Bionic black phosphorus nanomaterial and preparation and application thereof

Abstract

The application discloses a kind of bionic black phosphorus nanomaterial and its preparation and application, including the following steps: S1, the preparation of black phosphorus nanosheet;S2, the preparation of carboxylated black phosphorus nanosheet;S3, the preparation of composite black phosphorus nanomaterial;S4, the preparation of bionic black phosphorus nanomaterial.The structural core of the bionic black phosphorus nanomaterial is composed of black phosphorus nanosheet, which can be covalently connected to the amino-modified Anti miR 33 nucleic acid inhibitor through its terminal carboxyl group after modification by double-end carboxyl polyethylene glycol, to achieve the purpose of protecting nucleic acid structure and improving delivery efficiency, and then the material is coated with cell membrane derived from M2c phenotype macrophages, thereby endowing the material with active targeting ability in inflammatory areas.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomimetic black phosphorus nanomaterial and its preparation and application. Background Technology

[0002] In recent years, black phosphorus nanosheets (BPNSs) have gradually attracted attention in kidney disease research, especially in the areas of acute kidney injury (AKI) and chronic kidney disease (CKD), where numerous exploratory studies have been conducted. BPNSs possess a layered structure, high specific surface area, and abundant reactive sites. Their biodegradability allows them to be gradually converted into non-toxic phosphates in vivo, a superior property compared to metal nanomaterials. BPNSs have shown significant potential in anti-oxidation, anti-inflammation, cell fate regulation, and as a drug delivery platform.

[0003] Previous studies have shown that BPNSs can reduce oxidative stress in AKI models by scavenging large amounts of reactive oxygen species (ROS) generated during injury. For example, experiments have shown that BPNSs can effectively reduce ROS levels in kidney tissue in vivo and alleviate acute damage caused by ischemia-reperfusion (IRI). The mechanism mainly relies on the ROS-scavenging ability of intermediate products generated during the oxidation of phosphorus in black phosphorus, thereby mitigating oxidative stress-induced cell damage. Furthermore, BPNSs can affect inflammation-related signaling pathways, such as the NF-κB and MAPK pathways, reducing the expression levels of pro-inflammatory factors and thus decreasing the excessive infiltration of immune cells in the kidney, a finding validated in several small animal experiments.

[0004] Beyond their direct anti-damage effects, BPNSs are increasingly recognized as an excellent drug delivery platform. Their highly reactive surfaces can form stable composite structures with various polymers, peptides, and nucleic acids through simple chemical methods, enabling them to carry nucleic acid drugs, anti-inflammatory drugs, or small molecule inhibitors. For example, the BPNSs PEG system has been used to deliver siRNA, demonstrating good delivery efficiency and safety in a mouse inflammation model. BPNSs provide a physical barrier for nucleic acids, reducing their degradation rate by nucleases in body fluids. Their two-dimensional sheet-like structure also facilitates efficient uptake of materials by macrophages, thereby enhancing intracellular biological effects.

[0005] Despite the foundation these technologies provide, several key bottlenecks remain: 1. While existing BPNSs have shown potential in basic research on AKI / CKD, most BPNSs lack active targeting capabilities in their basic structure, typically relying on passive leakage or macrophage phagocytosis, thus limiting their local enrichment in the kidney; 2. Current nucleic acid delivery systems cannot maintain nucleic acid integrity in complex body fluid environments, resulting in low delivery efficiency; 3. The interaction between materials and immune cells lacks proactive design, making it difficult to precisely regulate macrophage function; 4. Although BPNSs alone can alleviate oxidative stress, they are insufficient to address complex immunometabolic disorders in AKI / CKD, such as impaired macrophage cholesterol efflux and persistent inflammation caused by lipid accumulation. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a biomimetic black phosphorus nanomaterial, its preparation, and its application. The core structure consists of black phosphorus nanosheets, which are coated with bi-terminal carboxyl polyethylene glycol. The terminal carboxyl groups can be covalently linked to an amino-modified Anti miR 33 nucleic acid inhibitor to protect the nucleic acid structure and improve delivery efficiency. Subsequently, the material is coated with a cell membrane derived from M2c phenotype macrophages, thereby endowing the material with active targeting capabilities to inflamed areas.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing biomimetic black phosphorus nanomaterials includes the following steps:

[0009] S1. In a glove box after deoxygenation by introducing inert gas, black phosphorus blocks are cut into thin slices, adhered to a conductive substrate, and then electrochemically exfoliated to obtain black phosphorus nanosheets.

[0010] S2. Disperse black phosphorus nanosheets in ultrapure water, adjust the pH, then add double-ended carboxyl polyethylene glycol, stir and react. After the reaction is complete, centrifuge, filter, wash and dry to obtain carboxylated black phosphorus nanosheets.

