Radix astragali and angelica composite carbon dot nanoscale enzyme, preparation method and application in renal ischemia-reperfusion injury

Abstract

The application belongs to the technical field of nanobiomedical materials, and particularly relates to a composite carbon dot nanenzyme based on radix astragali and angelica sinensis, a preparation method and application in renal ischemia-reperfusion injury. The nanenzyme takes radix astragali and angelica sinensis as a composite precursor and is synthesized by a hydrothermal method. The nanenzyme has a particle size of 3-5 nm. The surface of the nanenzyme is rich in nitrogen and oxygen functional groups, wherein the content of nitrogen is 6.0-8.5 at.%, and the content of oxygen is 24.0-28.0 at.%. The nanenzyme has superoxide dismutase-like activity. The nanenzyme has renal passive targeting ability, is enriched at the injury site, can significantly improve renal function, reduce renal tissue injury, inhibit immune cell infiltration and inflammatory response, has excellent biocompatibility in in-vivo and in-vitro toxicity evaluation, and has clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedical materials technology, specifically relating to a composite carbon dot nanoenzyme based on Astragalus membranaceus and Angelica sinensis, its preparation method, and its application in renal ischemia-reperfusion injury. Background Technology

[0002] Carbon dots (CDs), as zero-dimensional carbon-based nanomaterials, have seen rapid development in fields such as bioimaging, drug delivery, catalysis, and sensing since their accidental discovery in 2004, due to their ultra-small size (<10 nm), excellent optical properties, low toxicity, and surface functionalizability. Among them, the "carbon dot nanozymes" subclass, which possess enzyme-like activity, has become one of the most promising new members of the nanozyme family for clinical translation because it avoids the disadvantages of natural enzymes, such as easy inactivation, high cost, and demanding storage conditions.

[0003] Existing preparation routes for carbon dot nanozymes are mainly divided into two categories: "top-down" and "bottom-up". Top-down methods (laser ablation, arc discharge, electrochemical oxidation) produce carbon dots with high crystallinity, but have low yields and expensive equipment; bottom-up methods (hydrothermal, solvothermal, microwave) have become the mainstream due to their mild operation, high yields, and abundant surface functional groups. However, most studies still remain at the level of single carbon sources (citric acid, glucose, urea, etc.) or simple dopants (ethylenediamine, thiourea), resulting in limited catalytic activity, insufficient selectivity, and difficulty in finely controlling structure and performance.

[0004] In recent years, utilizing traditional Chinese medicine resources as carbon dot precursors has become a new trend: polysaccharides, flavonoids, alkaloids, and other naturally occurring substances in plants can simultaneously provide C, N, O, and S multi-component dopants and possess inherent pharmacological activities. However, existing literature only reports carbon dots from single medicinal materials, and there is no systematic research on the synergistic use of "medicinal pair" materials in carbon dot structure design. Furthermore, there is a lack of quantitative analysis of the relationship between "precursor ratio, heteroatom bonding mode, and catalytic activity."

[0005] On the other hand, the catalytic types of carbon dot nanozymes are generally concentrated in peroxidase-like (POD) or oxidase-like (OXD) enzymes, which require H2O2 or dissolved oxygen as substrates and are prone to inducing secondary oxidative stress in organisms; while superoxide dismutase-like (SOD) enzymes can remove superoxide anions (O2•) without additional oxidants. - While theoretically safer, the activity of existing SOD-type carbon dots is generally 2-3 orders of magnitude lower than that of natural enzymes, and there is a lack of specificity proof that they "only remove and do not produce reactive oxygen species".

[0006] In summary, the field still faces the following bottlenecks: 1. Limited precursor selection and lack of "drug pair" synergistic doping strategies; 2. Difficulty in controllably introducing highly efficient catalytic sites such as graphitic nitrogen and pyridine nitrogen; 3. Difficulty in achieving both small size (<5 nm) and high yield (>15%); 4. Low SOD-like activity and poor selectivity, and lack of scale-up processes; 5. Lack of quality evaluation and standard drafts for mass production.

[0007] Therefore, developing a carbon dot nanozyme that uses a pair of traditional Chinese medicines as a composite precursor and can achieve the synergistic effect of "high graphite nitrogen-high defects-small size" through a one-step hydrothermal process, and establishing corresponding scale-up processes and quality indicators, is of great practical significance for promoting the application of carbon dot nanozymes from the laboratory to clinical and industrial applications. Summary of the Invention

[0008] The purpose of this invention is to provide a composite carbon nanozyme based on Astragalus membranaceus and Angelica sinensis, its preparation method, and its application in renal ischemia-reperfusion injury. The carbon nanozyme prepared using the traditional Chinese medicine compound Astragalus membranaceus and Angelica sinensis as precursors has superoxide dismutase-like activity and can be used to scavenge reactive oxygen species, inhibit inflammatory responses, alleviate renal ischemia-reperfusion injury, and can also be used to prepare drugs or functional materials for the treatment of acute kidney injury.

[0009] Therefore, the present invention provides the following technical solution.

[0010] The first aspect of the present invention provides a composite carbon dot nanozyme based on Astragalus membranaceus and Angelica sinensis, wherein the nanozyme is synthesized by hydrothermal method using Astragalus membranaceus and Angelica sinensis as composite precursors; The nanozyme has a particle size of 3-5 nm; The surface of the nanozyme is rich in nitrogen and oxygen functional groups, wherein the nitrogen content is 6.0–8.5 at.% and the oxygen content is 24.0–28.0 at.%. The nanozyme exhibits superoxide dismutase-like activity, targeting superoxide anion radicals (O2•) at a concentration of 30 μg / mL. - The clearance rate of the substance is over 80%; and it does not exhibit peroxidase-like (POD) or oxidase-like (OXD) activity under physiological pH conditions (pH 7.4), thus avoiding the risk of secondary oxidative stress.

