A macrophage-inspired nanocomposite drug, its preparation method and application

Abstract

This invention belongs to the field of biochemistry, specifically relating to a macrophage-inspired biomimetic nanocomposite drug, its preparation method, and its application. The preparation method of this macrophage-inspired biomimetic nanocomposite drug includes the following steps: A) reducing chloroplatinic acid to platinum nanoparticles using a trisodium citrate reduction method; B) polymerizing the platinum nanoparticles obtained in step A using a cross-linking agent to obtain platinum nanozyme clusters; C) mixing the platinum nanozyme clusters obtained in step B with a macrophage membrane and sonicating to obtain the macrophage-inspired platinum nanozyme cluster composite drug. This drug can effectively promote kidney repair and can be used to alleviate inflammatory and oxidative stress responses after acute kidney injury, promoting the recovery of normal kidney function.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry, specifically relating to a macrophage-inspired biomimetic nanocomposite drug, its preparation method, and its application. Background Technology

[0002] Acute kidney injury (AKI) is a common clinical syndrome characterized by rapid loss of renal filtration function. Its development is a continuous, dynamic, and complex pathological process, with oxidative stress and inflammatory response being the main influencing factors. Currently, there is a lack of effective drug treatments in clinical practice, and existing small-molecule drugs are insufficient for sustained treatment of the kidneys and promotion of renal function recovery. Compared to the lack of specificity of small-molecule drugs, nanomedicines encapsulated in macrophage membranes can target and accumulate at damaged inflammatory sites, neutralize inflammatory cytokines, and thus control the development of inflammation. Furthermore, the core composed of platinum nanozyme clusters can actively scavenge reactive oxygen species, regulate the redox balance in damaged tissues, and responsively degrade into small-sized platinum nanozymes, which are then excreted through the kidneys.

[0003] Based on this, the inventors pre-constructed a cell-inspired nanocomposite drug with platinum nanozyme clusters encapsulated in macrophage membranes and possessing reactive oxygen species-responsive deformation properties, providing a novel approach for exploring the treatment of acute kidney injury in clinical practice. Summary of the Invention

[0004] This invention provides a macrophage biomimetic composite nanomedicine (MM-PtNCs), which is a macrophage biomimetic platinum nanoenzyme cluster composite drug (MM-PtNCs) obtained by cross-linking platinum nanoparticles obtained by chloroplatinic acid reduction and then encapsulating them through a macrophage membrane (MM).

[0005] The preparation method of the macrophage-inspired biomimetic composite nanomedicine of the present invention includes the following steps:

[0006] A. The chloroplatinic acid (H2PtCl6) was reduced to platinum nanoparticles (PtNPs) using the trisodium citrate (TCD) reduction method.

[0007] B. The platinum nanoparticles obtained in step A are then polymerized with a crosslinking agent to obtain platinum nanozyme clusters (PtNCs for short).

[0008] C. The platinum nanozyme clusters (PtNCs) obtained in step B are mixed with macrophage membranes (MM) and sonicated to prepare a macrophage biomimetic platinum nanozyme cluster composite drug.

[0009] In the above preparation method, step A must satisfy at least one of the following:

[0010] Step A involves preparing platinum nanoparticles by reducing chloroplatinic acid to ultrasmall platinum nanoparticles using the trisodium citrate reduction method. The specific steps include:

[0011] A1. Heat the chloroplatinic acid solution to boiling, then add the trisodium citrate solution. Mix the solutions and heat at 115-125℃ for 15-60 minutes. Stop heating and continue stirring until the reaction temperature drops to room temperature.

[0012] A2. The solution obtained in step a is concentrated by ultrafiltration to obtain platinum nanoparticles.

[0013] In step A1, the molar ratio of chloroplatinic acid to trisodium citrate is 1-3:5-9.

[0014] Preferably, the molar ratio of chloroplatinic acid to trisodium citrate in step A1 is 2:7.

[0015] Wherein, the concentration of the chloroplatinic acid solution in step A1 is 0.8-1.2 mmol / L; and the concentration of the sodium citrate solution is 30-40 mmol / L.

[0016] Preferably, the concentration of the chloroplatinic acid solution in step A1 is 1 mmol / L; and the concentration of the sodium citrate solution is 35 mmol / L.

[0017] The heating conditions for the mixed solution in step A1 are 120°C for 30 minutes.

[0018] In step A2, the ultrafiltration concentration is performed by treating the solution obtained in step a with an ultrafiltration centrifuge tube.

[0019] Furthermore, the solution obtained by the ultrafiltration concentration process in step A2 has a molecular weight cutoff of 10 kD.

[0020] The ultrafiltration centrifuge tube used in step A2 has a working volume of 15 mL and a molecular weight cutoff of 10 kD.

[0021] Preferably, the above method can be used to reduce chloroplatinic acid to ultrasmall platinum nanoparticles using the trisodium citrate reduction method, and the resulting ultrasmall nanoparticles have a particle size of 2-5 nm.

[0022] In the above preparation method, step B must satisfy at least one of the following:

[0023] Step B involves preparing platinum nanozyme clusters (PtNCs) by polymerizing ultra-small platinum nanozyme particles into large-sized platinum nanozyme clusters using a cross-linking agent. The specific steps include:

[0024] B1. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the crosslinking agent and activate for 1.5-3 hours;

[0025] B2. Add mercaptoethylamine and mercaptoethanol to platinum nanoparticles and stir the reaction at room temperature for 2-4 hours;

[0026] B3. Add the activated crosslinking agent obtained in step B1 to the reaction solution described in step B2, and continue stirring the reaction for 10-24 hours;

[0027] B4. After centrifuging the solution from step B3, add ultrapure water and sonicate to disperse it evenly to obtain platinum nanozyme clusters.

[0028] In step B1, the crosslinking agent is a reactive oxygen species-sensitive linker that is pre-activated and reactive oxygen species-responsive crosslinking agent after being treated with EDC and NHS.

[0029] Among them, reactive oxygen species sensitive linkers refer to thioketals containing two carboxyl groups (2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid, propane-2,2-dimethylbis(thio)]diacetic acid), linkers containing two carboxyl groups and a disulfide bond (2,2'-dithiodiacetic acid), and linkers containing two carboxyl groups and a diselenoside bond (3,3'-diselenodipropionic acid, diselenodiglycolic acid).

[0030] Among them, thioacetal, disulfide bond, and diselenyl bond are all sensitive to reactive oxygen species, and carboxyl groups can play a cross-linking role.

[0031] The activation time in step B1 is 2 hours.

[0032] The activation in step B1 is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is first added to the crosslinking agent and reacted for 0.75 h (i.e. 45 minutes), and then N-hydroxysuccinimide (NHS) is added and reacted for 1.5-3 h (i.e., the reaction time after adding NHS is 45 minutes to 135 minutes, or it can be expressed as a reaction time of 0.75-2.25 h).

[0033] Preferably, the activation in step B1 is as follows: first, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is added to the crosslinking agent and reacted for 0.75 h (i.e. 45 minutes), and then N-hydroxysuccinimide (NHS) is added and reacted for 2 h (i.e., the reaction time after adding NHS is 75 minutes).

[0034] In step B1, the molar ratio of the crosslinking agent, EDC, and NHS is 1-2:25-40:5-9.

[0035] Preferably, the molar ratio of the crosslinking agent, EDC and NHS in step B1 is 1:35:7.