[0011] S3. Add carboxylated black phosphorus nanosheets to MES buffer, then add EDC and NHS, stir and activate, then add terminal amino-modified Anti miR 33, and carry out a constant temperature reaction. After the reaction is completed, filter and wash, then add enzyme-free water to disperse and obtain a dispersion of composite black phosphorus nanomaterials.

[0012] S4. Mix the composite black phosphorus nanomaterial dispersion with the M2c macrophage membrane solution evenly, then perform ultrasonic treatment, and after the treatment is completed, perform extrusion fusion to obtain the biomimetic black phosphorus nanomaterial.

[0013] Preferably, the area of ​​the sheet in step S1 is 1-5 mm. 2 The dimensions of the black phosphorus nanosheets are: thickness of 1-10 nm and particle size of 50-150 nm.

[0014] Preferably, the specific method for electrochemical stripping in step S1 is as follows: at room temperature, under a constant voltage of -5V, using large pieces of black phosphorus as the cathode and a platinum sheet as the anode, stripping is performed in propylene carbonate and tetrabutylammonium hexafluorophosphate for 10-30 min, followed by sonication for 1-2 h. The stripped black phosphorus sheet is then dispersed in anhydrous acetone, centrifuged at 2000-3000 rpm for 10-20 min, and then centrifuged at 8000-9000 rpm for 10-15 min to collect the product.

[0015] Preferably, in step S2, the mass ratio of black phosphorus nanosheets, ultrapure water, and bicarboxyl-terminated polyethylene glycol is 2-3:1000:10-15, the pH is 8-9, the stirring reaction temperature is 20-30℃, and the time is 5-7h.

[0016] Preferably, the concentration of the MES buffer in step S3 is 20-30 mM / L, and the pH is 5.5-6.5; the stirring activation temperature is 20-30℃, and the time is 15-25 min.

[0017] Preferably, in step S3, the concentration of the terminal amino-modified Anti miR 33 is 10-20 μM / L; the mass-to-volume ratio of the carboxylated black phosphorus nanosheets, MES buffer, EDC, NHS, and terminal amino-modified Anti miR 33 is 1-2 mg:1 mL:5-6 mg:6-7 mg:1.5-2 mL; and the isothermal reaction is carried out at a temperature of 20-30°C for 5-7 hours.

[0018] Preferably, the preparation method of the M2c macrophage membrane solution in step S4 is as follows:

[0019] RAW264.7 macrophage cells in logarithmic growth phase were cultured in a 10 cm cell culture dish using 20 ng / mL... -1 After 48 hours of IL-10 induction, the number was (1-3)×10. 7 M2c macrophages were obtained by collecting cells per dish, then suspended in hypotonic medium containing 1 mM PMSF. After sonication for 2 min, the cells were centrifuged at 1500 rpm for 5 min to remove organelles. The supernatant was collected and further centrifuged at 15000 rpm for 30 min to collect the M2c macrophage membranes, which were then stored at -80℃ for later use. Membrane protein concentration was determined by BCA method: 0.5-1 mg / mL.

[0020] Preferably, the concentration of the composite black phosphorus nanomaterial dispersion in step S4 is 1-1.5 mg / mL, the volume ratio of the composite black phosphorus nanomaterial dispersion to the M2c macrophage membrane solution is 1:1.5-2.5, the frequency of the ultrasound is 30-40 kHz, the power is 100-150 W, and the time is 5-10 min.

[0021] This invention also protects a biomimetic black phosphorus material prepared by the method described above.

[0022] The present invention also protects the use of a biomimetic black phosphorus material as described above in the preparation of drugs for chronic kidney disease.

[0023] Preferably, the chronic kidney disease is diabetic nephropathy, hypertensive nephropathy, or IgA nephropathy.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The biomimetic black phosphorus nanomaterial provided by the present invention has a typical core-shell structure. The core is BPNSs, the outer layer is a composite layer formed by PEG and Anti miR 33, and the outermost layer is a biological interface composed of macrophage membrane. BPNSs can be gradually degraded into non-toxic phosphate in vivo, so that the material has good biosafety. PEG improves the stability of the material and blocks the adsorption of non-specific proteins, so that the material is more stable in vivo. Anti miR 33 can enter macrophages and play a role in regulating lipid metabolism when effectively protected. Macrophage membrane coating enables the material to accumulate in the kidney inflammation area and be preferentially phagocytosed by local macrophages.