[0011] In terms of structural characterization, the carbon dot nanozyme exhibits a graphene-like structure, with a broad diffraction peak at 22.8° in its X-ray diffraction (XRD) spectrum, corresponding to the (002) plane of graphitic carbon. Raman spectroscopy shows that the intensity ratio of the D band to the G band (ID / IG) is 0.85–1.05, indicating that the nanozyme material has a high defect density, which is beneficial for the exposure of active sites. X-ray photoelectron spectroscopy (XPS) analysis shows that the nitrogen element in the carbon dot nanozyme exists in three forms: pyridine nitrogen, pyrrole nitrogen / amino nitrogen, and graphitic nitrogen, with the relative content of graphitic nitrogen being 15%–25%. This structure is considered to be key to enhancing the simulated activity of SOD. The surface zeta potential of the carbon dot nanozyme is -20 to -30 mV, indicating that it has good colloidal stability in aqueous solution.

[0012] A second aspect of the present invention provides a method for preparing a composite carbon dot nanozyme based on Astragalus membranaceus and Angelica sinensis, the method comprising the following steps: S1: Add the mixed powder containing astragalus powder and angelica powder to water and disperse it by ultrasonication to obtain a suspension; S2: The resulting suspension is subjected to a hydrothermal reaction. After the reaction is complete, the suspension is centrifuged and the supernatant is collected. S3: The obtained supernatant was filtered through a membrane, ultrafiltered, dialyzed, and dried sequentially to obtain a composite carbon dot nanozyme.

[0013] In a preferred embodiment of the present invention, in step S1, the mass ratio of Astragalus powder to Angelica powder is (0~10):(0~10).

[0014] In a preferred embodiment of the present invention, in step S1, the mass ratio of Astragalus powder to Angelica powder is (0.2~5):1.

[0015] In a preferred embodiment of the present invention, in step S1, the mass ratio of Astragalus powder to Angelica powder is 1:1.

[0016] In a preferred embodiment of the present invention, in step S1, the amount of water added is 5 to 20 times the mass of the mixed powder.

[0017] In a preferred embodiment of the present invention, in step S1, the amount of water added is 10 to 15 times the mass of the mixed powder.

[0018] In a preferred embodiment of the present invention, in step S1, the ultrasonic dispersion time is 10~60 min.

[0019] In a preferred embodiment of the present invention, in step S1, the ultrasonic dispersion time is 30-40 min.

[0020] In a preferred embodiment of the present invention, in step S2, the hydrothermal reaction is carried out in a hydrothermal reactor lined with polytetrafluoroethylene, and the hydrothermal reaction conditions are: temperature of 150~200℃ and time of 4~10 h.

[0021] In a preferred embodiment of the present invention, in step S2, the hydrothermal reaction conditions are: temperature of 170~190℃ and time of 5~7 h.

[0022] In a preferred embodiment of the present invention, in step S2, the centrifugation conditions are: rotation speed of 8000~10000 rpm and time of 10~15 min.

[0023] In a preferred embodiment of the present invention, in step S2, the centrifugation conditions are: a rotation speed of 9000 rpm and a time of 12 min.

[0024] In a preferred embodiment of the present invention, in step S3, the filter membrane size is 0.22 µm.

[0025] In a preferred embodiment of the present invention, in step S3, the molecular weight cutoff of the ultrafiltration is 3 kDa.

[0026] In a preferred embodiment of the present invention, in step S3, the molecular weight cutoff for dialysis is 3500 Da, and the dialysis time is 2 to 4 days.

[0027] In a preferred embodiment of the present invention, in step S3, the dialysis time is 3 days, during which the ultrapure water is replaced every 8 hours.

[0028] In a preferred embodiment of the present invention, in step S3, the drying is freeze drying, and the freeze drying time is 24~48 h.

[0029] In a preferred embodiment of the present invention, in step S3, the freeze-drying time is 36 h.

[0030] In a preferred embodiment of the present invention, in step S3, the particle size of the obtained composite carbon dot nanozyme is 3.5 to 4.5 nm, and the yield can reach more than 15%.

[0031] A third aspect of the present invention provides the use of the Astragalus-Angelica-based composite carbon dot nanozyme as described above, or the Astragalus-Angelica-based composite carbon dot nanozyme prepared according to the preparation method described above, in the preparation of a product for targeted treatment of acute kidney injury.

[0032] In a preferred embodiment of the present invention, the acute kidney injury is acute kidney injury caused by ischemia-reperfusion.

[0033] A fourth aspect of the present invention provides a pharmaceutical composition for treating acute kidney injury, comprising: (i) the Astragalus-Angelica composite carbon dot nanozyme as described above, or the Astragalus-Angelica composite carbon dot nanozyme prepared according to the preparation method described above; and (ii) Pharmaceutically acceptable carriers or excipients.

[0034] In a preferred embodiment of the present invention, the effective dose of the composite carbon dot nanozyme in the pharmaceutical composition is 1~10 mg / kg body weight, preferably 4~6 mg / kg body weight.

[0035] In a preferred embodiment of the present invention, the route of administration of the drug composition includes intraperitoneal injection, intravenous injection, or oral administration.

[0036] A fifth aspect of the present invention provides a method for treating acute kidney injury, comprising administering to a subject in need an effective dose of the Astragalus-Angelica-based composite carbon dot nanozyme as described above, or the Astragalus-Angelica-based composite carbon dot nanozyme prepared according to the preparation method described above, or the pharmaceutical composition described above.

[0037] In a preferred embodiment of the present invention, the effective dose is 1~10 mg / kg body weight, preferably 4~6 mg / kg body weight.