[0036] The mercaptoethylamine and mercaptoethanol mentioned in step B2 are in a molar ratio of 2-5:4-8.

[0037] Preferably, in step B2, the mercaptoethylamine and mercaptoethanol are in a molar ratio of 4:6.

[0038] The platinum nanoparticles described in step B2 are mixed with mercaptoethylamine and mercaptoethanol in the following proportions:

[0039] Platinum nanoparticles 0.8-1.2 mg, mercaptoethylamine 0.2-0.8 × 10⁻⁸ mg -3 mmol, mercaptoethanol 0.4-0.8*10 -3 mmol.

[0040] Preferably, the platinum nanoparticles described in step B2 are mixed with mercaptoethylamine and mercaptoethanol in the following ratio:

[0041] Platinum nanoparticles 0.96 mg, mercaptoethylamine 0.4 x 10⁻⁶ -3 mmol, mercaptoethanol 0.6*10 -3 mmol.

[0042] The stirring reaction time in step B2 is 3 hours.

[0043] In step B3, the concentration of the crosslinking agent is 0.06-0.6 mmol / L.

[0044] Preferably, the concentration of the crosslinking agent in step B3 is 0.3 mmol / L.

[0045] The continuous stirring reaction time in step B3 is 12 hours.

[0046] In step B4, the centrifugation speed is 12000-15000 r / min and the centrifugation time is 5-20 minutes.

[0047] The centrifugation speed in step B4 is 15000 r / min, and the centrifugation time is 10 minutes.

[0048] In step B4, the amount of ultrapure water added is the same as the volume ratio of the solution in step B3 to ultrapure water, which is 1-3:10-30.

[0049] Preferably, the amount of ultrapure water added in step B4 is 1:10, which is the volume ratio of the solution in step B3 to the ultrapure water.

[0050] Using the above method, large-sized platinum nanozyme clusters were obtained by cross-linking polymerization. These clusters were small, uniform spheres with a particle size of 40-50 nm.

[0051] In the above preparation method, step C must satisfy at least one of the following:

[0052] The macrophage membrane described in step C was extracted using a hypotonic lysis and gradient centrifugation method.

[0053] a. Collect mouse RAW264.7 cells, wash with phosphate buffer, resuspend in hypotonic solution for lysis, and then homogenize the cell suspension thoroughly using a Dunns homogenizer.

[0054] b. Centrifuge the homogenate to collect the supernatant, homogenize the precipitate again and centrifuge, then combine the two supernatants;

[0055] c. After centrifuging the supernatant once at low speed, take the supernatant and centrifuge it once at high speed to obtain a colorless and transparent liquid, which is the extracted cell membrane. Resuspend it in sterile deionized water to obtain the macrophage cell membrane solution.

[0056] In step a, the phosphate buffer solution has a pH of 7.2-7.4; the hypotonic solution is tris(hydroxymethyl)aminomethane hydrochloride buffer with a concentration of 10 mmol / L.

[0057] In step b, the centrifugal force is 3000-3500g and the centrifugation time is 5-10 minutes.

[0058] Preferably, the centrifugal force in step b is 3200g and the centrifugation time is 5 minutes.

[0059] In step c, the centrifugal forces during the two centrifugations are 20,000g and 100,000g, respectively, and the centrifugation times are 15-25 minutes and 80-100 minutes, respectively.

[0060] Preferably, in step c, the centrifugal forces during the two centrifugations are 20,000g and 100,000g, respectively, and the centrifugation times are 20 minutes and 90 minutes, respectively.

[0061] The colorless and transparent liquid obtained by centrifugation twice in step c is the extracted cell membrane. It is resuspended in sterile deionized water and stored at -80℃ for later use.

[0062] In step C, the platinum nanozyme clusters ((PtNCs)) obtained in step B are mixed with macrophage membranes (MM) in solution form, wherein the volume ratio of macrophage membrane solution to platinum nanocluster solution is 1-3:1.

[0063] Preferably, the volume ratio of macrophage membrane solution to platinum nanocluster solution is 1:1.

[0064] In step C, the mass concentration ratio of the macrophage membrane solution to the platinum nanoclusters solution is 1-6:1.

[0065] Preferably, the mass concentration ratio of macrophage membrane solution to platinum nanoclusters solution is 3:1.

[0066] In step C, the macrophage membrane is mixed with platinum nanoclusters and then sonicated under ice-water bath conditions. The sonication conditions are: sonicate for 1-3 minutes, stop for 1-3 minutes, and repeat 2-5 times.

[0067] Preferably, the ultrasound conditions in step C are: ultrasound for 1 minute, stop for 1 minute, and repeat 3 times.

[0068] The present invention also provides the application of the above-mentioned macrophage biomimetic nanocomposite drug in the preparation of a drug for treating acute kidney injury.

[0069] This invention provides a macrophage-inspired biomimetic nanocomposite drug. Platinum nanozyme clusters, linked by a reactive oxygen species (ROS)-responsive cross-linking agent, can cope with oxidative stress and degrade into ultra-small platinum nanoparticles after scavenging ROS, continuously exerting ROS scavenging effects and being excreted through the kidneys. Furthermore, after being encapsulated by a macrophage membrane, the pro-inflammatory cytokines on the membrane surface allow this nanocomposite drug to target damaged and inflamed kidneys, neutralizing inflammatory factors and thus controlling inflammation. The antioxidant capacity of the platinum nanozyme clusters and the anti-inflammatory activity of the macrophage membrane enable this drug to effectively promote kidney repair. It can be used to alleviate the inflammatory response and oxidative stress response after acute kidney injury, promoting the recovery of normal kidney function. Attached Figure Description

[0070] Figure 1 Flowchart of the preparation method of macrophage biomimetic composite nanomedicine of the present invention.

[0071] Figure 2 TEM images of PtNCs synthesized with different amounts of crosslinking agent.

[0072] Figure 3 TEM images of MM-PtNCs prepared with different MM to PtNCs ratios.

[0073] Figure 4 PtNCs and MM-PtNCs for ABTS + ·and DPPH· · Screenshot showing the result of the cleanup.

[0074] in: Figure 4 a is ABTS + • Screenshot showing the cleanup results Figure 4 b is DPPH· · Screenshot showing the result of the cleanup.

[0075] Figure 5 TEM image of PtNCs after co-incubation with H2O2.

[0076] Figure 6 The effect of MM-PtNCs on neutralizing pro-inflammatory cytokines TNF-α, IL-6, and IL-1β.

[0077] in: Figure 6 Figure a shows the effect of neutralizing the pro-inflammatory cytokine TNF-α. Figure 6 b shows the effect of neutralizing the pro-inflammatory cytokine IL-6. Figure 6 c shows the effect of neutralizing the pro-inflammatory cytokine IL-1β.

[0078] Figure 7 Cell viability graph of HK-2 cells protected by MM-PtNCs from reactive oxygen species damage.

[0079] Figure 8 Serum creatinine levels in AKI model mice within 7 days after treatment.

[0080] Figure 9 Urea levels in AKI model mice within 7 days after treatment. Detailed Implementation

[0081] The present invention will be explained and described below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0082] In the following embodiments, the macrophage biomimetic composite nanomedicine of the present invention is abbreviated as MM-PtNCs; platinum nanozyme clusters are abbreviated as PtNCs; platinum nanoparticles are abbreviated as PtNPs; macrophage membranes are abbreviated as MM; trisodium citrate is abbreviated as TCD; chloroplatinic acid by reduction method is abbreviated as H2PtCl6; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is abbreviated as EDC; and N-hydroxysuccinimide is abbreviated as NHS.