[0026] (2) The biomimetic black phosphorus nanomaterials provided by this invention first use liquid-phase ultrasonic exfoliation to obtain uniform black phosphorus nanosheets. By controlling the exfoliation time, temperature and centrifugation speed, a two-dimensional sheet structure with a thickness of 1-10 nm can be obtained. Then, the carboxyl groups of PEG molecules are electrostatically adsorbed with the phosphorus groups formed after oxidation of the black phosphorus surface, so that PEG is uniformly covered on the surface of BPNSs (BP@PEG), ensuring its stability in water and physiological environments. Afterwards, the carboxyl groups at the end of PEG are activated by EDC / NHS chemical coupling, so that they form a stable amide bond with Anti-miR-33 with amino modification (PBP@Anti), making the nucleic acid firmly attached. The material adheres to the surface and is not easily detached or degraded by enzymes. To ultimately obtain a biomimetic interface with active targeting, ultrasonic membrane disruption and stepwise extrusion were used to process the M2c macrophage membrane, forming nanoscale vesicles that coat the outer layer of PBP@Anti (PBP@Anti@M2c). The entire structure was formed under mild conditions to ensure that the functionality of cell membrane proteins was not damaged. At the same time, the final particle size of the biomimetic black phosphorus nanomaterial was controlled at 50-150 nm, with a moderately negative charge potential, which helps to avoid the adsorption of non-specific proteins in the blood and improves in vivo stability. The spectral characteristics of BPNSs can be verified by Raman spectroscopy, and the membrane protein components can be identified by Western blotting to confirm whether the M2c-specific proteins are maintained during the coating process. In addition, the typical bilayer structure of the inner and outer membranes can be observed by transmission electron microscopy, further demonstrating the integrity of the biomimetic material structure.

[0027] (3) The biomimetic black phosphorus nanomaterial provided by the present invention can be understood as a multi-level synergistic mechanism. First, during the systemic circulation process, the presence of macrophage membranes improves the stability and immune escape ability of the material in vivo, making it difficult for the liver and spleen system to clear it quickly. Second, the chemokines, adhesion molecules and immune microenvironment of the inflamed kidney are highly matched with the macrophage membrane, enabling the material to actively adsorb onto the lesion area. After contact with the material, local macrophages will phagocytose, causing BPNSs and Anti miR 33 to be released into the cells. BPNSs can directly clear excess ROS and reduce oxidative stress. At the same time, it can inhibit multiple inflammatory signaling pathways, thereby reducing the expression of pro-inflammatory factors. Anti miR 33 can restore the cholesterol efflux level of macrophages and promote the transformation of macrophages from an inflammatory phenotype to a repair phenotype from a metabolic level. This plays a key role in terminating inflammation and promoting damage repair. Thus, the material restores the function of damaged macrophages through a dual-mechanism synergistic approach, which helps to improve the inflammatory environment of the kidney and reduce the occurrence of CKD fibrosis.

[0028] (4) This invention overcomes the problem of lack of targeting in traditional drug therapy. By introducing the M2c macrophage membrane, the material can be significantly enriched in the inflamed area of ​​the kidney, increasing the local drug concentration and thus greatly improving the therapeutic effect. The material has a composite therapeutic mechanism, which can simultaneously regulate inflammation, oxidative stress and macrophage cholesterol metabolism, no longer limited to a single target or a single pathway. This multimodal treatment approach is more in line with the complexity of the pathological process and is more in line with the future development direction of precision medicine. Through PEG modification and cell membrane coating, Anti-miR-33 can be significantly protected in the circulating environment, enabling it to be successfully delivered into the macrophage, solving the problem of low delivery efficiency of traditional nucleic acid drugs. At the same time, the material has good immunocompatibility and biodegradability, reducing the risk of long-term accumulation and immune stimulation. Furthermore, the preparation method used in this invention has good scalability, and the material composition is derived from a widely validated biomaterial system, making it more feasible in future industrialization and clinical transformation. Attached Figure Description

[0029] Figure 1 The images show transmission electron microscopy (TEM) images of the BPNSs, M2c films, and PBP@Anti@M2c films prepared in Example 1 of this invention.

[0030] Figure 2 The images show the BPNSs prepared in Example 1 of this invention and their transmission electron microscopy (TEM) images after H2O2 treatment.

[0031] Figure 3 The images show the Raman spectra of BPNSs, BP@PEG, and PEG prepared in Example 1 of this invention.

[0032] Figure 4 Zeta potential diagrams of BPNSs, BP@PEG, PBP@Anti, M2c, and PBP@Anti@M2c prepared in Example 1 of this invention.

[0033] Figure 5 Hydrodynamic diameter diagrams of BPNSs, BP@PEG, PBP@Anti, M2c, and PBP@Anti@M2c prepared in Example 1 of this invention.

[0034] Figure 6 The C1s high-resolution XPS spectra of BPNSs and BP@PEG prepared in Example 1 of this invention are shown.

[0035] Figure 7 The hemolysis rate of erythrocytes by different concentrations of PBP@Anti@M2c prepared in Example 1 of the invention is shown in the figure.

