Macrophage bionic nano composite medicine as well as preparation method and application thereof

Abstract

The invention belongs to the field of biochemistry, and particularly relates to a macrophage bionic nano composite medicine as well as a preparation method and application thereof. The preparation method of the macrophage bionic composite nano-drug comprises the following steps: A, reducing chloroplatinic acid into platinum nanoparticles by adopting a trisodium citrate reduction method; b, polymerizing the platinum nanoparticles obtained in the step A through a cross-linking agent to obtain a platinum nano enzyme cluster; and C, mixing the platinum nano-enzyme cluster obtained in the step B with a macrophage membrane, and performing ultrasonic treatment to prepare the macrophage bionic platinum nano-enzyme cluster composite drug. The medicine provided by the invention can effectively promote kidney repair, can be applied to slow down inflammatory response and oxidative stress response after acute kidney injury, and can promote kidney function to recover to normal.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, and specifically relates to a macrophage bionic nanocomposite drug and a preparation method and application thereof. Background Art

[0002] Acute kidney injury is a common clinical syndrome characterized by rapid loss of renal filtration function. Its occurrence and development is a continuous, dynamically developing complex pathological process, and oxidative stress and inflammatory response are its main influencing factors. At present, there is a lack of effective drug treatments in clinical practice, and existing small molecule drugs are difficult to continuously treat the kidneys and promote the recovery of renal function. Compared with the lack of specificity of small molecule drugs, nanodrugs wrapped by macrophage membranes can target and enrich in damaged inflammatory sites, neutralize inflammatory cytokines, and thus control the development of inflammation. In addition, 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 excreted from the body through the kidneys.

[0003] On this basis, the inventors pre-constructed a cell-bionic nanocomposite drug with a platinum nanozyme cluster wrapped in a macrophage membrane and having reactive oxygen responsive deformation properties, which provides a new idea for the clinical treatment of acute kidney injury. Summary of the invention

[0004] The present invention provides a macrophage biomimetic composite nanomedicine (MM-PtNCs for short), which is a platinum nanozyme cluster (PtNCs for short) formed by cross-linking platinum nanoparticles obtained by reduction with chloroplatinic acid, and then wrapped by a macrophage membrane (MM for short) to obtain a macrophage biomimetic platinum nanozyme cluster composite medicine (MM-PtNCs for short).

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

[0006] A. Using trisodium citrate (TCD) reduction method to reduce chloroplatinic acid (H2PtCl6) into platinum nanoparticles (PtNPs);

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

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

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

[0010] Step A: The method for preparing platinum nanoparticles is to reduce chloroplatinic acid into ultra-small platinum nanoparticles by a trisodium citrate reduction method, which specifically includes the following steps:

[0011] A1. Heat the chloroplatinic acid solution to boiling, then add the trisodium citrate solution, mix the solution, heat at 115-125°C 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] Wherein, the molar ratio of chloroplatinic acid to trisodium citrate in step A1 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 described in step A1 is 0.8-1.2 mmol / L; 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; the concentration of the sodium citrate solution is 35 mmol / L.

[0017] Wherein, the heating condition of the mixed solution in step A1 is heating at 120° C. for 30 min.

[0018] Wherein, the ultrafiltration concentration in step A2 uses an ultrafiltration centrifuge tube to process the solution obtained in step a.

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

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

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

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

[0023] Step B: The preparation method of platinum nanozyme clusters (PtNCs) is to polymerize ultra-small platinum nanozyme particles into large-sized platinum nanozyme clusters through a cross-linking agent, which specifically includes the following steps:

[0024] B1. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the crosslinker for activation for 1.5-3h;

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

[0026] B3, adding the activated cross-linking agent obtained in step B1 to the reaction solution in step B2, and continuously stirring the reaction for 10-24 hours;

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

[0028] Among them, the cross-linking agent in step B1 is an active oxygen-sensitive linker, which is treated with EDC and NHS to obtain a pre-activated active oxygen-responsive cross-linking agent.

[0029] Among them, the active oxygen sensitive linker refers to a thioacetal containing two carboxyl groups (2,2'-[propane-2,2-diylbis(thio)]diacetic acid), propane-2,2-diylbis(thio)]diacetic acid), a linker containing two carboxyl groups and a disulfide bond (2,2'-dithiodiacetic acid) and a linker containing two carboxyl groups and a diselenide bond (3,3'-diselendipropionic acid, diselendiglycolic acid).

[0030] Among them, thioketal, disulfide bond, and diselenide bond are all sensitive to active oxygen, and the carboxyl group can play a cross-linking role.