[0038] In a preferred embodiment of the present invention, the administration route includes intraperitoneal injection, intravenous injection, or oral administration.

[0039] By employing the above technical solution, the present invention has at least the following advantages: 1. Innovative material design: This invention is the first to use a traditional Chinese medicine "pair" (Astragalus membranaceus-Angelica sinensis) as a composite precursor to synthesize composite carbon dot nanozymes through a one-step hydrothermal method, achieving multi-component synergistic doping and significantly improving the SOD-like activity and biocompatibility of the prepared nanozymes.

[0040] 2. Green and safe preparation process: In the preparation process of the nanozyme of the present invention, only water is used as solvent, and no chemical reagents are added. The reaction conditions are mild and meet the requirements of green chemistry. The obtained carbon dot nanozyme has no POD / OXD activity under physiological conditions, avoiding secondary oxidative damage.

[0041] 3. Excellent therapeutic effect: The composite carbon nanoparticle nanozyme prepared in this invention significantly alleviates renal ischemia-reperfusion injury through a multi-link action network of "scavenging ROS-inhibiting AP-1-reducing chemokines-blocking immune infiltration", and its effect is superior to carbon dots from single medicinal materials.

[0042] 4. Simple and easy-to-implement scalability potential: The preparation method of the composite carbon dot nanozyme of the present invention is simple, the raw materials are widely available, and it is easy to scale up production, providing a new strategy for the application of nanozymes in the treatment of acute kidney injury.

[0043] 5. Excellent targeting and safety: The nanozyme of this invention has passive targeting ability to the kidneys, and it is enriched and enhanced at the site of injury, which can significantly improve kidney function, reduce kidney tissue damage, and inhibit immune cell infiltration and inflammatory response; in vivo and in vitro toxicity evaluation shows that it has excellent biocompatibility and has clinical translation potential.

[0044] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0045] Figure 1 A flowchart illustrating the synthesis of the nanozyme AM-AS@CDs of the present invention and a schematic diagram illustrating its mechanism of action in the treatment of renal ischemia-reperfusion injury are shown.

[0046] Figure 2 The structural characterization of the nanoenzyme materials AM-AS@CDs, AM@CDs, and AS@CDs of the present invention is shown; wherein, a is the UV-Vis absorption spectrum of the three nanoenzyme materials CDs; (b, c) are the fluorescence excitation / emission spectra of the three nanoenzyme materials CDs, respectively; d is the hydrodynamic size distribution of AM@CDs, e is the hydroenzyme material of AS@CDs, and f is the hydrodynamic size distribution of AM-AS@CDs; g is the high-resolution transmission electron microscopy (HRTEM) image of AM-AS@CDs, with the inset showing the corresponding particle size distribution; h is the X-ray diffraction (XRD) pattern of the three nanoenzyme materials CDs; i is the Raman spectrum of the three nanoenzyme materials CDs; and j is the Zeta potential of the three nanoenzyme materials CDs.

[0047] Figure 3 The full infrared and X-ray photoelectron spectroscopy (XPS) spectra of the nanoenzyme materials AM-AS@CDs, AM@CDs, and AS@CDs of the present invention are shown; wherein, (ac) is the FT-IR spectrum of the three nanoenzyme materials CDs; (df) is the XPS full scan spectrum of the three nanoenzyme materials CDs (including the atomic percentage of elemental composition); (gi) is the high-resolution C 1s XPS spectrum of the three nanoenzyme materials CDs; and (jl) is a bar chart of the atomic percentages of C, O, and N elements in the three nanoenzyme materials CDs.

[0048] Figure 4The comparison of SOD-like activities (WST-1 method) and verification by electron paramagnetic resonance (EPR) superoxide radical scavenging of the nanoenzyme materials AM-AS@CDs, AM@CDs, and AS@CDs of the present invention are shown. In this diagram, (a) shows the SOD-like activity of the three nanoenzyme materials CDs; (b) shows the EPR spectra of the three nanoenzyme materials CDs, indicating no hydroxyl radicals were generated; (c, d) show the superoxide radical scavenging activity verified by DPPH. • (c) and ABTS +• (d) The free radical scavenging ability of the three nanozyme materials CDs was evaluated by assay; (e, f) are heat maps of the antioxidant properties of the three nanozyme materials CDs and a summary of their free radical types; (gi) shows the SOD-like activity of composite AM-AS@CDs synthesized with different precursor mass ratios (AM and AS = 5:1, 2:1, 1:1, 1:2, 1:5), of which the 1:1 ratio was selected as the optimal ratio for subsequent studies; j is a schematic diagram of the surface functional group passivation experiment; (k, l) show the successful synthesis of surface-modified CDs confirmed by FT-IR spectroscopy: AM-AS@CDs-PS / AM-AS@CDs-PS-Hy (k) and AM-AS@CDs-SPI (l).

[0049] Figure 5 The nanozyme materials AM-AS@CDs, AM@CDs, and AS@CDs of the present invention are shown to exhibit OXD-like and POD-like activities; wherein, Figure a on the left represents oxidase activity, and Figure b on the right represents peroxidase activity; none of these three carbon dots exhibit OXD and POD activities, indicating that they do not generate additional ROS.

[0050] Figure 6 The in vitro safety of AM-AS@CDs with different precursor mass ratios is shown; where ah represents the relative survival rate of NRK-52E and HK-2 cells after exposure to different concentrations of AM-AS@CDs, AM@CDs, or AS@CDs within 24 hours.