[0083] The preparation method of the macrophage biomimetic composite nanomedicine of the present invention is as follows: Figure 1 It includes the following steps:

[0084] A. The chloroplatinic acid (H2PtCl6) was reduced to platinum nanoparticles (PtNPs) using the trisodium citrate (TCD) reduction method.

[0085] B. The platinum nanoparticles obtained in step A are then polymerized with a crosslinking agent to obtain platinum nanozyme clusters (PtNCs for short).

[0086] C. The platinum nanozyme clusters (PtNCs) obtained in step B are mixed with macrophage membranes (MM) and sonicated to prepare a macrophage biomimetic platinum nanozyme cluster composite drug.

[0087] The preparation method of the macrophage-inspired biomimetic nanocomposite drug of the present invention includes the following steps:

[0088] a. Heat the chloroplatinic acid solution to boiling, then add the sodium citrate solution. After 30 minutes, stop heating and continue stirring until the reaction temperature drops to room temperature. The concentration of the chloroplatinic acid solution is 1 mmol / L and the volume is 200 mL. The concentration of the sodium citrate solution is 35 mmol / L and the volume is 20 mL.

[0089] b. Subsequently, the prepared solution was concentrated by ultrafiltration using an ultrafiltration centrifuge tube to obtain platinum nanoparticles; the working volume of the ultrafiltration tube was 15 mL, and the molecular weight cutoff was 10 kD.

[0090] In the preparation method of the macrophage biomimetic nanocomposite drug of the present invention, the preparation method of the platinum nanoclusters includes the following steps:

[0091] a. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the crosslinking agent and activate for 2 hours; the molar ratio of EDC to NHS is 4-5:1;

[0092] b. Add mercaptoethylamine and mercaptoethanol to platinum nanoparticles and stir the reaction at room temperature for 3 hours; the concentration of the platinum nanoparticles is 2.4 mg / mL and the volume is 400 μL, the concentration of mercaptoethylamine is 10 mmol / L and the volume is 40 μL; the concentration of mercaptoethanol in step b is 10 mmol / L and the volume is 60 μL.

[0093] c. Add a pre-activated reactive oxygen species-responsive crosslinking agent to the above solution and stir continuously for 12 hours; the concentration of the crosslinking agent is 0.3 mmol / L and the volume is 200 μL;

[0094] d. After centrifuging the sample, add 100 μL of ultrapure water and disperse evenly by ultrasonication to obtain platinum nanozyme clusters; the centrifugation speed is 15000 r / min and the centrifugation time is 10 minutes.

[0095] In the preparation method of the macrophage biomimetic nanocomposite drug of the present invention, the macrophage membrane solution preparation method includes the following steps:

[0096] a. Collect mouse RAW264.7 cells, wash with phosphate buffer, resuspend in hypotonic solution for lysis, and homogenize the cell suspension thoroughly using a Durns homogenizer; the pH of the phosphate buffer is 7.2-7.4; the hypotonic solution is tris(hydroxymethyl)aminomethane hydrochloride buffer with a concentration of 10 mmol / L.

[0097] b. After centrifuging the homogenate, collect the supernatant, homogenize the precipitate again, and centrifuge. Combine the supernatants obtained from the two centrifugations. The centrifugal force is 3200g and the centrifugation time is 5 minutes.

[0098] c. After centrifuging the supernatant once at low speed, take the supernatant and centrifuge it once at high speed to obtain a colorless and transparent liquid, which is the extracted cell membrane. Resuspend it in sterile deionized water. During the two centrifugations, the centrifugation forces are 20,000g and 100,000g, and the centrifugation times are 20 minutes and 90 minutes, respectively.

[0099] The preparation method of the macrophage biomimetic nanocomposite drug of the present invention includes the following steps: mixing macrophage membranes with a platinum nanocluster solution, sonicating the mixture under ice-water bath conditions for 1 minute, stopping for 1 minute, repeating this process 3 times, and then centrifuging and resuspending to obtain the macrophage biomimetic platinum nanocluster composite drug. The volume ratio of the macrophage membrane to the platinum nanocluster solution is 1:1, and the mass concentration ratio is 3:1.

[0100] The chloroplatinic acid (H2PtCl6), mercaptoethylamine, and mercaptoethanol used in the embodiments of this invention were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), sodium citrate, and catalase (CAT) were purchased from Sigma-Aldrich, USA; 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS) was purchased from Roche; and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was purchased from Alfa Aesar. DMEM / F-12, DMEM, fetal bovine serum, and penicillin-streptomycin solution (double antibiotic) were purchased from Gibico, USA. TNF-α, IL-1β, and IL-6 cytokines were purchased from Preprotein. BCA protein concentration assay kits, mouse tumor necrosis factor α ELISA kits (Mouse TNF-α ELISA Kit), mouse interleukin-1β ELISA kits (Mouse IL-1β ELISA Kit), and mouse interleukin-6 ELISA kits (Mouse IL-6 ELISA Kit) were purchased from Shanghai Yisheng Biotechnology Co., Ltd. Rabbit anti-mouse interleukin-6 polyclonal antibody (Anti-IL-6 Rabbit pAb), rabbit anti-mouse interleukin-1β polyclonal antibody (Anti-IL-1β Rabbit pAb), and rabbit anti-mouse tumor necrosis factor (Anti-TNF-α Rabbit pAb) were purchased from Abcam. Tetramethylazoazole (MTT) was purchased from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Human renal cortical proximal tubule epithelial cells (HK-2 cells) were purchased from the American Type Culture Collection (ATCC). SPF (Specific Pathogen Free) grade C57BL / 6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0101] Example 1

[0102] 1. Preparation of ultrasmall platinum nanoparticles:

[0103] A chloroplatinic acid solution (1 mmol / L, 200 mL) was heated to boiling at 120 °C, followed by the addition of a sodium citrate solution (35 mmol / L, 20 mL). The reaction was allowed to proceed for 30 minutes. Heating was stopped when the color changed from yellow to brownish-black, and the mixture was stirred until it reached room temperature. The resulting solution was then subjected to ultrafiltration centrifugation using a 10 kDa ultrafiltration centrifuge tube with a sample volume of 15 mL at 3500 rpm for 10 minutes. Deionized water was added, and the mixture was centrifuged under the same conditions. The final volume was adjusted to 5 mL to obtain ultrafine platinum nanoparticles.

[0104] 2. Activation of the crosslinking agent:

[0105] Weigh 1.7 mg of crosslinking agent (propane-2,2-dimethylbis(thio)diacetic acid), 38 mg of EDC, and 5.8 mg of NHS. Add 1 mL of DMSO to each, sonicate to dissolve, and pipette the entire 1 mL crosslinking agent solution into a 50 mL centrifuge tube. Add another 17 mL of DMSO and sonicate again. Add 1 mL each of EDC and NHS to the centrifuge tube sequentially and place in a dark, 37°C, 200 rpm shaker for 2 hours. Take 2 mL of the obtained solution and add 8 mL of DMSO. The activated crosslinking agent is obtained.

