digested, and collected, and quantitatively analyzed by flow cytometry.

[0091] Results: (1) Figure 3As shown in (b, d), compared with the HMPB-Pt group, M1 macrophages have a stronger uptake ability for HMPB-Pt@MM; while M0 macrophages have a stronger uptake ability for HMPB-Pt. The same result was obtained by flow cytometry. Therefore, HMPB-Pt@MM can significantly increase the uptake level of inflammatory macrophages and reduce the uptake ability of reticuloendothelial cells, further proving that biomimetic integrated nanozymes can enhance the therapeutic effect of integrated nanozymes on gouty arthritis.

[0092] (2) Figure 3 As shown in (c, e), HMPB-Pt@MM has stronger adhesion to inflammatory endothelial cells than HMPB-Pt group. Therefore, HMPB-Pt@MM can target the inflammatory site and improve the anti-inflammatory effect.

[0093] Example 4: In vitro anti-inflammatory level of biomimetic integrated nanozymes

[0094] Methods: (1) An in vitro gout model was constructed using RAW264.7 cells: after incubation with 500 ng / ml LPS for 6 hours, the cells were stimulated with 200 μg / ml MSU for 2 hours. Then, the serum-free DMEM high-glucose medium containing each group of preparations was replaced and incubated for 12 hours. The medium was then discarded, washed with PBS, and incubated with the ROS probe DCFH-DA for 20 minutes. The cellular ROS level was qualitatively observed and photographed using an inverted fluorescence microscope. Another group was treated with the same operation, washed, digested, and collected, and quantitatively analyzed and detected by flow cytometry.

[0095] (2) In vitro gout model construction and preparation incubation were the same as (1). The medium containing the preparation was discarded, and the cells were washed with PBS and incubated with PE-CD86 antibody for 15 minutes. After washing with PBS three times, the cells were digested and collected, and the fluorescence level was detected by flow cytometry.

[0096] (3) In vitro gout model construction and preparation incubation were the same as (1). The culture medium containing the preparation was discarded, and the cells were washed with PBS three times, digested and collected, and the expression levels of HIF-1α and CD86 proteins were detected by Western blot analysis.

[0097] (4) The in vitro gout model was constructed and the preparation incubation was the same as (1). The cell supernatant was collected and the expression level of IL-1β was detected by ELISA kit.

[0098] Results: (1) Figure 3 As shown in (g, f), the in vitro gout model highly expressed ROS. After treatment with different preparations, the ROS level of cells could be reduced to varying degrees, among which HMPB-Pt@MM had the best effect.

[0099] (2) Figure 3As shown in (h), there are nearly 30% M1 macrophages in the in vitro gout model. After treatment with different preparations, the infiltration level of M1 macrophages can be reduced to varying degrees. The infiltration level of M1 macrophages in the HMPB-Pt@MM group is the lowest, indicating its better anti-inflammatory effect.

[0100] (3) Figure 3 As shown in (i, j, k), the in vitro gout model highly expressed HIF-1α and CD86 proteins. The expression levels of HIF-1α and CD86 proteins could be reduced to varying degrees after treatment with different preparations, among which HMPB-Pt@MM had the best reduction effect, indicating that the bionic integrated nanozyme can effectively alleviate the inflammatory hypoxic environment and reduce the infiltration of M1 macrophages.

[0101] (4) Figure 3 As shown in (l), the in vitro gout model highly expressed IL-1β, and different preparations could reduce IL-1β to varying degrees after treatment, among which HMPB-Pt@MM had the best reduction effect, indicating that the bionic integrated nanozyme can effectively relieve inflammation in vitro.

[0102] Example 5: In vivo pharmacodynamic evaluation of biomimetic integrated nanozymes

[0103] Methods: (1) In vivo distribution experiment: First, a mouse gout model was constructed: 50 μl MSU (20 mg / ml) was injected into the right ankle joint, and 50 μl saline was injected into the left ankle joint at the same time. After 8 hours, the right ankle joint was obviously swollen. Model mice with a right sole / left sole thickness ratio between 1.2 and 1.9 were selected for the experiment. FITC-labeled HMPB-Pt and HMPB-Pt@MM were injected into gout mice by intravenous injection (FITC 1 mg / kg), and the distribution of the preparations was observed by IVIS in vivo imaging at different time points.

[0104] (2) In vivo efficacy test: The mouse gout model was constructed in the same way as (1). The model mice with a right foot / left foot thickness ratio between 1.2 and 1.9 were randomly divided into 6 groups, 6 mice in each group, namely the control group (normal saline group), indomethacin (Indo) group, Pt nanozyme group, HMPB group, HMPB-Pt group, and HMPB-Pt@MM group. The experimental design is as follows: Figure 4 (c) as shown:

[0105] (i) Drugs were administered at time 0, and the dose was 3 mg / kg.

[0106] (ii) At 4 hours, the thickness of the mouse paws was measured and the swelling elimination rate was calculated.

[0107] (iii) At 16 hours, the thickness of the mouse paw was measured and the swelling elimination rate was calculated. The inflammation score of the right ankle joint was scored and photographed. The right ankle joint tissue was obtained for sectioning, hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) staining.

[0108] (iv) At 16 and 40 hours, 0.6 mg MSU was injected into the right ankle joint of mice in each group to simulate the recurrence of gout.

[0109] (v) At 24 and 48 hours, the thickness of the mouse paws was measured and the swelling elimination rate was calculated to evaluate the relapse inhibition ability of each group of preparations.