[0031] Wherein, the activation time in step B1 is 2h.

[0032] Among them, the activation described in step B1 is: first add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to the cross-linking agent and react for 0.75h (i.e. 45 minutes), and then add N-hydroxysuccinimide (NHS) to react for 1.5-3h (i.e. the reaction time after adding NHS is 45 minutes to 135 minutes, or expressed as a reaction time of 0.75-2.25h).

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

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

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

[0036] In step B2, the molar ratio of mercaptoethylamine to mercaptoethanol is 2-5:4-8.

[0037] Preferably, in step B2, the molar ratio of mercaptoethylamine to mercaptoethanol is 4:6.

[0038] In step B2, the platinum nanoparticles are mixed with mercaptoethylamine and mercaptoethanol according to the following ratio:

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

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

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

[0042] Wherein, the stirring reaction time described in step B2 is 3 hours.

[0043] Wherein, the concentration of the cross-linking agent described in step B3 is 0.06-0.6mmol / L.

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

[0045] Wherein, the continuous stirring reaction time described in step B3 is 12h.

[0046] Wherein, the centrifugal speed described in step B4 is 12000-15000 r / min, and the centrifugal time is 5-20 minutes.

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

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

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

[0050] The above method is used to cross-link and polymerize to obtain large-sized platinum nanozyme clusters, which are small and uniform spheres with a particle size of 40-50nm.

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

[0052] In step C, the macrophage membrane is extracted by hypotonic lysis and gradient centrifugation.

[0053] a. Collect mouse RAW264.7 cells, wash with phosphate buffer, resuspend and lyse with hypotonic solution, and then fully homogenize the cell suspension with a Dounce homogenizer;

[0054] b. The homogenate is centrifuged to collect the supernatant, the precipitate is homogenized again and centrifuged, and the two supernatants are combined;

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

[0056] Wherein, the pH value of the phosphate buffer in step a is 7.2-7.4; the hypotonic fluid component is tris(hydroxymethyl)aminomethane hydrochloride buffer with a concentration of 10 mmol / L.

[0057] Wherein, the centrifugal force in step b 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] Wherein, during the two centrifugation processes in step c, the centrifugal forces are 20000g and 100000g respectively, and the centrifugation times are 15-25 minutes and 80-100 minutes respectively.

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

[0061] The colorless transparent liquid obtained by two centrifugations in step c is the extracted cell membrane, which is resuspended with sterile deionized water and stored at -80°C for later use.

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

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

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

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

[0066] In step C, after the macrophage membrane and the platinum nanoclusters are mixed, the ultrasonic conditions are ultrasonicated in an ice water bath, and the ultrasonic conditions are: ultrasonicate for 1-3 minutes, stop for 1-3 minutes, and repeat 2-5 times.

[0067] Preferably, the ultrasonic condition in step C is ultrasonication for 1 minute, stop for 1 minute, and repeat 3 times.

[0068] The present invention also provides the use of the macrophage bionic nanocomposite drug in preparing a drug for treating acute kidney injury.

[0069] The present invention provides a macrophage biomimetic nanocomposite drug, in which the platinum nanozyme clusters connected by a cross-linking agent responsive to active oxygen can cope with oxidative stress, and can also be degraded into ultra-small platinum nanoparticles after removing active oxygen, continuously play an active oxygen removal role, and can be excreted from the body through the kidneys. On this basis, after being wrapped by the macrophage membrane, the pro-inflammatory cytokines on the surface of the membrane enable the nanocomposite drug to target the damaged and inflamed kidneys, neutralize inflammatory factors, and thus control the development of inflammation. The antioxidant capacity of the platinum nanozyme clusters and the anti-inflammatory activity of the macrophage membrane enable the drug to effectively promote kidney repair. It can be used to slow down the inflammatory response and oxidative stress response after acute kidney injury, and promote the recovery of renal function to normal. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Flow chart of the preparation method of the macrophage bionic composite nanomedicine of the present invention.

[0071] Figure 2 TEM images of PtNCs synthesized with different cross-linker dosages.

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

[0073] Figure 4 Effect of PtNCs and MM-PtNCs on ABTS + · and DPPH· · Clear the rendering.

[0074] in: Figure 4 a is ABTS + Clear the effect map, Figure 4 b is DPPH· · Clear the rendering.

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

[0076] Figure 6 Figure 3 Effects of MM-PtNCs in neutralizing proinflammatory cytokines TNF-α, IL-6, and IL-1β.

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

[0078] Figure 7 Figure 4. Cell viability of HK-2 cells protected from reactive oxygen species by MM-PtNCs.