[0051] Figure 7 The experimental results of the nanozyme materials AM-AS@CDs, AM@CDs, and AS@CDs of the present invention in clearing ROS (fluorescence microscopy and flow cytometry) and inhibiting apoptosis (Annexin V / PI staining) in renal tubular epithelial cells are shown. Among them, a is a fluorescence micrograph of ROS in NRK-52E cells under indicated treatment (scale bar is 50 μm); b is the flow cytometry analysis of ROS levels in NRK-52E cells; c is the quantitative quantification of relative ROS levels normalized to the control group; d and e are representative flow cytometry images and quantitative analysis of apoptosis in NRK-52E cells.

[0052] Figure 8The study presents serum CRE and BUN levels, Lcn2 / Kim-1 mRNA expression, H&E staining and scoring of renal tissue, and the tissue distribution of AM-AS@CDs in various groups of a mouse model of renal ischemia-reperfusion injury. Specifically, a is a timeline diagram illustrating the prevention and treatment regimens for different CDs treatment groups; b and c are assessments of serum creatinine and blood urea nitrogen 48 hours after reperfusion, respectively; d and e are the mRNA expression levels of Lcn2 and Kim-1 in renal tissue of each group; f is a representative histological image with H&E staining (scale bar 50 μm); g is the renal tissue injury score; h is the time-dependent accumulation of AM-AS@CDs in the kidney; i is the in vitro fluorescence image of the major organs removed from I / R mice 4 hours after AM-AS@CDs administration; and j is the quantitative analysis of fluorescence intensity of the organs shown in figure i.

[0053] Figure 9 The results show the changes in neutrophils, macrophages, and T cells in peripheral blood and kidney tissue analyzed by flow cytometry; the mRNA expression of chemokines Ccl2, Ccl7, Ccl20, and Cxcl1 was detected by qRT-PCR; where a is a representative flow cytometry atlas and quantitative analysis of circulating neutrophils; b is a representative flow cytometry atlas and quantitative analysis of circulating T lymphocytes; c is a representative flow cytometry atlas and quantitative analysis of renal neutrophils; d is a representative flow cytometry atlas and quantitative analysis of renal macrophages; eh is the qRT-PCR results of chemokines Ccl2, Ccl7, Ccl20, and Cxcl1 in each group.

[0054] Figure 10 The results of RNA-seq analysis (volcano plot, cluster heatmap, and GSEA analysis) are shown; Nrf2 / HO-1 levels were detected by Western blot; and SOD1, SOD2, and SOD3 mRNA expression were detected by qRT-PCR. Specifically, a) is the volcano plot of differentially expressed genes between the I / R group and the sham-operated group; b) is the volcano plot of differentially expressed genes between I / R+AM-AS@CDs and I / R; c) is the heatmap of differentially expressed genes under the three treatments; d and e) are the GSEA analyses of the sham-operated group and the I / R group; f and g) are the immunoblotting analyses of Nrf2 and Hmox1 expression in each group; and h) is the qRT-PCR results of SOD1, SOD2, and SOD3 in different treatment groups.

[0055] Figure 11The results show the Venn diagram of differentially expressed genes from RNA-seq analysis and the heatmap of differentially expressed genes from GO / KEGG enrichment analysis; qRT-PCR was used to detect the mRNA expression of Fosl1, S100a8, S100a9, and c-Jun; Western blot was used to detect c-Jun, Fosl1, and their phosphorylation levels; a) shows the intersection of differentially expressed genes between the I / R group and the Sham group, and between the I / R + AM-AS@CDs group and the I / R group; b) shows the GO enrichment analysis of differentially expressed genes in the intersection; c) shows the KEGG pathway enrichment analysis of genes upregulated in the I / R group within the differentially expressed gene intersection; d) shows the expression heatmap of selected genes in the three groups; e) shows the qRT-PCR results of Fosl1, S100a8, S100a9, and c-Jun in different treatment groups; f) shows the representative immunoblotting analysis of Fosl1, p-Fosl1, c-Jun, and pc-Jun expression in each group.

[0056] Figure 12 The changes in renal inflammation levels after ischemia-reperfusion therapy are shown; where a and b are WB analyses of NF-κB expression in each group; c and d are qRT-PCR results of TNF-α and IL-1β in different treatment groups.

[0057] Figure 13 The biocompatibility analysis of AM-AS@CDs in mice is shown; where a is a representative H&E stained histological image (scale bar 50 μm); b is the body weight of mice in different treatment groups; c is the ratio of bilateral kidney weight to body weight; d and e are the assessments of serum creatinine and blood urea nitrogen; f and g are the assessments of serum alanine aminotransferase and aspartate aminotransferase. Detailed Implementation

[0058] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0059] Example 1: Preparation of Astragalus-Angelica Composite Carbon Dot Nanozymes (AM-AS@CDs) The dried roots of Astragalus membranaceus and Angelica sinensis were pulverized separately using a ball mill and passed through a 200-mesh sieve to obtain Astragalus membranaceus powder and Angelica sinensis powder, respectively. 1.0 g of Astragalus membranaceus powder and 1.0 g of Angelica sinensis powder (mass ratio 1:1) were weighed, mixed, and 20 mL of ultrapure water was added. The mixture was then ultrasonically dispersed at 200 W and 40 kHz for 30 min to obtain a suspension. The resulting suspension was transferred to a 50 mL stainless steel hydrothermal reactor lined with polytetrafluoroethylene and reacted at 180℃ for 6 h. After the reaction, the mixture was allowed to cool naturally to obtain the reaction solution. The reaction solution was then centrifuged at 9000 rpm for 12 min, and the supernatant was collected. The supernatant was filtered sequentially through a 0.22 μm aqueous filter membrane and purified by ultrafiltration centrifuge tubes (3 kDa, 4000 rpm for 20 min). The purified supernatant was then transferred to a dialysis bag (3500 Da) and dialyzed in 2 L of ultrapure water for 3 days, with the water changed every 8 h, to obtain the dialysate. The obtained dialysate was pre-frozen at -80℃ and then dried in a freeze dryer for 36 h to obtain a brownish-yellow fluffy powder, which is the composite carbon dot nanoenzyme based on Astragalus membranaceus and Angelica sinensis, hereinafter referred to as AM-AS@CDs or AM-AS@CDs (1:1).