[0036] Figure 8The images show the PAS and Masson staining results of kidney tissue from UUO model mice under different treatment interventions. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0039] The bicarboxyl-terminated polyethylene glycol was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a molecular weight of M. W =4000; The amino-modified Anti miR 33 was purchased from Aoke Biotechnology Co., Ltd., and the sequence of this Anti miR 33 is as follows: TGCAATGCAACTACAATGCAC.

[0040] Example 1

[0041] A method for preparing biomimetic black phosphorus nanomaterials includes the following steps:

[0042] S1. Preparation of black phosphorus nanosheets: Black phosphorus blocks were cut into 5mm pieces in a glove box after being deoxygenated by purging with inert gas. 2 Thin sheets of black phosphorus were adhered to a conductive substrate. Then, at room temperature and a constant voltage of -5V, the sheets were exfoliated in propylene carbonate and tetrabutylammonium hexafluorophosphate for 20 min, using a large piece of black phosphorus as the cathode and a platinum sheet as the anode. The exfoliated sheets were then sonicated for 1.5 h. The exfoliated black phosphorus sheets were dispersed in anhydrous acetone, centrifuged at 2500 rpm for 15 min, and then centrifuged at 8500 rpm for 13 min. The product was collected to obtain black phosphorus nanosheets, denoted as BPNSs.

[0043] S2. Preparation of carboxylated black phosphorus nanosheets: 25 mg of black phosphorus nanosheets were dispersed in 10 mL of ultrapure water, and the pH was adjusted to 8.5. Then, 130 mg of double-terminated carboxyl polyethylene glycol was added, and the mixture was stirred at 25 °C for 6 h. After the reaction was completed, the mixture was centrifuged, filtered, washed, and dried to obtain carboxylated black phosphorus nanosheets, denoted as BP@PEG.

[0044] S3. Preparation of composite black phosphorus nanomaterial dispersion: 15 mg of carboxylated black phosphorus nanosheets were added to 10 mL of 25 mM / L MES buffer, followed by 55 mg EDC and 65 mg NHS. The mixture was stirred and activated at room temperature for 20 min. Then, 1.5 mL of 10 μM / L terminal amino-modified Anti miR 33 was added, and the mixture was reacted at 25 °C for 6 h. After the reaction was completed, the mixture was filtered and washed, and then dispersed in enzyme-free water to obtain the composite black phosphorus nanomaterial dispersion, denoted as PBP@Anti.

[0045] S4. Preparation of biomimetic black phosphorus nanomaterials: RAW264.7 macrophage cell line was subjected to 20 ng / mL... -1 M2c macrophages were obtained after 48 hours of IL-10 induction, and then the M2c macrophages were suspended in a solution containing 1 mM In the hypotonic medium of PMSF, the cells were sonicated for 2 min and then centrifuged at 1500 rpm for 5 min to remove organelles. The supernatant was collected and further centrifuged at 15000 rpm for 30 min to collect the M2c macrophage membrane, which was stored at -80℃ at a concentration of 0.8 mg / mL and designated as M2c for later use. The composite black phosphorus nanomaterial dispersion with a concentration of 1.3 mg / mL was mixed evenly with the M2c macrophage membrane solution at a volume ratio of 1:2. The mixture was then sonicated at 35 kHz and 130 W for 8 min. After treatment, the mixture was fused 20 times using a micro extruder, followed by centrifugation at 5000 rpm for 30 min to remove the uncoated membrane. Finally, the mixture was centrifuged at 10000 rpm for 15 min to obtain the biomimetic black phosphorus nanomaterial, designated as PBP@Anti@M2c.

[0046] Example 2

[0047] A method for preparing biomimetic black phosphorus nanomaterials includes the following steps:

[0048] S1. Preparation of black phosphorus nanosheets: Black phosphorus blocks were cut into 1mm pieces in a glove box after being deoxygenated by purging with inert gas. 2 Thin sheets of black phosphorus were adhered to a conductive substrate. Then, at room temperature and under a constant voltage of -5V, using a large piece of black phosphorus as the cathode and a platinum sheet as the anode, the sheets were exfoliated in propylene carbonate and tetrabutylammonium hexafluorophosphate for 10 min. After sonication for 1 h, the exfoliated black phosphorus sheets were dispersed in anhydrous acetone, centrifuged at 2000 rpm for 10 min, and then centrifuged at 8000 rpm for 10 min. The product was collected to obtain black phosphorus nanosheets, denoted as BPNSs.