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

[0080] Fig. 9 Urea levels in AKI model mice within 7 days after treatment. DETAILED DESCRIPTION

[0081] Below in conjunction with embodiment, the scheme of the present invention will be explained and described.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if no specific technology or condition is indicated, it is carried out according to the technology or condition described in the document in this area or according to the product specification.The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained by commercial purchase.

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

[0083] The preparation method of the macrophage biomimetic composite nano drug of the present invention is as follows Figure 1 , including the following steps:

[0084] A. Using trisodium citrate (TCD) reduction method to reduce chloroplatinic acid (H2PtCl6) into platinum nanoparticles (PtNPs);

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

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

[0087] In the method for preparing the macrophage bionic nanocomposite drug of the present invention, the method for preparing the ultra-small platinum nanoparticles comprises the following steps:

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

[0089] b. The obtained solution is then ultrafiltered and concentrated using an ultrafiltration centrifuge tube to obtain platinum nanoparticles; the working volume of the ultrafiltration tube is 15 mL, and the molecular weight cutoff is 10 kD.

[0090] In the method for preparing the macrophage bionic nanocomposite drug of the present invention, the method for preparing the platinum nanoclusters comprises the following steps:

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

[0092] b. Add mercaptoethylamine and mercaptoethanol to the platinum nanoparticles, and stir the reaction at room temperature for 3 hours; the concentration of the platinum nanoparticles is 2.4 mg / mL, 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 the pre-activated active oxygen responsive cross-linking agent to the above solution and continue stirring to react for 12 hours; the concentration of the cross-linking agent is 0.3 mmol / L and the volume is 200 μL;

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

[0095] In the method for preparing the macrophage bionic nanocomposite drug of the present invention, the method for preparing the macrophage membrane solution comprises the following steps:

[0096] a. Collect mouse RAW264.7 cells, wash with phosphate buffer, resuspend and lyse with hypotonic solution, and then use a Dounce homogenizer to fully homogenize the cell suspension; the pH value of the phosphate buffer is 7.2-7.4; the hypotonic solution component is tris(hydroxymethyl)aminomethane hydrochloride buffer with a concentration of 10mmo / L;.

[0097] b. After the homogenate is centrifuged, the supernatant is collected, the precipitate is homogenized again and centrifuged, and the supernatants obtained from the two centrifugations are combined; the centrifugal force is 3200 g, and the centrifugation time is 5 minutes.

[0098] c. After the supernatant is centrifuged once at low speed, it is taken out and centrifuged once at high speed to obtain a colorless transparent liquid, which is the extracted cell membrane, which is resuspended with sterile deionized water; during the two centrifugation processes, the centrifugal force is 20000g and 100000g respectively, and the centrifugation time is 20 minutes and 90 minutes respectively.

[0099] In the preparation method of the macrophage biomimetic nanocomposite drug of the present invention, the macrophage biomimetic platinum nanozyme cluster composite drug preparation method comprises the following steps: mixing the macrophage membrane with the platinum nanocluster solution, ultrasonicating the mixed solution under ice water bath conditions, ultrasonicating for 1 minute, stopping for 1 minute, repeating 3 times, centrifuging and resuspending to obtain the macrophage biomimetic platinum nanozyme cluster composite drug. Wherein, the volume ratio of the macrophage membrane to the platinum nanocluster solution is 1:1, and the mass concentration ratio is 3:1.

[0100] Chloroplatinic acid (H2PtCl6), mercaptoethylamine, and mercaptoethanol used in the examples of the present invention were purchased from Shanghai McLean 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 antibody) were purchased from Gibico, USA. TNF-α, IL-1β, and IL-6 cytokines were purchased from Preprotein, and BCA protein concentration assay kit, mouse tumor necrosis factor α detection kit (Mouse TNF-α ELISA Kit) kit, mouse interleukin-1β detection kit (Mouse IL-1β ELISA Kit), and mouse interleukin-6 detection kit (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, USA. MTT was purchased from Sigma-Aldrich, USA. 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 ultra-small platinum nanoparticles:

[0103] Heat chloroplatinic acid solution (1mmol / L, 200mL) to boiling at 120℃, then add sodium citrate solution (35mmol / L, 20mL), react for 30 minutes, stop heating when the reaction color changes from yellow to brown-black, and continue stirring to room temperature. Use 15mL sample volume, 10kDa ultrafiltration centrifuge tube to ultrafiltration centrifuge the obtained solution at a speed of 3500r / min for 10 minutes; add deionized water, centrifuge under the same conditions, and finally adjust the volume to 5mL to obtain ultrasmall platinum nanoparticles.