[0060] The yield of AM-AS@CDs was calculated to be approximately 18% (based on the total mass of raw materials).

[0061] Example 2: AM-AS@CDs (1:2) composite carbon dot nanozyme prepared based on Astragalus membranaceus and Angelica sinensis The only difference between this embodiment and Example 1 is that the mass ratio of Angelica sinensis to Astragalus membranaceus was adjusted to 1:2 during the preparation of the nanozyme, while the rest remained the same as in Example 1, resulting in Astragalus membranaceus-Angelica sinensis composite carbon dot nanozyme, hereinafter referred to as AM-AS@CDs (1:2).

[0062] Example 3: AM-AS@CDs (1:5) composite carbon dot nanozymes prepared based on Astragalus membranaceus and Angelica sinensis The only difference between this embodiment and Example 1 is that the mass ratio of Angelica sinensis to Astragalus membranaceus was adjusted to 1:5 during the preparation of the nanozyme, while the rest remained the same as in Example 1, resulting in Astragalus membranaceus-Angelica sinensis composite carbon dot nanozyme, hereinafter referred to as AM-AS@CDs (1:5).

[0063] Example 4: AM-AS@CDs (2:1) composite carbon dot nanozyme prepared based on Astragalus membranaceus and Angelica sinensis The only difference between this embodiment and Example 1 is that the mass ratio of Angelica sinensis to Astragalus membranaceus was adjusted to 2:1 during the preparation of the nanozyme, while the rest remained the same as in Example 1, resulting in Astragalus membranaceus-Angelica sinensis composite carbon dot nanozyme, hereinafter referred to as AM-AS@CDs (2:1).

[0064] Example 5: AM-AS@CDs (5:1) composite carbon dot nanozyme prepared based on Astragalus membranaceus and Angelica sinensis The only difference between this embodiment and Example 1 is that the mass ratio of Angelica sinensis to Astragalus membranaceus was adjusted to 5:1 during the preparation of the nanozyme, while the rest remained the same as in Example 1, resulting in Astragalus membranaceus-Angelica sinensis composite carbon dot nanozyme, hereinafter referred to as AM-AS@CDs (5:1).

[0065] Comparative Example 1: Preparation of Astragalus-based single-carbon nanoparticle nanozymes (AM@CDs) The only difference between this comparative example and Example 1 is that Angelica sinensis was replaced with an equal amount of Astragalus membranaceus in the preparation of the nanozyme. The rest is the same as in Example 1, resulting in a single carbon dot nanozyme based on Astragalus membranaceus, hereinafter referred to as AM@CDs.

[0066] Comparative Example 2: Preparation of single-carbon nanozymes (AS@CDs) based on Angelica sinensis The only difference between this comparative example and Example 1 is that Astragalus membranaceus was replaced with an equal amount of Angelica sinensis in the preparation of the nanozyme. The rest is the same as in Example 1, resulting in a single carbon dot nanozyme based on Angelica sinensis, hereinafter referred to as AS@CDs.

[0067] Experimental Example 1: Structural Characterization of Nanozyme Materials The nanozyme materials AM-AS@CDs, AM@CDs, and AS@CDs prepared in Example 1 and Comparative Examples 1-2 were characterized by the following structures: (1) The morphology of AM-AS@CDs was observed by transmission electron microscopy (TEM), and the particle size distribution was statistically analyzed; (2) X-ray diffraction (XRD) analysis of crystal structure; (3) Raman spectroscopy is used to assess the degree of defect; (4) X-ray photoelectron spectroscopy (XPS) analysis of elemental composition and chemical states; (5) Recording optical properties using ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy; (6) Zeta potentiometer measures surface charge.

[0068] See results Figure 2 and 3 .

[0069] like Figure 2 and 3As shown, the AM-AS@CDs synthesized in Example 1 of this invention have uniform particle size (average 4.2 nm), graphitized structure, and high defect density. Their surfaces are rich in CN, C=O, and other functional groups. The nitrogen content is 7.55 at.%, the oxygen content is 26.04 at.%, and the zeta potential is -25.3 mV. Furthermore, the AM@CDs and AS@CDs synthesized in Comparative Examples 1 and 2 of this invention also have uniform particle size (average 4.4 and 3.4 nm, respectively), graphitized structure, and low defect density. Their surfaces are rich in CN, C=O, and other functional groups. The nitrogen content is 2.82 at.% and 1.52 at.%, respectively, the oxygen content is 14.65 at.% and 16.18 at.%, respectively, and the zeta potentials are -24.9 mV and -20.2 mV, respectively.

[0070] Experimental Example 2: Determination of SOD-like Activity of Nanozyme Materials The SOD-like activity of the nanozyme materials AM-AS@CDs, AM@CDs, and AS@CDs prepared in Example 1 and Comparative Examples 1-2 was determined using an SOD detection kit (WST-method, WST-1 detection kit, purchased from Tongren Chemical). The specific steps included: Add 20 μL of the test sample (1 mg / mL), 200 μL of WST-1 working solution, and 20 μL of enzyme working solution to a 96-well plate, incubate at 37°C for 20 min, and then measure the absorbance at 450 nm. Follow the kit instructions, and set up blank and positive controls. Results are shown below. Figure 4 .