[0106] 3. Preparation of platinum nanoclusters:

[0107] Mercaptoethylamine (10 mmol / L, 40 μL) and mercaptoethanol (10 mmol / L, 60 μL) were added to PtNPs (2.4 mg / mL, 400 μL). After stirring at room temperature for 3 hours, the solution was brought to a final volume of 1 mL with PBS (pH 7.4). A pre-activated crosslinking agent (200 μL) was then added to the solution, and the mixture was stirred for another 12 hours. After the reaction was complete, the sample was centrifuged at 15000 rpm for 10 minutes, and then 100 μL of ultrapure water was added. The mixture was then ultrasonically dispersed to obtain platinum nanoclusters.

[0108] Example 2

[0109] 1. Preparation of ultrasmall platinum nanoparticles:

[0110] A chloroplatinic acid solution (1 mmol / L, 200 mL) was heated to boiling at 120 °C, followed by the addition of a sodium citrate solution (35 mmol / L, 20 mL). The reaction was allowed to proceed for 30 minutes. Heating was stopped when the color changed from yellow to brownish-black, and the mixture was stirred until it reached room temperature. The resulting solution was then subjected to ultrafiltration centrifugation using a 10 kDa ultrafiltration centrifuge tube with a sample volume of 15 mL at 3500 rpm for 10 minutes. Deionized water was added, and the mixture was centrifuged under the same conditions. The final volume was adjusted to 5 mL to obtain ultrafine platinum nanoparticles.

[0111] 2. Activation of the crosslinking agent:

[0112] Weigh 1.7 mg of crosslinking agent (propane-2,2-dimethylbis(thio)diacetic acid), 38 mg of EDC, and 5.8 mg of NHS. Add 1 mL of DMSO to each, sonicate to dissolve, and pipette 1 mL of the crosslinking agent solution into a 50 mL centrifuge tube. Add another 17 mL of DMSO and sonicate again. Add 1 mL each of EDC and NHS to the centrifuge tube sequentially and place in a dark 37°C, shake at 200 rpm for 2 hours. The activated crosslinking agent is obtained.

[0113] 3. Preparation of platinum nanoclusters:

[0114] Mercaptoethylamine (10 mmol / L, 40 μL) and mercaptoethanol (10 mmol / L, 60 μL) were added to ultrasmall platinum nanoparticles (2.4 mg / mL, 400 μL). After stirring at room temperature for 3 hours, the solution was brought to a final volume of 1 mL with PBS (pH 7.4). A pre-activated crosslinking agent (200 μL) was then added to the solution, and the mixture was stirred for another 12 hours. After the reaction was complete, the sample was centrifuged at 15000 rpm for 10 minutes, and then 100 μL of ultrapure water was added. The mixture was then ultrasonically dispersed to obtain platinum nanoclusters.

[0115] Example 3

[0116] 1. Preparation of ultrasmall platinum nanoparticles:

[0117] A chloroplatinic acid solution (1 mmol / L, 200 mL) was heated to boiling at 120 °C, followed by the addition of a sodium citrate solution (35 mmol / L, 20 mL). The reaction was allowed to proceed for 30 minutes. Heating was stopped when the color changed from yellow to brownish-black, and the mixture was stirred until it reached room temperature. The resulting solution was then subjected to ultrafiltration centrifugation using a 10 kDa ultrafiltration centrifuge tube with a sample volume of 15 mL at 3500 rpm for 10 minutes. Deionized water was added, and the mixture was centrifuged under the same conditions. The final volume was adjusted to 5 mL to obtain ultrafine platinum nanoparticles.

[0118] 2. Activation of the crosslinking agent:

[0119] 3.4 mg crosslinking agent (propane-2,2-dimethylbis(thio)diacetic acid), 76 mg EDC, and 10.6 mg NHS were dissolved in 1 mL of DMSO by sonication. 1 mL of the crosslinking agent solution was then added to a 50 mL centrifuge tube by pipette. 17 mL of DMSO was added and sonicated again. 1 mL of EDC and 1 mL of NHS were added to the centrifuge tube and the tube was placed in a dark place at 37°C and shaken at 200 rpm for 2 hours.

[0120] 3. Preparation of platinum nanoclusters:

[0121] Mercaptoethylamine (10 mmol / L, 40 μL) and mercaptoethanol (10 mmol / L, 60 μL) were added to PtNPs (2.4 mg / mL, 400 μL). After stirring at room temperature for 3 hours, the solution was brought to a final volume of 1 mL with PBS (pH 7.4). A pre-activated crosslinking agent (200 μL) was then added to the solution, and the mixture was stirred for another 12 hours. After the reaction was complete, the sample was centrifuged at 15000 rpm for 10 minutes, and then 100 μL of ultrapure water was added. The mixture was then ultrasonically dispersed to obtain platinum nanoclusters.

[0122] Example 4

[0123] A 250 μL macrophage membrane solution (6 mg / mL) was mixed with a 250 μL platinum nanoclusters (1 mg / mL) prepared according to Example 2. The mixture was then sonicated in an ice-water bath. The sonication was repeated three times, with a 1-minute interval between cycles. The sample was then centrifuged at 10,000 rpm for 10 minutes and resuspended in 100 μL of PBS to obtain the cell-inspired composite nanomedicine.

[0124] Example 5

[0125] A 3 mg / mL, 250 μL macrophage membrane solution was mixed with a 1 mg / mL, 250 μL platinum nanoclusters (prepared according to the method in Example 2), and the mixture was sonicated in an ice-water bath. The sonication was repeated three times, with a 1-minute interval between cycles. The sample was then centrifuged at 10,000 rpm for 10 minutes and resuspended in 100 μL of PBS to obtain the cell-inspired composite nanomedicine.

[0126] Example 6

[0127] A 250 μL macrophage membrane solution (2 mg / mL) was mixed with a 250 μL platinum nanoclusters (1 mg / mL) prepared according to Example 2. The mixture was then sonicated in an ice-water bath. The sonication was repeated three times, with a 1-minute interval between cycles. The sample was then centrifuged at 10,000 rpm for 10 minutes and resuspended in 100 μL of PBS to obtain the cell-inspired composite nanomedicine.

[0128] Example 7

[0129] A 1 mg / mL, 250 μL macrophage membrane solution was mixed with a 1 mg / mL, 250 μL platinum nanoclusters (prepared according to the method in Example 2), and the mixture was sonicated in an ice-water bath. The sonication was repeated three times, with a 1-minute interval between cycles. The sample was then centrifuged at 10,000 rpm for 10 minutes and resuspended in 100 μL of PBS to obtain the cell-inspired composite nanomedicine.

[0130] Example 8: Investigation of Crosslinking Agent Dosage

[0131] The PtNCs prepared in Examples 1, 2, and 3 were dropped onto an ultrathin copper mesh to prepare samples. The morphology of the nanoparticles was examined by transmission electron microscopy to determine the optimal amount of crosslinking agent. The results are as follows: Figure 2As shown, Examples 1, 2, and 3 exhibit different morphological characteristics. In Example 1, although spherical clusters are formed, many small particles remain uncrosslinked into spherical shapes and do not separate from the formed clusters, resulting in uneven dispersion of the final solution. In Example 3, PtNPs fail to aggregate into regular, uniform spherical particles, leading to poor solution stability and unfavorable in vivo drug delivery due to excessively large particle size. In contrast, the crosslinked PtNCs obtained in Example 2 exhibit the optimal morphology, consisting of uniformly sized spherical particles with a particle size of 40-50 nm.