[0049] S2. Preparation of carboxylated black phosphorus nanosheets: 20 mg of black phosphorus nanosheets were dispersed in 10 mL of ultrapure water, the pH was adjusted to 9, and then 100 mg of double-terminated carboxyl polyethylene glycol was added. The mixture was stirred at 20 °C for 7 h. After the reaction was completed, the mixture was centrifuged, filtered, washed and dried to obtain carboxylated black phosphorus nanosheets, denoted as BP@PEG.

[0050] S3. Preparation of composite black phosphorus nanomaterial dispersion: 10 mg of carboxylated black phosphorus nanosheets were added to 10 mL of 20 mM / L MES buffer, followed by 50 mg EDC and 60 mg NHS. The mixture was stirred and activated at room temperature for 15 min. Then, 1.5 mL of 15 μM / L terminal amino-modified Anti miR 33 was added, and the mixture was reacted at 20 °C for 7 h. After the reaction was completed, the mixture was filtered and washed, and then enzyme-free water was added for dispersion to obtain composite black phosphorus nanomaterial dispersion, denoted as PBP@Anti.

[0051] S4. Preparation of biomimetic black phosphorus nanomaterials: RAW264.7 macrophage cell line was subjected to 20 ng / mL... -1 M2c macrophages were obtained after 48 hours of IL-10 induction, and then the M2c macrophages were suspended in a solution containing 1 mM In the hypotonic medium of PMSF, the cells were sonicated for 2 min and then centrifuged at 1500 rpm for 5 min to remove organelles. The supernatant was collected and further centrifuged at 15000 rpm for 30 min to collect the M2c macrophage membrane, which was stored at -80℃ at a concentration of 0.8 mg / mL and designated as M2c for later use. The composite black phosphorus nanomaterial dispersion with a concentration of 1 mg / mL was mixed evenly with the M2c macrophage membrane solution at a volume ratio of 1:1.5. The mixture was then sonicated at 30 kHz and 100 W for 10 min. After treatment, the mixture was fused 20 times using a micro extruder, followed by centrifugation at 5000 rpm for 30 min to remove the uncoated membrane. Finally, the mixture was centrifuged at 10000 rpm for 15 min to obtain the biomimetic black phosphorus nanomaterial, designated as PBP@Anti@M2c.

[0052] Example 3

[0053] A method for preparing biomimetic black phosphorus nanomaterials includes the following steps:

[0054] S1. Preparation of black phosphorus nanosheets: Black phosphorus blocks were cut into 3mm pieces in a glove box after being deoxygenated by purging with inert gas. 2Thin sheets of black phosphorus were adhered to a conductive substrate. Then, at room temperature and under a constant voltage of -5V, using a large piece of black phosphorus as the cathode and a platinum sheet as the anode, the sheets were exfoliated in propylene carbonate and tetrabutylammonium hexafluorophosphate for 30 min. After sonication for 2 h, the exfoliated black phosphorus sheets were dispersed in anhydrous acetone, centrifuged at 3000 rpm for 20 min, and then centrifuged at 9000 rpm for 15 min. The product was collected to obtain black phosphorus nanosheets, denoted as BPNSs.

[0055] S2. Preparation of carboxylated black phosphorus nanosheets: 30 mg of black phosphorus nanosheets were dispersed in 10 mL of ultrapure water, and the pH was adjusted to 9. Then, 150 mg of double-terminated carboxyl polyethylene glycol was added, and the mixture was stirred at 30 °C for 5 h. After the reaction was completed, the mixture was centrifuged, filtered, washed, and dried to obtain carboxylated black phosphorus nanosheets, denoted as BP@PEG.

[0056] S3. Preparation of composite black phosphorus nanomaterial dispersion: 20 mg of carboxylated black phosphorus nanosheets were added to 10 mL of 30 mM / L MES buffer, followed by 60 mg of EDC and 70 mg of NHS. The mixture was stirred and activated at room temperature for 25 min. Then, 2 mL of 10 μM / L terminal amino-modified Anti miR 33 was added, and the mixture was reacted at 30 °C for 5 h. After the reaction was completed, the mixture was filtered and washed, and then enzyme-free water was added for dispersion to obtain composite black phosphorus nanomaterial dispersion, denoted as PBP@Anti.

[0057] S4. Preparation of biomimetic black phosphorus nanomaterials: RAW264.7 macrophage cell line was subjected to 20 ng / mL... -1 M2c macrophages were obtained after 48 hours of IL-10 induction, and then the M2c macrophages were suspended in a solution containing 1 mM In the hypotonic medium of PMSF, the cells were sonicated for 2 min and then centrifuged at 1500 rpm for 5 min to remove organelles. The supernatant was collected and further centrifuged at 15000 rpm for 30 min to collect the M2c macrophage membrane, which was stored at -80℃ at a concentration of 0.8 mg / mL and designated as M2c for later use. The composite black phosphorus nanomaterial dispersion with a concentration of 1.5 mg / mL was mixed evenly with the M2c macrophage membrane solution at a volume ratio of 1:2.5. The mixture was then sonicated at 40 kHz and 150 W for 5 min. After treatment, the mixture was fused 20 times using a micro extruder, followed by centrifugation at 5000 rpm for 30 min to remove the uncoated membrane. Finally, the mixture was centrifuged at 10000 rpm for 15 min to obtain the biomimetic black phosphorus nanomaterial, designated as PBP@Anti@M2c.