[0071] like Figure 4 As shown, the AM-AS@CDs prepared in Example 1 of this invention exhibit antioxidant activity (O2 scavenging) ·- ABTS +· and DPPH · The antioxidant capacity of the composite carbon dots prepared in Examples 1-5 was superior to that of the single-drug carbon dots in Comparative Examples 1 and 2. In addition, the antioxidant capacity of the composite carbon dots prepared in different ratios in Examples 1-5 was tested. The results showed that the AM-AS@CDs (Example 1) prepared with the mass ratio of Astragalus membranaceus to Angelica sinensis was the best.

[0072] Experimental Example 3: Evaluation of POD / OXD-like Activity of Nanozymes This embodiment uses the TMB colorimetric method to evaluate the POD / OXD-like activity of the nanozyme materials AM-AS@CDs, AM@CDs, and AS@CDs prepared in Example 1 and Comparative Examples 1-2, respectively. The specific steps include: (1) OXD activity: 30 μL of the test sample (1 mg / mL) and 50 μL of TMB (10 mM) were added to a 1 mL reaction system, and the mixture was replenished with PBS buffer at pH 7.4. The mixture was incubated at 25 °C for 20 min, and the absorbance was measured at 652 nm to evaluate its OXD activity.

[0073] (2) An additional H2O2 (10 mM) group was set up to evaluate POD activity: 30 μL of the test sample (1 mg / mL), 50 μL of TMB (10 mM), and 100 μL of H2O2 (10 mM) were added to a 1 mL reaction system, and the mixture was made up with PBS buffer at pH 7.4. The mixture was incubated at 25 °C for 20 min, and then the absorbance was measured at 652 nm to evaluate the POD activity. The detection results are shown below. Figure 5 .

[0074] like Figure 5 As shown, the three nanozymes AM-AS@CDs, AM@CDs, and AS@CDs did not show a significant increase in absorbance in the absence of H2O2, indicating no OXD activity; the absorbance also did not change significantly after the addition of H2O2, indicating that none of the three nanozymes had POD activity. These results confirm that the AM-AS@CDs prepared in this invention will not catalyze the generation of additional ROS under physiological conditions.

[0075] Experimental Example 4: Cytotoxicity Assay of Nanozymes NRK-52E and HK-2 cells (derived from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were seeded in 96-well plates and cultured for 24 h. Then, different concentrations (0–100 μg / mL) of the nanozymes AM-AS@CDs, AM@CDs, and AS@CDs prepared in Examples 1–5 and Comparative Examples 1–2 were added to the cells, followed by further culturing for 24 h. Cell viability was then assessed using the CCK-8 assay. Results are shown below. Figure 6 .

[0076] like Figure 6 As shown, the cell viability of each concentration group was >90%, indicating that the nanozymes all have good cell compatibility.

[0077] Experimental Example 5: Intracellular ROS Scavenging and Anti-apoptosis Experiments of Nanozymes 1. Detection of intracellular ROS scavenging efficiency of nanozymes NRK-52E cells were seeded in 12-well plates and divided into several groups: a blank control group (corresponding to Control(-) in the figure), a model group (containing 200 μM H2O2, corresponding to Control(+) in the figure), an AM-AS@CDs(+) group (pre-incubated with 30 μg / mL AM-AS@CDs prepared in Example 1 for 2 h, followed by 200 μM H2O2), an AM-AS@CDs(-) group (pre-incubated with 30 μg / mL AM-AS@CDs prepared in Example 1 for 2 h), an AM@CDs(+) group (pre-incubated with 30 μg / mL AM@CDs prepared in Comparative Example 1 for 2 h, followed by 200 μM H2O2), an AM@CDs(-) group (pre-incubated with 30 μg / mL AM@CDs prepared in Comparative Example 1 for 2 h), and an AS@CDs(+) group (pre-incubated with 30 μg / mL AS@CDs prepared in Comparative Example 2 for 2 h, followed by 200 μM H2O2). The groups were divided into three groups: μM group and AS@CDs(-) group (pre-incubated with 30 μg / mL AS@CDs prepared in Comparative Example 2 for 2 h). After H2O2 treatment for 1 h, each group was incubated with DCFH-DA probe (10 μM) for 20 min, washed with PBS, observed and photographed under a fluorescence microscope, and quantitatively analyzed by flow cytometry.

[0078] 2. Detection of anti-apoptotic properties of nanozymes NRK-52E cells were seeded in 12-well plates and divided into several groups: a blank control group (corresponding to Control(-) in the figure), a model group (containing 200 μM H2O2, corresponding to Control(+) in the figure), an AM-AS@CDs(+) group (pre-incubated with 30 μg / mL AM-AS@CDs prepared in Example 1 for 2 h, followed by 200 μM H2O2), an AM-AS@CDs(-) group (pre-incubated with 30 μg / mL AM-AS@CDs prepared in Example 1 for 2 h), an AM@CDs(+) group (pre-incubated with 30 μg / mL AM@CDs prepared in Example 2 for 2 h, followed by 200 μM H2O2), an AM@CDs(-) group (pre-incubated with 30 μg / mL AM@CDs prepared in Comparative Example 1 for 2 h), and an AS@CDs(+) group (pre-incubated with 30 μg / mL AS@CDs prepared in Example 3 for 2 h, followed by 200 μM H2O2). The cells were divided into two groups: μM group and AS@CDs(-) group (pre-incubated with 30 μg / mL AS@CDs prepared in Comparative Example 2 for 2 h). After 24 h of H2O2 treatment, cells were collected, stained with Annexin V-Alexa Fluor 647 / PI kit, and the apoptosis rate was detected by flow cytometry. The results are shown in the figure. Figure 7 .