[0132] In the process of screening crosslinking agents, the inventors investigated a variety of reactive oxygen species-sensitive linkers. Crosslinking agents capable of achieving the aforementioned crosslinking effect include thioketals containing two carboxyl groups (2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid, propane-2,2-dimethylbis(thio)]diacetic acid), linkers containing two carboxyl groups and a disulfide bond (2,2'-dithiodiacetic acid), and linkers containing two carboxyl groups and a diselenate bond (3,3'-diselenodipropionic acid, diselenodiglycolic acid). Because thioketals, disulfide bonds, and diselenate bonds are sensitive to reactive oxygen species, and the carboxyl groups can play a crosslinking role, these crosslinking agents can be used in the preparation of this invention.

[0133] Example 9: Screening of the concentration ratio of MM and PtNCs solution

[0134] The composite nanomedicines prepared in Examples 4, 5, 6, and 7 were dropped onto an ultrathin copper mesh to prepare samples. The morphology of the nanoparticles was examined using transmission electron microscopy to determine the optimal mixing ratio. The results are as follows: Figure 3 As shown, Examples 4, 5, 6, and 7 exhibit different morphological characteristics, while the composite nanomedicine in Example 5 has a uniformly thick and completely encapsulated membrane structure. The inventors ultimately used the ratio (3:1) from Example 5 as the subsequent usage ratio.

[0135] Example 10 Antioxidant Experiment (ABTS Free Radical Scavenging Ability)

[0136] 5 mL of ABTS (2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt) stock solution (7.4 mmol / L) was mixed with 88 μL of K₂S₂O₈ stock solution (2.6 mmol / L) and allowed to stand for 12–16 hours to prepare the ABTS working solution. 0.4 mL of the ABTS working solution was diluted with ethanol to obtain an absorbance of 0.74 ± 0.02 at 734 nm at room temperature. 0.2 mL of the above solution was mixed with the nanomedicine solutions (0.1–1 μg / mL, 10 μL) prepared in Examples 2 and 5, respectively. The absorbance at 734 nm was then measured using a chemiluminescence immunoassay reader (SpectraMax ABS Plus) and the changes were recorded over 10 minutes.

[0137] The free radical scavenging capacity of ABTS is calculated by the following formula:

[0138] Clearance rate (%) = [(A0-A i [) / A0]×100, where: A0 is the value of ABTS added only, without sample. + Absorbance of · A i For adding samples and ABTS + · Absorbance.

[0139] Test results: such as Figure 4 As shown in Figure a, the nanomedicines prepared in Examples 2 and 5 showed ABTS free radical scavenging rates of 88.3% and 89.5% at a concentration of 1 μg / mL, respectively. This indicates that the nanoclusters and composite nanomedicines have a good scavenging effect on ABTS free radicals and have antioxidant properties.

[0140] Example 11 Antioxidant Experiment (DPPH Free Radical Scavenging Ability)

[0141] First, prepare a 40 μM DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) working solution. Dilute the working solution with ethanol to adjust its concentration so that its absorbance at 517 nm is approximately 0.9. Mix the working solution and the sample, and record the absorbance at 517 nm. The measurement and calculation methods for DPPH free radical scavenging ability are the same as above.

[0142] Test results: such as Figure 4 As shown in b, the nanomedicines prepared in Examples 2 and 5 showed DPPH free radical scavenging rates of 82.6% and 81.9% at a concentration of 1 μg / mL, respectively. This indicates that the nanoclusters and composite nanomedicines have a good scavenging effect on DPPH free radicals and have antioxidant effects.

[0143] Example 12 Responsive Deformation Experiment

[0144] After incubating 20 μg / mL PtNCs with 20 μM H2O2 solution for 30 minutes, the nanoclusters were removed by centrifugation. The morphological changes were examined by transmission electron microscopy to evaluate the reactive oxygen species-responsive deformation properties of PtNCs.

[0145] Test results: such as Figure 5 As shown, large nanoclusters responsively degrade and transform into ultrasmall platinum nanoparticles under the action of hydrogen peroxide.

[0146] Example 13 Anti-inflammatory experiment

[0147] Recombinant mouse IL-1β, TNF-α, or IL-6 were mixed with different concentrations of MM and MM-PtNCs, respectively, and then incubated at 37°C for 2 hours. The nanocomposites were then removed by centrifugation at 100,000g for 30 minutes, and the supernatant was collected. The levels of pro-inflammatory cytokines IL-1β, TNF-α, or IL-6 in the supernatant were quantitatively analyzed using an ELISA kit.

[0148] Test results: such as Figure 6 As shown, after co-incubating the cell membrane with pro-inflammatory cytokines, the cytokine content in the solution gradually decreased with the increase of membrane concentration, indicating that macrophage membranes can indeed neutralize pro-inflammatory cytokines and exert an anti-inflammatory effect.

[0149] Example 14 Evaluation of the ability of MM-PtNCs to protect cells from reactive oxygen species damage

[0150] The ability of nanocomplexes to maintain cell viability under high reactive oxygen species (ROS) conditions was evaluated using the MTT assay. HK-2 cells were seeded in 96-well plates. After 24 hours, the culture medium was replaced with solutions containing different concentrations of PtNCs or MM-PtNCs (12.5–200 μg / mL), with an equal volume of medium solution without nanoclusters serving as a control. After 15 minutes, to simulate a high ROS environment in vivo, H2O2 was added to the culture medium to a final concentration of 500 μM. After 24 hours, cell viability was measured using the MTT assay: MTT solution (5 mg / mL, 20 μL / well) was added directly to the 96-well plate under dark conditions. The plate was incubated at 37°C for 3 hours, the supernatant was discarded, 150 μL of dimethyl sulfoxide was added, and the plate was shaken for 10 minutes. The nanoparticles were then removed by centrifugation at 4000 rpm for 5 minutes to avoid affecting the measurement. Finally, the supernatant was aspirated and the absorbance at 570 nm was measured using an ELISA reader.

[0151] Test results: After incubation for 24 hours with different concentrations of nanoclusters in a 500 μM H2O2 culture environment, the viability of HK-2 cells was as follows: Figure 7 As shown, compared to the control group without any treatment, the cell viability of the experimental group with only H2O2 added decreased to 59.01%. However, with the addition of PtNCs and MM-PtNCs, cell viability improved. At a concentration of 25 μg / mL, PtNCs and MM-PtNCs achieved cell viability of 92.37% and 92.52%, respectively. Furthermore, with MM-PtNCs at concentrations of 50-200 μg / mL, cell viability exceeded 100%, and H2O2 did not affect cell growth. These results indicate that nanoclusters can effectively alleviate the damage caused by H2O2 to cells and maintain cell growth.

[0152] Example 15: Evaluation of the in vivo therapeutic effect of MM-PtNCs

[0153] Bilateral renal pedicles were clamped with non-traumatic arterial clamps for 35 minutes, and then the clamps were removed to allow blood reperfusion, thus establishing an AKI model. On the day the mouse AKI model was successfully established, the mice were treated, with the following groupings and drug dosages: MM group (30 mg / kg); PtNCs group (15 mg / kg); MM-PtNCs group (15 mg / kg); and a sham-operated group (Sham group) and a group receiving an equal volume of physiological saline after modeling (Saline group) as controls. Blood and kidneys were collected from mice in each group on days 1, 3, and 7 after treatment. The treatment effect was mainly assessed using blood biochemical indicators—serum creatinine (CREA) and urea (UREA).