[0104] 2. Activation of cross-linking agent:

[0105] Weigh 1.7 mg of crosslinker (propane-2,2-diylbis(thio)]diacetic acid), 38 mg of EDC, and 5.8 mg of NHS, add 1 mL of DMSO respectively, dissolve by ultrasound, add all 1 mL of crosslinker solution into a 50 mL centrifuge tube with a pipette, continue to add 17 mL of DMSO and ultrasound once; add 1 mL of EDC and NHS into the centrifuge tube in turn and put it in the dark at 37°C, shake on a shaker at 200 rpm for 2 hours. Take 2 mL of the obtained solution and add 8 mL of DMSO. Get the activated crosslinker.

[0106] 3. Preparation of platinum nanoclusters:

[0107] Add mercaptoethylamine (10mmol / L, 40μL) and mercaptoethanol (10mmol / L, 60μL) to PtNPs (2.4mg / mL, 400μL), stir and react at room temperature for 3 hours, and then dilute to 1mL with PBS (pH 7.4). Add the pre-activated crosslinker (200μL) to the above solution and continue stirring and reacting for 12 hours. After the reaction, centrifuge the sample at 15000r / min for 10 minutes, add 100μL of ultrapure water, and disperse evenly by ultrasonication to obtain platinum nanoclusters.

[0108] Example 2

[0109] 1. Preparation of ultra-small platinum nanoparticles:

[0110] Heat chloroplatinic acid solution (1mmol / L, 200mL) to boiling at 120℃, then add sodium citrate solution (35mmol / L, 20mL), react for 30 minutes, stop heating when the reaction color changes from yellow to brown-black, and continue stirring to room temperature. Use 15mL sample volume, 10kDa ultrafiltration centrifuge tube to ultrafiltration centrifuge the obtained solution at a speed of 3500r / min for 10 minutes; add deionized water, centrifuge under the same conditions, and finally adjust the volume to 5mL to obtain ultrasmall platinum nanoparticles.

[0111] 2. Activation of cross-linking agent:

[0112] Weigh 1.7 mg of crosslinker (propane-2,2-diylbis(thio)]diacetic acid), 38 mg of EDC, and 5.8 mg of NHS, add 1 mL of DMSO respectively, dissolve by ultrasonication, add all 1 mL of crosslinker solution into a 50 mL centrifuge tube with a pipette, continue to add 17 mL of DMSO and ultrasonicate once; add 1 mL of EDC and NHS into the centrifuge tube in turn and place it in a dark 37°C shaker at 200 rpm for 2 hours to obtain the activated crosslinker.

[0113] 3. Preparation of platinum nanoclusters:

[0114] Add mercaptoethylamine (10mmol / L, 40μL) and mercaptoethanol (10mmol / L, 60μL) to ultra-small platinum nanoparticles (2.4mg / mL, 400μL), stir and react at room temperature for 3 hours, and then dilute to 1mL with PBS (pH 7.4). Add the pre-activated cross-linking agent (200μL) to the above solution and continue stirring and reacting for 12 hours. After the reaction, centrifuge the sample at 15000r / min for 10 minutes, add 100μL ultrapure water, and disperse evenly by ultrasound to obtain platinum nanoclusters.

[0115] Example 3

[0116] 1. Preparation of ultra-small platinum nanoparticles:

[0117] Heat chloroplatinic acid solution (1mmol / L, 200mL) to boiling at 120℃, then add sodium citrate solution (35mmol / L, 20mL), react for 30 minutes, stop heating when the reaction color changes from yellow to brown-black, and continue stirring to room temperature. Use 15mL sample volume, 10kDa ultrafiltration centrifuge tube to ultrafiltration centrifuge the obtained solution at a speed of 3500r / min for 10 minutes; add deionized water, centrifuge under the same conditions, and finally adjust the volume to 5mL to obtain ultrasmall platinum nanoparticles.

[0118] 2. Activation of cross-linking agent:

[0119] 3.4 mg of cross-linker (propane-2,2-diylbis(thio)]diacetic acid), 76 mg of EDC, and 10.6 mg of NHS were added with 1 mL of DMSO respectively, and dissolved by ultrasound. 1 mL of the cross-linker solution was added into a 50 mL centrifuge tube with a pipette, and 17 mL of DMSO was added and ultrasound was continued. 1 mL of EDC and NHS were added into the centrifuge tube in turn and placed in a dark 37°C shaker at 200 rpm for 2 hours.