[0079] like Figure 7As shown, the ROS scavenging efficacy test results indicated that AM-AS@CDs significantly reduced the H2O2-induced ROS fluorescence intensity (p < 0.001), with better effects than single-drug carbon dots (AM@CDs and AS@CDs). The anti-apoptotic assay results showed that the total apoptosis rate in the model group was 32.1±3.5%, which decreased to 12.3±2.1% in the AM-AS@CDs group (p < 0.001). The AM@CDs and AS@CDs groups had apoptosis rates of 24.5±2.8% and 23.1±2.6%, respectively, demonstrating that the composite carbon dots possess stronger anti-apoptotic capabilities.

[0080] Experimental Example 6: Therapeutic Effect of Nanozymes on Ischemia-Reperfusion Kidney Injury 1. Experimental Methods 1.1 Animal model construction and dosing regimen 1.1.1 Animal Model Construction After at least 7 days of acclimatization, 8-week-old male C57BL / 6J mice (purchased from Huachuang Xinno Pharmaceutical Technology Co., Ltd., Animal License No. SYXK2021(Su)-0007) were anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution and placed on a 37°C heated surgical board. A 1.5–2 cm incision was made along the midline of the abdomen using surgical scissors, and the bilateral renal vessels were carefully dissected under a microscope. The bilateral renal pedicles were clamped with non-traumatic microvascular clamps for 25 minutes; the renal pedicles changed from bright red to dark purple, indicating successful ischemia. After the clamps were removed, the kidneys returned to bright red, confirming reperfusion. After confirming no bleeding or organ damage, the abdomen was closed, the kidneys were gently repositioned, and the incision was sutured in layers, thus obtaining the I / R injury model mouse.

[0081] Sham surgery group: The difference between this group of mice and the I / R injury model is that the mice only exposed the renal blood vessels without clamping them.

[0082] 1.1.2 Dosing regimen Male C57BL / 6J mice (8 weeks old) were randomly divided into 5 groups: sham operation group, model group (I / R), I / R+AM@CDs group, I / R+AS@CDs group, and I / R+AM-AS@CDs group, with 8 mice in each group. The mice in each group were then treated as follows: I / R+AM-AS@CDs group: One day before surgery, mice were injected intraperitoneally with AM-AS@CDs (5 mg / kg, dissolved in 100 μL PBS) prepared in Example 1. Then, an I / R injury model was constructed according to the method described in 1.1.1 above. On the day of surgery and one day after surgery, mice were injected intraperitoneally again with AM-AS@CDs (5 mg / kg, dissolved in 100 μL PBS) prepared in Example 1. I / R+AM@CDs group: The only difference between this group and the I / R+AM-AS@CDs group is that the intraperitoneal injection drug is AM@CDs prepared in Comparative Example 1, and the rest are the same. I / R+AS@CDs group: The only difference between this group and the I / R+AM-AS@CDs group is that the intraperitoneal injection drug is AS@CDs prepared in comparative example 2, and the rest are the same. I / R group: The only difference between this group and the I / R+AM-AS@CDs group is that the intraperitoneal injection drug is PBS, otherwise they are the same. Sham group: The only difference between this group and the I / R group is that the mice only exposed the renal blood vessels without clamping them.

[0083] Serum and kidney tissue were collected from each group of mice 48 hours after surgery for testing.

[0084] 2. Detection and Result Analysis 2.1 Renal function and histological assessment Serum creatinine (CRE) (catalog number: ADS-W-FM034, purchased from Jiangsu Aidisheng Biotechnology Co., Ltd.) and blood urea nitrogen (BUN) (catalog number: C013-2-1, purchased from Nanjing Jiancheng Bioengineering Institute) were measured using a kit. Total RNA was extracted from kidney tissue, and the mRNA levels of Lcn2 and Kim-1 were detected by qRT-PCR. Paraffin sections of kidney tissue were stained with H&E, and the degree of renal tubular damage was assessed according to the Paller score. Results are shown below. Figure 8 .

[0085] like Figure 8 As shown, compared with the model group, mice treated with AM-AS@CDs (5 mg / kg, intraperitoneal injection, once each on the day before, day of, and day after reperfusion) had serum creatinine (CRE) and blood urea nitrogen (BUN) levels reduced by 42%–55% and 38%–50%, respectively (p<0.001), and the mRNA expression of kidney injury molecules Lcn2 and Kim-1 was downregulated by 45%–65% (p < 0.01). H&E staining of kidney tissue showed that the model group exhibited widespread renal tubular dilation, vacuolar degeneration, and brush border detachment, with a Paller score of 5.2 ± 0.4; the AM-AS@CDs treatment group showed significantly reduced pathological changes, with a Paller score decreasing to 2.1 ± 0.3 (p < 0.001).

[0086] 2.2 Analysis of Immune Cell Infiltration Single-cell suspensions were collected from peripheral blood and kidney tissue, stained with fluorescent antibodies (CD45, CD11b, Ly6G, F4 / 80, CD3, CD4, CD8), and analyzed by flow cytometry to determine the proportions of neutrophils, macrophages, and T cells. Results are shown below. Figure 9 a-9d.

[0087] like Figure 9 As shown in a-9d, flow cytometry analysis revealed that AM-AS@CDs treatment significantly reduced peripheral blood neutrophils (Ly6G). + CD11b + ) and total T cells (CD3) + The proportion of neutrophils (Ly6G) decreased by 38% and 30%, respectively, p < 0.01; the number of neutrophils infiltrating the kidney tissue (Ly6G) decreased. + CD11b + ) and macrophages (F4 / 80) + CD11b + The number of [items] decreased by 45% and 50% respectively (p < 0.001).