[0154] Test results: The CREA value of AKI model mice within 7 days after treatment was as follows: Figure 8 As shown, the CREA values ​​at all time points in the Sham group were not significantly different, so its average value (47.32±2.88) μmol / L was used as a control. The CREA value in the Saline group increased sharply on postoperative day 1, reaching five times that of the Sham group ((257.83±17.57) μmol / L), and remained twice that of the Sham group on postoperative day 7. The CREA value in the MM group rose rapidly on postoperative day 1 and then rapidly decreased, returning to normal levels on day 7 (46.84±12.59) μmol / L. Although both MM and PtNCs showed some therapeutic effect, the MM-PtNCs group showed a better therapeutic effect, with no significant difference in CREA levels between the MM and Sham groups at any of the three postoperative time points.

[0155] The UREA values ​​of AKI model mice within 7 days after treatment are as follows: Figure 9As shown, the changes in UEA values ​​exhibited similar trends. The average total UEA level in the Sham group over 7 days was (8.78±0.58) mmol / L. In the Saline group, it reached (82.86±5.11) mmol / L on day 1 post-surgery, remained almost unchanged on day 3, and was still at a relatively high level of (42.38±11.20) mmol / L on day 7. Both the PtNCs and MM groups showed varying degrees of increase post-surgery, but recovered to lower levels on day 7, at (18.5±3.06) mmol / L and (16.81±5.84) mmol / L, respectively. Compared to the higher UEA levels on day 1 in the first three experimental groups, the UEA level in the MM-PtNCs group was only (18.81±7.17) mmol / L on day 1, and by day 7, the UEA had decreased to (9.32±0.94) mmol / L, showing no significant difference from the Sham group. This indicates that the nano-bionic system of the present invention exhibits good effects in resisting kidney damage and protecting kidney function.

[0156] In summary, the macrophage-inspired biomimetic nanocomposite drug of this invention can address oxidative stress through platinum nanozyme clusters linked by reactive oxygen species-responsive cross-linking agents. After scavenging reactive oxygen species, it degrades into ultra-small platinum nanoparticles, continuously exerting its reactive oxygen species scavenging effect, and can be excreted through the kidneys. This invention provides a novel clinical application strategy for alleviating inflammatory and oxidative stress responses following acute kidney injury and promoting the recovery of normal kidney function.

Claims

1. A macrophage-inspired biomimetic composite nanomedicine, characterized in that: It is a macrophage-inspired platinum nanozyme cluster composite drug obtained by cross-linking platinum nanoparticles obtained by chloroplatinic acid reduction, followed by encapsulation in a macrophage membrane; the preparation method includes the following steps: A. Chloroplatinic acid was reduced to platinum nanoparticles using the trisodium citrate reduction method; B. The platinum nanoparticles obtained in step A are then polymerized with a crosslinking agent to obtain platinum nanozyme clusters; C. Mix the platinum nanozyme clusters obtained in step B with the macrophage membrane and sonicate to prepare a macrophage biomimetic platinum nanozyme cluster composite drug. in: Step B involves preparing platinum nanozyme clusters by polymerizing ultra-small platinum nanozyme particles into large-sized platinum nanozyme clusters using a cross-linking agent, and includes the following steps: B1. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the crosslinking agent and activate for 1.5-3 hours; B2. Add mercaptoethylamine and mercaptoethanol to platinum nanoparticles and stir the reaction at room temperature for 2-4 hours; B3. Add the activated crosslinking agent obtained in step B1 to the reaction solution described in step B2, and continue stirring the reaction for 10-24 hours. B4. After centrifuging the solution from step B3, add ultrapure water and disperse it evenly by ultrasonication to obtain platinum nanozyme clusters. Wherein, the crosslinking agent mentioned in step B1 is a reactive oxygen species-sensitive linker obtained by treating 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a pre-activated reactive oxygen species-responsive crosslinking agent; the reactive oxygen species-sensitive linker refers to a thioketal linker containing two carboxyl groups, a linker containing two carboxyl groups and a disulfide bond, and a linker containing two carboxyl groups and a diselenide bond; In step B2, the mercaptoethylamine and mercaptoethanol are in a molar ratio of 2-5:4-8. In step B2, the platinum nanoparticles, mercaptoethylamine, and mercaptoethanol are mixed in the following proportions: Platinum nanoparticles 0.8-1.2 mg, mercaptoethylamine 0.2-0.8 × 10⁻⁸ mg -3 mmol, mercaptoethanol 0.4-0.8*10 -3 mmol; Wherein, the concentration of the crosslinking agent mentioned in step B3 is 0.06-0.6 mmol / L; In step B4, the centrifugation speed is 12000-15000 r / min and the centrifugation time is 5-20 minutes. In step B4, the amount of ultrapure water added is the same as the volume ratio of the solution in step B3 to ultrapure water, which is 1-3:10-30.

2. The macrophage-inspired biomimetic composite nanomedicine according to claim 1, characterized in that: Step A involves preparing platinum nanoparticles by reducing chloroplatinic acid to ultrasmall platinum nanoparticles using the trisodium citrate reduction method. The specific steps include: A1. Heat the chloroplatinic acid solution to boiling, then add the trisodium citrate solution. Mix the solutions and heat at 115-125℃ for 15-60 minutes. Stop heating and continue stirring until the reaction temperature drops to room temperature. A2. The solution obtained in step a is concentrated by ultrafiltration to obtain platinum nanoparticles; Wherein: the molar ratio of chloroplatinic acid to trisodium citrate in step A1 is 1-3:5-9; Wherein, the concentration of the chloroplatinic acid solution in step A1 is 0.8-1.2 mmol / L; and the concentration of the sodium citrate solution is 30-40 mmol / L; In step A2, the ultrafiltration concentration is performed by treating the solution obtained in step a with an ultrafiltration centrifuge tube.

3. The macrophage-inspired biomimetic composite nanomedicine according to claim 2, characterized in that: Step A must satisfy at least one of the following: In step A1, the molar ratio of chloroplatinic acid to trisodium citrate is 2:

7. Wherein, the concentration of the chloroplatinic acid solution in step A1 is 1 mmol / L; and the concentration of the sodium citrate solution is 35 mmol / L; The heating conditions for the mixed solution in step A1 are: heating at 120°C for 30 minutes; The solution obtained by ultrafiltration concentration in step A2 has a molecular weight cutoff of 10 kD. The ultrafiltration centrifuge tube used in step A2 has a working volume of 15 mL and a molecular weight cutoff of 10 kD. The above method can be used to reduce chloroplatinic acid to ultra-small platinum nanoparticles using trisodium citrate reduction, and the resulting ultra-small nanoparticles have a particle size of 2-5 nm.

4. The macrophage-inspired biomimetic composite nanomedicine according to claim 1, characterized in that: Step B must satisfy at least one of the following: The thioketal linker containing two carboxyl groups is 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, or propane-2,2-diylbis(thio)]diacetic acid; The linker containing two carboxyl groups and a disulfide bond is 2,2'-dithiodiacetic acid; The linker containing two carboxyl groups and a diselenium bond is 3,3'-diselenodipropionic acid or diselenodiethanolic acid; The activation time in step B1 is 2 hours. The activation in step B1 is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is first added to the crosslinking agent and reacted for 0.75 h, and then N-hydroxysuccinimide is added and reacted for 1.5-3 h. The molar ratio of the crosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step B1 is 1-2:25-40:5-9. In step B2, the mercaptoethylamine and mercaptoethanol are in a molar ratio of 2-5:4-8. In step B2, the platinum nanoparticles, mercaptoethylamine, and mercaptoethanol are mixed in the following proportions: Platinum nanoparticles 0.8-1.2 mg, mercaptoethylamine 0.2-0.8 × 10⁻⁸ mg -3 mmol, mercaptoethanol 0.4-0.8*10 -3 mmol; The stirring reaction time in step B2 is 3 hours; Wherein, the concentration of the crosslinking agent mentioned in step B3 is 0.06-0.6 mmol / L; The continuous stirring reaction time in step B3 is 12 hours. In step B4, the centrifugation speed is 12000-15000 r / min and the centrifugation time is 5-20 minutes. In step B4, the amount of ultrapure water added is the same as the volume ratio of the solution in step B3 to ultrapure water, which is 1-3:10-30. Among them, large-sized platinum nanozyme clusters were obtained by cross-linking and polymerization using the above method. These clusters are small, uniform spheres with a particle size of 40-50 nm.