[0058] The samples prepared in Example 1 were subjected to comprehensive quality characteristics and functional model verification, as detailed below:

[0059] (1) Transmission electron microscopy: 1 mg mL -1 Concentrations of BPNSs, M2c membrane dispersions, and PBP@Anti@M2c ultrapure water dispersions were dropped onto a copper mesh and dried at room temperature in a desiccator. The samples were then stored in the desiccator. The copper mesh containing the samples was fixed in a sample holder, and the accelerating voltage was 200 kV. Observation and imaging were performed using a transmission electron microscope. The results are as follows: Figure 1 .

[0060] (2) Transmission electron microscopy experiments of BPNSs and H2O2-treated samples: 1 mg of BPNSs was added to 1 mL of ultrapure water or 1 mL of H2O2 and allowed to stand at room temperature in the dark for 24 hours. The resulting system was then dropped onto a copper mesh and dried at room temperature in a desiccator. The sample-loaded copper mesh was fixed in a sample holder with an accelerating voltage of 200 kV and observed and photographed using a transmission electron microscope. The results are as follows: Figure 2 .

[0061] (3) Raman spectroscopy: 1 mg mL -1 Concentrated concentrations of BPNSs and BP@PEG ultrapure aqueous dispersions, along with PEG, were loaded onto the center of a clean quartz substrate. After being allowed to dry naturally, suitable areas were located under an optical microscope using a 532 nm laser wavelength, with a wavenumber range of 300-4000 cm⁻¹. -1 The result is as follows Figure 3 .

[0062] (4) Zeta potential and hydrodynamic diameter detection: BPNSs, BP@PEG, PBP@Anti, M2c membranes and PBP@Anti@M2c were placed in ultrapure water to form 1 mg mL -1 After thoroughly mixing the concentration dispersion by sonication for 20 minutes, the Zeta potential and hydrodynamic diameter were measured, and the results are as follows. Figure 4 and Figure 5 .

[0063] (5) XPS: Take 5 mg of the lyophilized powder of the sample to be tested, carefully load the powder onto the center of the sample stage, and scan to obtain the full spectrum, carbon spectrum, and fine spectrum of the target element of the sample X-ray photoelectron spectrum. The results are as follows: Figure 6 .

[0064] (6) Hemolysis test: Fresh anticoagulated whole blood from C57BL / 6 mice was centrifuged at 1500 rpm for 5 min. The supernatant and leukocyte layer were discarded, and the red blood cells were washed three times with sterile physiological saline until the supernatant was clear. The washed red blood cells were prepared into a 2% (v / v) red blood cell suspension with physiological saline. Different concentrations of PBP@Anti@M2c were added and gently mixed, and the suspension was incubated at 37℃ for 1 h. After incubation, the suspension was centrifuged at 3000 rpm for 5 min to precipitate the unruptured red blood cells. 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 540 nm using a spectrophotometer. The results are as follows. Figure 7 .

[0065] (7) Establishment of UUO model in experimental animals: Male C57BL / 6 mice (8 weeks old, weighing 20–25g) were fasted for 4–6 hours before surgery. They were anesthetized with 1% sodium pentobarbital, their abdomens were shaved, disinfected with povidone-iodine, and fixed in a supine position. A 1cm longitudinal incision was made approximately 0.5cm lateral to the left midline of the abdomen, and the subcutaneous tissue and muscle were dissected layer by layer. The intestines were gently pushed aside to expose the left kidney and ureter. The ureter was double-ligated with 5-0 silk suture approximately 5mm below the renal pelvis. The muscle layer and skin were sutured layer by layer, and 1mL of warm saline was injected subcutaneously to prevent dehydration. The mice were placed on a warming pad to awaken, and analgesics were administered. Their mental state, activity, and wound healing were observed daily.