[0120] 3. Preparation of platinum nanoclusters:

[0121] Add mercaptoethylamine (10mmol / L, 40μL) and mercaptoethanol (10mmol / L, 60μL) to PtNPs (2.4mg / mL, 400μL), stir and react at room temperature for 3 hours, and then dilute to 1mL with PBS (pH 7.4). Add the pre-activated crosslinker (200μL) to the above solution and continue stirring and reacting for 12 hours. After the reaction, centrifuge the sample at 15000r / min for 10 minutes, add 100μL of ultrapure water, and disperse evenly by ultrasonication to obtain platinum nanoclusters.

[0122] Example 4

[0123] 6 mg / mL, 250 μL of macrophage membrane solution was mixed with 1 mg / mL, 250 μL of platinum nanoclusters (prepared according to the method of Example 2), and the mixture was ultrasonicated in an ice-water bath. Ultrasonication was performed for 1 minute, and then stopped for 1 minute, and repeated 3 times. The sample was then centrifuged at 10000 r / min for 10 minutes, and then 100 μL of PBS was added for re-suspending to obtain a cell-mimetic composite nanomedicine.

[0124] Example 5

[0125] 3 mg / mL, 250 μL of macrophage membrane solution was mixed with 1 mg / mL, 250 μL of platinum nanoclusters (prepared according to the method of Example 2), and the mixture was ultrasonicated in an ice-water bath. Ultrasonication was performed for 1 minute, and then stopped for 1 minute, and repeated 3 times. The sample was then centrifuged at 10000 r / min for 10 minutes, and then 100 μL of PBS was added for re-suspending to obtain a cell-mimetic composite nanomedicine.

[0126] Example 6

[0127] 2mg / mL, 250μL of macrophage membrane solution was mixed with 1mg / mL, 250μL of platinum nanoclusters (prepared according to the method of Example 2), and the mixture was ultrasonicated in an ice-water bath. Ultrasonication was performed for 1 minute, and then stopped for 1 minute, and repeated 3 times. The sample was then centrifuged at 10000r / min for 10 minutes, and then 100μL of PBS was added for re-suspending to obtain a cell-mimetic composite nanomedicine.

[0128] Example 7

[0129] 1 mg / mL, 250 μL of macrophage membrane solution was mixed with 1 mg / mL, 250 μL of platinum nanoclusters (prepared according to the method of Example 2), and the mixture was ultrasonicated in an ice-water bath. Ultrasonication was performed for 1 minute, and then stopped for 1 minute, and repeated 3 times. The sample was then centrifuged at 10000 r / min for 10 minutes, and then 100 μL of PBS was added for re-suspending to obtain a cell-mimetic composite nanomedicine.

[0130] Example 8 Investigation of the amount of crosslinking agent used

[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. Figure 2As shown: Examples 1, 2, and 3 respectively exhibit different morphological characteristics. In Example 1, although spherical clusters are formed, there are still many small particles that are not cross-linked into spheres and are not separated from the formed clusters, which will cause the final solution to be unevenly dispersed. In Example 3, PtNPs cannot be agglomerated into regular, uniform spherical particles, which will also lead to poor solution stability, and the particles are too large to be conducive to in vivo administration. The PtNCs obtained by cross-linking in Example 2 present the best morphology, which is a spherical shape of uniform size with a particle size of 40-50nm.

[0132] In the process of screening cross-linking agents, the inventors investigated a variety of active oxygen-sensitive linkers, and the cross-linking agents that can achieve the above cross-linking effect include thioketal containing two carboxyl groups (2,2'-[propane-2,2-diylbis(thio)]diacetic acid), propane-2,2-diylbis(thio)]diacetic acid), containing two carboxyl groups and a disulfide bond (2,2'-dithiodiacetic acid) and containing two carboxyl groups and a diselenide bond (3,3'-diselendipropionic acid, diselendiglycolic acid). The above cross-linking agents are active oxygen-sensitive because the thioketal, disulfide bond, and diselenide bond are active oxygen-sensitive, and the carboxyl group can play a cross-linking role and can be applied to the preparation of the present invention.

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

[0134] The composite nanoparticles prepared in Examples 4, 5, 6, and 7 were dripped onto an ultra-thin copper mesh to prepare samples. The morphology of the nanoparticles was examined by transmission electron microscopy to determine the optimal mixing ratio. Figure 3 As shown: Examples 4, 5, 6, and 7 exhibit different morphological characteristics, and the composite nanomedicine in Example 5 has a film structure with uniform thickness and complete wrapping. The inventors finally used the ratio (3:1) in Example 5 as the subsequent use ratio.