5. The macrophage-inspired biomimetic composite nanomedicine according to claim 3, characterized in that: Step B must satisfy at least one of the following: The reactive oxygen-sensitive linker is propane-2,2-dimethylbis(thio)diacetic acid; The activation in step B1 is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is first added to the crosslinking agent and reacted for 0.75 h, and then N-hydroxysuccinimide is added and reacted for 2 h. The molar ratio of the crosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step B1 is 1:35:

7. In step B2, the mercaptoethylamine and mercaptoethanol are in a molar ratio of 4:

6. In step B2, the platinum nanoparticles, mercaptoethylamine, and mercaptoethanol are mixed in the following proportions: Platinum nanoparticles 0.96 mg, mercaptoethylamine 0.4 x 10⁻⁶ -3 mmol, mercaptoethanol 0.6*10 -3 mmol; Wherein, the concentration of the crosslinking agent mentioned in step B3 is 0.3 mmol / L; The centrifugation speed in step B4 is 15000 r / min, and the centrifugation time is 10 minutes; In step B4, the amount of ultrapure water added is 1:10, which is the volume ratio of the solution in step B3 to the ultrapure water.

6. The macrophage-inspired biomimetic composite nanomedicine according to claim 1, characterized in that: The macrophage membrane described in step C is extracted using a hypotonic lysis and gradient centrifugation method, including the following steps: a. Collect mouse RAW264.7 cells, wash with phosphate buffer, resuspend in hypotonic solution for lysis, and then homogenize the cell suspension thoroughly using a Dunns homogenizer. b. Centrifuge the homogenate to collect the supernatant, homogenize the precipitate again and centrifuge, then combine the two supernatants; c. After centrifuging the supernatant once at low speed, take the supernatant and centrifuge it once at high speed to obtain a colorless and transparent liquid, which is the extracted cell membrane. Resuspend it in sterile deionized water to obtain the macrophage cell membrane solution.

7. The macrophage-inspired biomimetic composite nanomedicine according to claim 6, characterized in that: Step C must satisfy at least one of the following: In step a, the phosphate buffer solution has a pH of 7.2-7.4; the hypotonic solution is tris(hydroxymethyl)aminomethane hydrochloride buffer with a concentration of 10 mmol / L. In step b, the centrifugal force is 3000-3500g and the centrifugation time is 5-10 minutes; In step c, the centrifugal forces during the two centrifugations are 20,000g and 100,000g, respectively, and the centrifugation times are 15-25 minutes and 80-100 minutes, respectively. In step c, the colorless and transparent liquid obtained by centrifugation twice is the extracted cell membrane, which is resuspended in sterile deionized water and stored at -80℃ for later use. In step C, the platinum nanozyme clusters (PtNCs) obtained in step B are mixed with macrophage membranes (MM) in solution form, with the volume ratio of macrophage membrane solution to platinum nanocluster solution being 1-3:

1. In step C, the mass concentration ratio of the macrophage membrane solution to the platinum nanocluster solution is 1-6:

1. In step C, the macrophage membrane is mixed with platinum nanoclusters and then sonicated under ice-water bath conditions. The sonication conditions are: sonicate for 1-3 minutes, stop for 1-3 minutes, and repeat 2-5 times.

8. The macrophage-inspired biomimetic composite nanomedicine according to claim 7, characterized in that: Step C must satisfy at least one of the following: In step b, the centrifugal force is 3200 g and the centrifugation time is 5 minutes. In step c, the centrifugal forces during the two centrifugations are 20,000g and 100,000g, respectively, and the centrifugation times are 20 minutes and 90 minutes, respectively. The volume ratio of the macrophage membrane solution to the platinum nanocluster solution was 1:

1. The mass concentration ratio of macrophage membrane solution to platinum nanoclusters solution was 3:

1. In step C, the ultrasound conditions are: ultrasound for 1 minute, stop for 1 minute, and repeat 3 times.

9. The method for preparing the macrophage-inspired biomimetic composite nanomedicine according to claim 1, characterized in that: Includes the following steps: A. Chloroplatinic acid was reduced to platinum nanoparticles using the trisodium citrate reduction method; B. The platinum nanoparticles obtained in step A are then polymerized with a crosslinking agent to obtain platinum nanozyme clusters; C. Mix the platinum nanozyme clusters obtained in step B with the macrophage membrane and sonicate to prepare a macrophage biomimetic platinum nanozyme cluster composite drug. Step B involves preparing platinum nanozyme clusters by polymerizing ultra-small platinum nanozyme particles into large-sized platinum nanozyme clusters using a cross-linking agent, and includes the following steps: B1. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the crosslinking agent and activate for 1.5-3 hours; B2. Add mercaptoethylamine and mercaptoethanol to platinum nanoparticles and stir the reaction at room temperature for 2-4 hours; B3. Add the activated crosslinking agent obtained in step B1 to the reaction solution described in step B2, and continue stirring the reaction for 10-24 hours. B4. After centrifuging the solution from step B3, add ultrapure water and disperse it evenly by ultrasonication to obtain platinum nanozyme clusters. Wherein, the crosslinking agent mentioned in step B1 is a reactive oxygen species-sensitive linker obtained by treating 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a pre-activated reactive oxygen species-responsive crosslinking agent; the reactive oxygen species-sensitive linker refers to a thioketal linker containing two carboxyl groups, a linker containing two carboxyl groups and a disulfide bond, and a linker containing two carboxyl groups and a diselenide bond; In step B2, the mercaptoethylamine and mercaptoethanol are in a molar ratio of 2-5:4-8. In step B2, the platinum nanoparticles, mercaptoethylamine, and mercaptoethanol are mixed in the following proportions: Platinum nanoparticles 0.8-1.2 mg, mercaptoethylamine 0.2-0.8 × 10⁻⁸ mg -3 mmol, mercaptoethanol 0.4-0.8*10 -3 mmol; Wherein, the concentration of the crosslinking agent mentioned in step B3 is 0.06-0.6 mmol / L; In step B4, the centrifugation speed is 12000-15000 r / min and the centrifugation time is 5-20 minutes. In step B4, the amount of ultrapure water added is the same as the volume ratio of the solution in step B3 to ultrapure water, which is 1-3:10-30.