[0066] Animal experiment grouping and intervention: Eight-week-old male C57BL / 6 mice were randomly divided into five groups: Control group, UUO group, UUO+PBP@Anti group, UUO+PBP@M2c group, and UUO+PBP@Anti@M2c group. Intervention was performed 2 hours after model establishment. Mice were fixed to a tail vein injection device. The mouse's tail was held in place by the thumb, index, and middle fingers of one hand, while the tail was gently wiped with an alcohol swab to make the veins more visible. A sterile insulin syringe was then inserted parallel to the tail vein. After blood was aspirated, the corresponding intervention fluid was injected into the mouse's tail vein. The mice were administered sterile PBS via tail vein injection in the Contro1 group, UUO group, AKI+PBP@Anti group, and M2c group. The AKI+PBP@Anti group received a PBP@Anti suspension prepared with sterile PBS via tail vein injection, and the AKI+PBP@Anti@M2c group received a PBP@Anti@M2c suspension prepared with sterile PBS via tail vein injection. Injections were administered twice weekly for 14 consecutive days. Afterward, the mice were euthanized and their tissues were collected. Results are as follows: Figure 8 .

[0067] from Figure 1As can be seen, BPNSs exhibits a single-layer lamellar structure, while the M2c membrane has a three-dimensional circular shape. After extrusion, it is successfully wrapped around the surface of PBP@Anti, forming a complete and continuous membrane structure.

[0068] from Figure 2 Normal BPNSs exhibit a single-layer lamellar structure, while BPNSs treated with H2O2 develop pores on their surface due to oxidation and etching, indicating BPNSs degradation.

[0069] from Figure 3 As can be seen, the Raman spectrum of the original black phosphorus nanosheets (BPNSs) shows a depth of approximately 360 cm⁻¹. -1 (Ag1), 440cm -1 (B2g) and 470cm -1 The characteristic peak at (Ag2) indicates successful exfoliation and preservation of its inherent crystal structure. After PEGylation, the BP@PEG sample retained these characteristic peaks, indicating that surface modification did not significantly alter the electronic structure of BPNSs. Furthermore, a peak at 3400–3700 cm⁻¹ appeared in both BP@PEG and pure PEG. -1 The broad peaks within the range correspond to OH stretching vibrations, further confirming the presence of a PEG coating on the nanosheet surface.

[0070] from Figure 4 As can be seen, the original BPNSs had a highly negative Zeta potential (approximately -42 mV), reflecting strong electrostatic repulsion and excellent colloidal stability in water. After PEGylation, the Zeta potential decreased to approximately -28 mV, indicating that the surface charge was partially shielded by the neutral PEG chains. Subsequent binding with Anti-miR-33 and M2c films led to a further reduction in negative charge, to approximately -30 to -35 mV, consistent with the introduction of additional functional components. Despite this change, all modified nanoparticles maintained a Zeta potential above -30 mV, indicating sufficient stability to prevent aggregation during storage and in biological environments.

[0071] from Figure 5 As can be seen, the original BPNSs had a hydrodynamic diameter of approximately 150 nm, demonstrating good colloidal stability in aqueous solution. After PEGylation, the particle size increased to approximately 200 nm, due to the formation of a hydrophilic polymer shell. Upon binding with Anti-miR-33, the size increased slightly to approximately 210 nm, likely due to the attachment of bioactive molecules. The final PBP@Anti@M2c exhibited an even larger hydrodynamic diameter of approximately 280 nm, consistent with the coating of the M2c film. The gradual increase in size at each modification step indicates successful hierarchical surface engineering without aggregation.

[0072] from Figure 6 As can be seen, the original black phosphorus nanosheets (BPNSs) exhibit a main peak at approximately 284.5 eV, which can be attributed to sp in graphite carbon or residual organic solvents. 2 Carbon; after PEGylation modification, in addition to the main peak, a new peak appeared at 285 eV in the C1s spectrum, corresponding to C–O and Cn bonds respectively, confirming the successful modification of polyethylene glycol.

[0073] from Figure 7 As can be seen, within the concentration range of 20-60 μg / mL, the hemolysis rate of PBP@Anti@M2c was less than 1%, indicating good blood compatibility at low to moderate concentrations. When the concentration increased to 80 and 100 μg / mL, the hemolysis rate increased slightly (approximately 1.5% and 2.5%, respectively), but remained far lower than the positive control (distilled water, 100% hemolysis). The figure shows the color changes of the supernatant in each group: the water treatment group was dark red, indicating complete hemolysis, while the supernatant of the PBP@Anti@M2c group was basically colorless or very pale, further confirming its low hemolytic toxicity. The overall results indicate that PBP@Anti@M2c possesses good biocompatibility at physiologically relevant concentrations and is suitable for potential in vivo applications.