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

[0136] 5mL ABTS (2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt) stock solution (7.4mmol / L) was mixed with 88μL K2S2O8 stock solution (2.6mmol / L), and allowed to stand for 12-16 hours to prepare ABTS working solution. 0.4mL ABTS working solution was taken and diluted with ethanol so that its absorbance at 734nm at room temperature was 0.74±0.02. 0.2mL of the above solution was mixed with the nanodrug (0.1-1μg / mL, 10μL) solution prepared in Examples 2 and 5, respectively. Then, a chemiluminescence full-wavelength microplate reader (SpectraMax ABS Plus) was used for wavelength detection, and the change in absorbance at 734nm wavelength was recorded within 10 minutes.

[0137] The free radical scavenging ability of ABTS was calculated by the following formula:

[0138] Clearance rate (%) = [(A0-A i ) / A0]×100, where A0 means no sample is added and only ABTS is added + Absorbance: A i For adding samples and ABTS + ·Absorbance.

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

[0140] Example 11 Antioxidant test (DPPH free radical scavenging ability)

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

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

[0143] Example 12 Responsive deformation experiment

[0144] After 20 μg / mL PtNCs were incubated with 20 μM H2O2 solution for 30 min, the nanoclusters were removed by centrifugation, and the changes in their morphology were investigated by transmission electron microscopy to evaluate the active oxygen responsive deformation properties of PtNCs.

[0145] Test results: Figure 5 As shown, under the action of hydrogen peroxide, the large-sized nanoclusters responsively degrade and transform into ultra-small platinum nanoparticles.

[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, and then centrifuged at 100000g for 30 minutes to remove the nanocomplexes and collect the supernatant. The content of IL-1β, TNF-α or IL-6 proinflammatory cytokines in the supernatant was quantitatively analyzed using an Elisa kit.

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

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

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

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

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

[0153] The AKI model was established by clamping the bilateral renal pedicles with non-damaging arterial clamps for 35 minutes, then removing the vascular clamps to allow blood to be reperfused. On the day when the mouse AKI model was successfully established, the mice were treated, and the group settings and drug doses were as follows: MM group (30 mg / kg); PtNCs group (15 mg / kg); MM-PtNCs (15 mg / kg) group; the sham group (Sham group) and the group given an equal volume of saline after modeling (Saline group) were used as controls. The blood and kidneys of mice in each group were collected on the 1st, 3rd, and 7th days after treatment, and the therapeutic effect was mainly evaluated by blood biochemical indicators-serum creatinine (CREA) and urea (UREA).

[0154] Test results: CREA values ​​of AKI model mice within 7 days after treatment were as follows Figure 8 As shown in the figure, the values ​​of the Sham group at all time points were similar, so the average value (47.32±2.88)μmol / L was used as the control. The CREA value of the Saline group increased sharply on the first day after surgery, reaching 5 times that of the Sham group ((257.83±17.57)μmol / L), and its value was still twice that of the Sham group on the 7th day after surgery. The CREA value of the MM group rose rapidly on the first day after surgery and then fell rapidly, and returned to normal levels on the 7th day, (46.84±12.59)μmol / L. Although both MM and PtNCs showed certain therapeutic effects, the MM-PtNCs group showed a better therapeutic effect, and its CREA levels at three time points after surgery were not significantly different from those of the Sham group.

[0155] UREA values ​​of AKI model mice within 7 days after treatment Fig. 9As shown in the figure, the changes in UREA values ​​showed a similar trend. The total average UREA level in the Sham group was (8.78±0.58)mmol / L on the 7th day, and that in the Saline group reached (82.86±5.11)mmol / L on the first day after surgery, which remained almost unchanged on the 3rd day. On the 7th day, it was still at a high level of (42.38±11.20)mmol / L. The PtNCs group and the MM group both increased to varying degrees after surgery, but returned to a lower level on the 7th day, which was (18.5±3.06)mmol / L and (16.81±5.84)mmol / L, respectively. Compared with the higher UREA values ​​on the first day of the first three experimental groups, the UREA value of the MM-PtNCs group on the first day was only (18.81±7.17)mmol / L, and on the 7th day, UREA had dropped to (9.32±0.94)mmol / L, which was not significantly different from the results of 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 biomimetic nanocomposite drug of the present invention can respond to oxidative stress through platinum nanozyme clusters connected by cross-linking agents responsive to active oxygen, and can also be degraded into ultra-small platinum nanoparticles after removing active oxygen, continuously play an active oxygen removal role, and can be excreted from the body through the kidneys. The present invention provides a new clinical application scheme for alleviating inflammatory reactions and oxidative stress reactions after acute kidney injury and promoting the recovery of renal function to normal.