10. The method for preparing macrophage-inspired biomimetic composite nanomedicine according to claim 9, characterized in that: Step A involves preparing platinum nanoparticles by reducing chloroplatinic acid to ultrasmall platinum nanoparticles using the trisodium citrate reduction method. The specific steps include: A1. Heat the chloroplatinic acid solution to boiling, then add the trisodium citrate solution. Mix the solutions and heat at 115-125℃ for 15-60 minutes. Stop heating and continue stirring until the reaction temperature drops to room temperature. A2. The solution obtained in step a is concentrated by ultrafiltration to obtain platinum nanoparticles; Wherein: the molar ratio of chloroplatinic acid to trisodium citrate in step A1 is 1-3:5-9; Wherein, the concentration of the chloroplatinic acid solution in step A1 is 0.8-1.2 mmol / L; and the concentration of the sodium citrate solution is 30-40 mmol / L; In step A2, the ultrafiltration concentration is performed by treating the solution obtained in step a with an ultrafiltration centrifuge tube.

11. The method for preparing macrophage-inspired biomimetic composite nanomedicine according to claim 10, characterized in that: Step A must satisfy at least one of the following: In step A1, the molar ratio of chloroplatinic acid to trisodium citrate is 2:

7. Wherein, the concentration of the chloroplatinic acid solution in step A1 is 1 mmol / L; and the concentration of the sodium citrate solution is 35 mmol / L; The heating conditions for the mixed solution in step A1 are: heating at 120°C for 30 minutes; The solution obtained by ultrafiltration concentration in step A2 has a molecular weight cutoff of 10 kD. The ultrafiltration centrifuge tube used in step A2 has a working volume of 15 mL and a molecular weight cutoff of 10 kD. Using the above method, chloroplatinic acid can be reduced to ultra-small platinum nanoparticles by the trisodium citrate reduction method, and the resulting ultra-small nanoparticles have a particle size of 2-5 nm.

12. The method for preparing macrophage-inspired biomimetic composite nanomedicine according to claim 9, characterized in that: Step B must satisfy at least one of the following: The thioketal linker containing two carboxyl groups is 2,2'-[propane-2,2-diylbis(thio)]diacetic acid, or propane-2,2-diylbis(thio)]diacetic acid; The linker containing two carboxyl groups and a disulfide bond is 2,2'-dithiodiacetic acid; The linker containing two carboxyl groups and a diselenium bond is 3,3'-diselenodipropionic acid or diselenodiethanolic acid; The activation time in step B1 is 2 hours. The activation in step B1 is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is first added to the crosslinking agent and reacted for 0.75 h, and then N-hydroxysuccinimide is added and reacted for 1.5-3 h. The molar ratio of the crosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step B1 is 1-2:25-40:5-9. In step B2, the mercaptoethylamine and mercaptoethanol are in a molar ratio of 2-5:4-8. In step B2, the platinum nanoparticles, mercaptoethylamine, and mercaptoethanol are mixed in the following proportions: Platinum nanoparticles 0.8-1.2 mg, mercaptoethylamine 0.2-0.8 × 10⁻⁸ mg -3 mmol, mercaptoethanol 0.4-0.8*10 -3 mmol; The stirring reaction time in step B2 is 3 hours; Wherein, the concentration of the crosslinking agent mentioned in step B3 is 0.06-0.6 mmol / L; The continuous stirring reaction time in step B3 is 12 hours. In step B4, the centrifugation speed is 12000-15000 r / min and the centrifugation time is 5-20 minutes. In step B4, the amount of ultrapure water added is the same as the volume ratio of the solution in step B3 to ultrapure water, which is 1-3:10-30. Among them, large-sized platinum nanozyme clusters were obtained by cross-linking and polymerization using the above method. These clusters are small, uniform spheres with a particle size of 40-50 nm.

13. The method for preparing macrophage-inspired biomimetic composite nanomedicine according to claim 12, characterized in that: Step B must satisfy at least one of the following: The reactive oxygen-sensitive linker is propane-2,2-dimethylbis(thio)diacetic acid; The activation in step B1 is as follows: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is first added to the crosslinking agent and reacted for 0.75 h, and then N-hydroxysuccinimide is added and reacted for 2 h. The molar ratio of the crosslinking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step B1 is 1:35:

7. In step B2, the mercaptoethylamine and mercaptoethanol are in a molar ratio of 4:

6. In step B2, the platinum nanoparticles, mercaptoethylamine, and mercaptoethanol are mixed in the following proportions: Platinum nanoparticles 0.96 mg, mercaptoethylamine 0.4 x 10⁻⁶ -3 mmol, mercaptoethanol 0.6*10 -3 mmol; Wherein, the concentration of the crosslinking agent mentioned in step B3 is 0.3 mmol / L; The centrifugation speed in step B4 is 15000 r / min, and the centrifugation time is 10 minutes; In step B4, the amount of ultrapure water added is 1:10, which is the volume ratio of the solution in step B3 to the ultrapure water.

14. The method for preparing macrophage-inspired biomimetic composite nanomedicine according to claim 9, characterized in that: The macrophage membrane described in step C is extracted using a hypotonic lysis and gradient centrifugation method, including the following steps: a. Collect mouse RAW264.7 cells, wash with phosphate buffer, resuspend in hypotonic solution for lysis, and then homogenize the cell suspension thoroughly using a Dunns homogenizer. b. Centrifuge the homogenate to collect the supernatant, homogenize the precipitate again and centrifuge, then combine the two supernatants; c. After centrifuging the supernatant once at low speed, take the supernatant and centrifuge it once at high speed to obtain a colorless and transparent liquid, which is the extracted cell membrane. Resuspend it in sterile deionized water to obtain the macrophage cell membrane solution.

15. The method for preparing macrophage-inspired biomimetic composite nanomedicine according to claim 14, characterized in that: Step C must satisfy at least one of the following: In step a, the phosphate buffer solution has a pH of 7.2-7.4; the hypotonic solution is tris(hydroxymethyl)aminomethane hydrochloride buffer with a concentration of 10 mmol / L. In step b, the centrifugal force is 3000-3500g and the centrifugation time is 5-10 minutes; In step c, the centrifugal forces during the two centrifugations are 20,000g and 100,000g, respectively, and the centrifugation times are 15-25 minutes and 80-100 minutes, respectively. In step c, the colorless and transparent liquid obtained by centrifugation twice is the extracted cell membrane, which is resuspended in sterile deionized water and stored at -80℃ for later use. In step C, the platinum nanozyme clusters (PtNCs) obtained in step B are mixed with macrophage membranes (MM) in solution form, with the volume ratio of macrophage membrane solution to platinum nanocluster solution being 1-3:

1. In step C, the mass concentration ratio of the macrophage membrane solution to the platinum nanocluster solution is 1-6:

1. In step C, the ultrasonic conditions after mixing the macrophage membrane with the platinum nanoclusters are as follows: ultrasonication in an ice-water bath for 1-3 minutes, followed by a 1-3 minute break, repeated 2-5 times.

16. The method for preparing macrophage-inspired biomimetic composite nanomedicine according to claim 14, characterized in that: Step C must satisfy at least one of the following: In step b, the centrifugal force is 3200 g and the centrifugation time is 5 minutes. In step c, the centrifugal forces during the two centrifugations are 20,000g and 100,000g, respectively, and the centrifugation times are 20 minutes and 90 minutes, respectively. The volume ratio of the macrophage membrane solution to the platinum nanocluster solution was 1:

1. The mass concentration ratio of macrophage membrane solution to platinum nanoclusters solution was 3:

1. In step C, the ultrasound conditions are: ultrasound for 1 minute, stop for 1 minute, and repeat 3 times.

17. The use of the macrophage-inspired biomimetic composite nanomedicine according to any one of claims 1-8 in the preparation of a medicament for treating acute kidney injury.

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