[0074] from Figure 8 It can be seen from this that Figure 8 PAS staining revealed changes in renal tubular structure: In the control group, renal tubules were neatly arranged with an intact basement membrane; in the UUO group, significant renal tubular dilation, atrophy, and epithelial cell shedding were observed, indicating renal injury; PBP@Anti and PBP@M2c groups partially improved renal tubular structural disorder; while the PBP@Anti@M2c bifunctional nanoparticle group showed the most significant protective effect, with renal tubular morphology approaching normal, indicating its potential for synergistic repair of renal tubular injury. Figure 8 b. Masson staining was used to assess the degree of fibrosis: the blue area represents collagen deposition. Extensive blue staining was observed in the UUO group, indicating severe interstitial fibrosis; single treatment with PBP@Anti and PBP@M2c slightly reduced fibrosis; while the combined treatment group with PBP@Anti@M2c significantly reduced collagen deposition, approaching control levels. This indicates that the nanosystem effectively inhibits the progression of renal fibrosis through targeted delivery and regulation of the immune microenvironment.

[0075] The above description is a further detailed explanation of the present invention in conjunction with specific implementation examples. It should not be considered that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the protection scope of the present invention.

[0076] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing biomimetic black phosphorus nanomaterials, characterized in that, Includes the following steps: S1. In a glove box after deoxygenation by introducing inert gas, black phosphorus blocks are cut into thin slices, adhered to a conductive substrate, and then electrochemically exfoliated to obtain black phosphorus nanosheets. S2. Disperse black phosphorus nanosheets in ultrapure water, adjust the pH, then add double-ended carboxyl polyethylene glycol, stir and react. After the reaction is complete, centrifuge, filter, wash and dry to obtain carboxylated black phosphorus nanosheets. S3. Add carboxylated black phosphorus nanosheets to MES buffer, then add EDC and NHS, stir and activate, then add terminal amino-modified Anti miR 33, and carry out a constant temperature reaction. After the reaction is completed, filter and wash, then add enzyme-free water to disperse and obtain a dispersion of composite black phosphorus nanomaterials. S4. Mix the composite black phosphorus nanomaterial dispersion with the M2c macrophage membrane solution evenly, then perform ultrasonic treatment, and after the treatment is completed, perform extrusion fusion to obtain the biomimetic black phosphorus nanomaterial. The preparation method of the M2c macrophage membrane solution in step S4 is as follows: The RAW264.7 macrophage cell line was subjected to 20 ng / mL -1 After IL-10 induction for 48 h, M2c macrophages were obtained. The M2c macrophages were then suspended in hypotonic medium containing 1 mM PMSF, sonicated for 2 min, and centrifuged at 1500 rpm for 5 min to remove organelles. The supernatant was collected and further centrifuged at 15000 rpm for 30 min to collect the M2c macrophage membranes, which were then stored at -80℃ for later use.

2. The preparation method according to claim 1, characterized in that, The area of ​​the thin sheet mentioned in step S1 is 1-5 mm. 2 The dimensions of the black phosphorus nanosheets are: thickness of 1-10 nm and particle size of 50-150 nm.

3. The preparation method according to claim 1, characterized in that, The specific method for electrochemical stripping described in step S1 is as follows: At room temperature, under a constant voltage of -5V, using large pieces of black phosphorus as the cathode and a platinum sheet as the anode, stripping is performed in propylene carbonate and tetrabutylammonium hexafluorophosphate for 10-30 min, followed by sonication for 1-2 h. The stripped black phosphorus sheets are then dispersed in anhydrous acetone, centrifuged at 2000-3000 rpm for 10-20 min, and then centrifuged at 8000-9000 rpm for 10-15 min. The product is then collected.

4. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of black phosphorus nanosheets, ultrapure water, and bicarboxyl-terminated polyethylene glycol is 2-3:1000:10-15, the pH is 8-9, the stirring reaction temperature is 20-30℃, and the time is 5-7h.

5. The preparation method according to claim 1, characterized in that, The concentration of the MES buffer in step S3 is 20-30 mM / L, and the pH is 5.5-6.5; the stirring activation temperature is 20-30℃, and the time is 15-25 min.

6. The preparation method according to claim 1, characterized in that, In step S3, the mass-to-volume ratio of carboxylated black phosphorus nanosheets, MES buffer, EDC, NHS, and terminal amino-modified Anti miR 33 is 1-2 mg:1 mL:5-6 mg:6-7 mg:1.5-2 mL, and the concentration of terminal amino-modified Anti miR 33 is 10-20 μM / L; the isothermal reaction is carried out at a temperature of 20-30 °C for 5-7 h.

7. The preparation method according to claim 1, characterized in that, In step S4, the concentration of the composite black phosphorus nanomaterial dispersion is 1-1.5 mg / mL, the volume ratio of the composite black phosphorus nanomaterial dispersion to the M2c macrophage membrane solution is 1:1.5-2.5, the frequency of the ultrasound is 30-40 kHz, the power is 100-150 W, and the time is 5-10 min.

8. A biomimetic black phosphorus material prepared by the method according to any one of claims 1-7.

9. The use of the biomimetic black phosphorus material as described in claim 8 in the preparation of a drug for chronic kidney disease.

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