Claims

1. Macrophage biomimetic composite nanomedicine, characterized by: It is a macrophage biomimetic platinum nanozyme cluster complex drug obtained by cross-linking platinum nanoparticles obtained by reduction with chloroplatinic acid, and then wrapping them with macrophage membranes.

2. The method for preparing the macrophage biomimetic composite nanomedicine according to claim 1, characterized in that: The steps include: A. Using trisodium citrate reduction method to reduce chloroplatinic acid into platinum nanoparticles; B. The platinum nanoparticles obtained in step A are polymerized with a crosslinker to obtain platinum nanozyme clusters; C. The platinum nanozyme clusters obtained in step B are mixed with macrophage membranes and subjected to ultrasound to prepare macrophage biomimetic platinum nanozyme cluster composite drugs.

3. The method for preparing the macrophage biomimetic composite nanomedicine according to claim 2, characterized in that: Step A: The method for preparing platinum nanoparticles is to reduce chloroplatinic acid into ultra-small platinum nanoparticles by a trisodium citrate reduction method, which specifically includes the following steps: A1. Heat the chloroplatinic acid solution to boiling, then add the trisodium citrate solution, mix the solution, heat at 115-125°C 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.

4. The method for preparing the macrophage biomimetic composite nanomedicine according to claim 3, characterized in that: Step A satisfies at least one of the following: Wherein, the molar ratio of chloroplatinic acid to trisodium citrate in step A1 is 1-3:5-9; Preferably, the molar ratio of chloroplatinic acid to trisodium citrate in step A1 is 2:7; Wherein, the concentration of the chloroplatinic acid solution in step A1 is 0.8-1.2 mmol / L; the concentration of the sodium citrate solution is 30-40 mmol / L; Preferably, the concentration of the chloroplatinic acid solution in step A1 is 1 mmol / L; the concentration of the sodium citrate solution is 35 mmol / L; Wherein, the heating condition of the mixed solution in step A1 is heating at 120° C. for 30 min; Wherein, the ultrafiltration concentration in step A2 uses an ultrafiltration centrifuge tube to process the solution obtained in step a; Furthermore, the solution obtained by ultrafiltration and 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 cut-off of 10 kD; Preferably, the above method can be used to reduce chloroplatinic acid into ultra-small platinum nanoparticles using trisodium citrate reduction method, and the obtained ultra-small nanoparticles have a particle size of 2-5 nm.

5. The method for preparing the macrophage biomimetic composite nanomedicine according to claim 2, characterized in that: Step B is a method for preparing platinum nanozyme clusters, which is to polymerize ultra-small platinum nanozyme particles into large-sized platinum nanozyme clusters through a cross-linking agent, and specifically includes the following steps: B1. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the crosslinker for activation for 1.5-3h; B2. Add mercaptoethylamine and mercaptoethanol to the platinum nanoparticles and stir the mixture at room temperature for 2-4 hours; B3, adding the activated cross-linking agent obtained in step B1 to the reaction solution in step B2, and continuously stirring the reaction for 10-24 hours; B4. After centrifuging the solution in step B3, add ultrapure water and disperse it evenly by ultrasonication to obtain platinum nanozyme clusters.