[0110] (vi) The ankle joints of mice in the HMPB-Pt and HMPB-Pt@MM groups were taken at 4, 24, and 48 hours, and the Pt element content was detected by ICP-MS.

[0111] Results: (1) Figure 4 As shown in (a, b, k), compared with HMPB-Pt, HMPB-Pt@MM can quickly and effectively target the right ankle joint of gouty arthritis 2 hours after administration, and can accumulate at the inflammation site for up to 48 hours.

[0112] (2) Figure 4 As shown in (d), 4 hours after administration, the swelling of the right sole of the model group and the positive drug Indo group continued to increase. Compared with them, the swelling of the right sole of the mice in the Pt nanozyme, HMPB and HMPB-Pt groups was relieved to a certain extent, eliminating 11.35%, 16.33% and 18.50% respectively. However, the HMPB-Pt@MM group showed an excellent rapid treatment effect for gout, reducing the degree of swelling by 47.04%, which is consistent with its rapid inflammation targeting ability in vivo. This shows that the biomimetic integrated nanozyme has the potential to be used as a drug for rapid relief of gouty arthritis.

[0113] (3) Figure 4 As shown in (e, f, g, h, i), 16 hours after administration, the swelling of the model group still increased. Although the positive drug Indo group reduced the swelling by 12.35%, the other nanozyme groups showed better therapeutic effects in swelling elimination, immune cell aggregation and M1 macrophage infiltration. In particular, the HMPB-Pt@MM group eliminated 74.38% of the swelling and significantly inhibited the infiltration of immune cells and M1 macrophages in the inflammatory site, showing excellent therapeutic effects.

[0114] (4) Figure 4As shown in (j, k), when the second MSU stimulation simulated the recurrence of gout, except for the positive drug Indo group, all preparations in each group could inhibit the recurrence of gouty arthritis to a certain extent. Because Indo is a small molecule drug, it cannot be targeted and accumulated at the site of inflammation and will be quickly cleared. The nanoformulation can accumulate at the site of inflammation due to its suitable particle size and continue to exert its efficacy. When the third MSU stimulation simulated the second recurrence of gout, only the HMPB-Pt@MM group could inhibit the second recurrence of gout. The ICP-MS test results also showed that HMPB-Pt@MM was retained in a large amount at the site of inflammation, indicating the ability of the bionic integrated nanozyme to inhibit the recurrence of gout.

[0115] In summary, the present invention combines platinum nanozymes, hollow Prussian blue and macrophage membranes for the first time, and prepares a combined preparation of the composition - bionic integrated nanozymes, which can not only quickly relieve the inflammation level of gouty arthritis, but also inhibit multiple recurrences of gout, and has good application prospects.

Claims

1. A biomimetic integrated nanozyme that effectively relieves gouty arthritis and inhibits recurrence, characterized in that: The bionic integrated nanozyme is composed of platinum nanozyme, hollow Prussian blue and macrophage membrane. The platinum nanozyme is prepared by chemical reduction of platinum tetrachloride, and the particle size and zeta potential are adjusted by adjusting the reaction temperature and time. The reaction time is 2-5 hours and the reaction temperature is 140-180°C. The hollow Prussian blue is prepared by a hydrothermal method, and the particle size can be controlled by the reaction time, reaction temperature and hydrochloric acid concentration. The reaction time is 3-12 hours, the reaction temperature is 37-160°C, and the hydrochloric acid concentration is 0.1-0.01M. The cell membrane is derived from RAW264.7 macrophage cell line, J774A.1 macrophage cell line, bone marrow-derived macrophages or peritoneal macrophages.

2. The biomimetic integrated nanozyme for effectively relieving gouty arthritis and inhibiting recurrence according to claim 1, characterized in that: The reducing agents in the platinum nanozyme are sodium citrate, ascorbic acid and polyvinyl pyrrolidone.

3. The biomimetic integrated nanozyme for effectively relieving gouty arthritis and inhibiting recurrence according to claim 1, characterized in that: The Prussian blue is obtained by thermal aging reaction of potassium ferrocyanide and polyvinyl pyrrolidone at 80°C. The particle size and shape can be adjusted by adjusting the ratio of the raw materials. The mass ratio of polyvinyl pyrrolidone to potassium ferrocyanide can be selected in the range of (11-23):

1.

4. The biomimetic integrated nanozyme for effectively relieving gouty arthritis and inhibiting recurrence according to claim 1, characterized in that: The mass ratio of the hollow Prussian blue to the platinum nanozyme is 5:1-1:

5.

5. The biomimetic integrated nanozyme for effectively relieving gouty arthritis and inhibiting recurrence according to claim 1, characterized in that: The mass ratio of the macrophage membrane to the platinum nanozyme is 1:(1-5).

6. The biomimetic integrated nanozyme for effectively relieving gouty arthritis and inhibiting recurrence according to claim 1, characterized in that: The bionic integrated nanozyme is prepared by loading the platinum nanozyme onto the surface and pores of the hollow Prussian blue through stirring.

7. The biomimetic integrated nanozyme for effectively relieving gouty arthritis and inhibiting recurrence according to claim 1, characterized in that: The biomimetic integrated nanozyme adopts a polycarbonate membrane co-extrusion method to coat the macrophage membrane on the surface of the integrated nanozyme, and the process includes: extruding the macrophage membrane solution and the integrated nanozyme solution through the polycarbonate membrane to obtain the biomimetic integrated nanozyme.

8. Use of the biomimetic integrated nanozyme that effectively relieves gouty arthritis and inhibits recurrence according to claim 1 in the preparation of a preparation for intravenous administration.

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