6. The method for preparing the macrophage biomimetic composite nanomedicine according to claim 5, characterized in that: Step B satisfies at least one of the following: The cross-linking agent in step B1 is an active oxygen-sensitive linker, which is treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain a pre-activated active oxygen-responsive cross-linking agent; The active oxygen 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; Preferably, the thioketal linker containing two carboxyl groups is 2,2'-[propane-2,2-diylbis(thio)]diacetic acid), propane-2,2-diylbis(thio)]diacetic acid; Preferably, the linker containing two carboxyl groups and a disulfide bond is 2,2'-dithiodiacetic acid; Preferably, the linker containing two carboxyl groups and a diselenide bond is 3,3'-diselenedipropionic acid or diselenediglycolic acid; Most preferably, the active oxygen sensitive linker is propane-2,2-diylbis(thio)]diacetic acid; The activation time in step B1 is 2h; The activation in step B1 is as follows: firstly add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the crosslinking agent and react for 0.75 h, and then add N-hydroxysuccinimide and react for 1.5-3 h; Preferably, the activation in step B1 is as follows: firstly add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the cross-linking agent and react for 0.75 h, and then add N-hydroxysuccinimide and react for 2 h; The molar ratio of the cross-linking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step B1 is 1-2:25-40:5-9; Preferably, the molar ratio of the cross-linking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step B1 is 1:35:7; In step B2, the molar ratio of mercaptoethylamine to mercaptoethanol is 2-5:4-8; Preferably, in step B2, the molar ratio of mercaptoethylamine to mercaptoethanol is 4:6; In step B2, the platinum nanoparticles are mixed with mercaptoethylamine and mercaptoethanol according to the following ratio: Platinum nanoparticles 0.8-1.2 mg, mercaptoethylamine 0.2-0.8*10 -3 mmol, mercaptoethanol 0.4-0.8*10 -3 mmol; Preferably, the platinum nanoparticles in step B2 are mixed with mercaptoethylamine and mercaptoethanol in the following ratio: Platinum nanoparticles 0.96 mg, mercaptoethylamine 0.4*10 -3 mmol, mercaptoethanol 0.6*10 -3 mmol; The stirring reaction time in step B2 is 3 hours; The cross-linking agent concentration in step B3 is 0.06-0.6 mmol / L; Preferably, the concentration of the cross-linking agent in step B3 is 0.3 mmol / L; The continuous stirring reaction time in step B3 is 12 hours; The centrifugal speed in step B4 is 12000-15000 r / min, and the centrifugal time is 5-20 minutes; The centrifugal speed in step B4 is 15000 r / min, and the centrifugal time is 10 minutes; The amount of ultrapure water added in step B4 is the volume ratio of the solution in step B3 to ultrapure water of 1-3:10-30; Preferably, the amount of ultrapure water added in step B4 is a volume ratio of the solution in step B3 to ultrapure water of 1:10; The above method is used to cross-link and polymerize to obtain large-sized platinum nanozyme clusters, which are small and uniform spheres with a particle size of 40-50nm.

7. The method for preparing the macrophage biomimetic composite nanomedicine according to claim 2, characterized in that: In step C, the macrophage membrane is extracted by hypotonic lysis and gradient centrifugation. a. Collect mouse RAW264.7 cells, wash with phosphate buffer, resuspend and lyse with hypotonic solution, and then fully homogenize the cell suspension with a Dounce homogenizer; b. The homogenate is centrifuged to collect the supernatant, the precipitate is homogenized again and centrifuged, and the two supernatants are combined; c. Centrifuge the supernatant once at a low speed and then centrifuge it again at a high speed to obtain a colorless transparent liquid, which is the extracted cell membrane. Resuspend it with sterile deionized water to obtain a macrophage membrane solution.

8. The method for preparing the macrophage biomimetic composite nanomedicine according to claim 7, characterized in that: Step C satisfies at least one of the following: The pH value of the phosphate buffer in step a is 7.2-7.4; the hypotonic solution component is tris(hydroxymethyl)aminomethane hydrochloride buffer with a concentration of 10 mmol / L; The centrifugal force in step b is 3000-3500g, and the centrifugation time is 5-10 minutes; Preferably, the centrifugal force in step b is 3200 g and the centrifugation time is 5 minutes; In the two centrifugation processes of step c, the centrifugal force is 20000g and 100000g respectively, and the centrifugation time is 15-25 minutes and 80-100 minutes respectively; Preferably, in the two centrifugation processes in step c, the centrifugal forces are 20000g and 100000g respectively, and the centrifugation time is 20 minutes and 90 minutes respectively; The colorless transparent liquid obtained by two centrifugations in step c is the extracted cell membrane, which is resuspended with sterile deionized water and stored at -80°C for later use; In step C, the platinum nanozyme clusters (PtNCs) obtained in step B are mixed with macrophage membranes (MM) in the form of a solution, and the volume ratio of the macrophage membrane solution to the platinum nanocluster solution is 1-3:1; Preferably, the volume ratio of the macrophage membrane solution to the platinum nanocluster solution is 1:1; In step C, the mass concentration ratio of the macrophage membrane solution to the platinum nanocluster solution is 1-6:1; Preferably, the mass concentration ratio of the macrophage membrane solution to the platinum nanocluster solution is 3:1; In step C, after the macrophage membrane and the platinum nanoclusters are mixed, the ultrasonic condition is ultrasonicated in an ice water bath, and the ultrasonic conditions are: ultrasonicate for 1-3 minutes, stop for 1-3 minutes, and repeat 2-5 times; Preferably, the ultrasonic condition in step C is ultrasonication for 1 minute, stop for 1 minute, and repeat 3 times.

9. Use of the macrophage biomimetic composite nanomedicine according to claim 1 in the preparation of a medicine for treating acute kidney injury.

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