[0088] 2.3 Expression of inflammatory factors and chemokines The mRNA levels of Ccl2, Ccl7, Ccl20, Cxcl1, TNF-α, and IL-1β in renal tissue were detected by qRT-PCR. Western blot was used to detect the protein expression of NF-κB p65, c-Jun, pc-Jun, Fosl1, and p-Fosl1; the results for Ccl2, Ccl7, Ccl20, and Cxcl1 are shown below. Figure 9 The detection results for e-9h; c-Jun, pc-Jun, Fosl1, and p-Fosl1 are shown in [link to results]. Figure 11 The detection results of e-11f; NF-κB p65, TNF-α, and IL-1β are shown in the figure. Figure 12 .

[0089] The results showed that transcriptome sequencing (RNA-seq) and real-time quantitative PCR confirmed that AM-AS@CDs downregulated the mRNA expression of chemokines Ccl2, Ccl7, Ccl20, and Cxcl1 by 40%–70% (p < 0.01). Western blot analysis showed that AM-AS@CDs inhibited the phosphorylation levels of transcription factors c-Jun and Fosl1 (decreasing by 50%–60%, p < 0.001), blocked AP-1 signaling pathway activation, and consequently reduced the production of downstream inflammatory factors TNF-α and IL-1β. These results indicate that AM-AS@CDs significantly inhibited the transcription of the aforementioned pro-inflammatory factors and chemokines, downregulated the phosphorylation level of c-Jun / Fosl1, and blocked the AP-1 signaling pathway.

[0090] 2.4 Transcriptome Sequencing Analysis Total RNA was extracted from kidney tissues of three groups (Sham, I / R, and I / R+AM-AS@CDs) and sequenced using Illumina. Differential gene screening (|log2FC|≥1, p<0.05) and GO, KEGG, and GSEA analyses were performed. The mRNA levels of SOD1, SOD2, and SOD3 in kidney tissues were detected using qRT-PCR. Nrf2 and Hmox1 protein expression were detected by Western blot. The results of differential gene screening, GO, and KEGG enrichment analyses are shown below. Figure 11 Results of GSEA, qPCR, and Western blot assays are shown in [link to results]. Figure 10 .

[0091] like Figure 10 and Figure 11 The results showed that AM-AS@CDs reversed the changes in the expression of inflammation and oxidative stress-related genes induced by I / R, and gene enrichment analysis confirmed that they significantly inhibited the AP-1 and chemokine signaling pathways.

[0092] 2.5 In vivo distribution and biocompatibility Eight-week-old male C57BL / 6J mice were intraperitoneally injected with AM-AS@CDs (5 mg / kg, once daily). Mice were sacrificed at different time points (0.5, 1, 2, 4, 8, and 24 h), and major organs were harvested for fluorescence intensity detection using a small animal in vivo imaging system. After 7 consecutive days of administration, serum ALT, AST, CRE, and BUN levels were measured, and major organs were harvested for H&E staining. Results are shown below. Figure 8 and Figure 13 .

[0093] like Figure 8 As shown in h-8j, AM-AS@CDs, utilizing their autofluorescence, accumulated in the kidneys 2–4 h after intraperitoneal injection, with fluorescence intensity 2.1 times that of the liver and 4.5 times that of the spleen; the signal in the kidney on the I / R injury side was 1.8 times higher than that on the healthy side, indicating its ability to target and accumulate in injury-induced pathways. Figure 13 As shown, administration for 7 consecutive days (5 mg / kg / d) did not cause any abnormalities in body weight, liver and kidney function indicators (ALT, AST, CRE, BUN), or histological abnormalities in major organs (heart, liver, spleen, lung, and kidney) in mice, demonstrating its good in vivo safety.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A composite carbon dot nanoenzyme based on Astragalus membranaceus and Angelica sinensis, characterized in that, The nanozyme was synthesized via a hydrothermal method using Astragalus membranaceus and Angelica sinensis as composite precursors. The nanozyme has a particle size of 3-5 nm; The surface of the nanozyme is rich in nitrogen and oxygen functional groups, wherein the nitrogen content is 6.0–8.5 at.% and the oxygen content is 24.0–28.0 at.%. The nanozyme exhibits superoxide dismutase-like activity.

2. The preparation method of the Astragalus-Angelica composite carbon dot nanozyme according to claim 1, characterized in that, The method includes the following steps: S1: Add the mixed powder containing astragalus powder and angelica powder to water and disperse it by ultrasonication to obtain a suspension; S2: The resulting suspension is subjected to a hydrothermal reaction. After the reaction is complete, the suspension is centrifuged and the supernatant is collected. S3: The obtained supernatant was filtered through a membrane, ultrafiltered, dialyzed, and dried sequentially to obtain a composite carbon dot nanozyme.

3. The preparation method according to claim 2, characterized in that, In step S1, the mass ratio of Astragalus powder to Angelica powder is (0~10):(0~10).

4. The preparation method according to claim 2, characterized in that, In step S1, the amount of water added is 5 to 20 times the mass of the mixed powder.

5. The preparation method according to claim 2, characterized in that, In step S1, the ultrasonic dispersion time is 10~60 min.

6. The preparation method according to claim 2, characterized in that, In step S2, the hydrothermal reaction is carried out in a hydrothermal reactor lined with polytetrafluoroethylene, and the hydrothermal reaction conditions are: temperature of 150~200℃ and time of 4~10 h.

7. The preparation method according to claim 2, characterized in that, In step S2, the centrifugation conditions are: rotation speed of 8000~10000 rpm and time of 10~15 min.

8. The preparation method according to claim 2, characterized in that, In step S3, the filter membrane size is 0.22 µm; The ultrafiltration has a molecular weight cutoff of 3 kDa; The molecular weight cutoff for the dialysis is 3500 Da, and the dialysis time is 2-4 days. The drying process is freeze drying, and the freeze drying time is 24~48 hours.

9. The application of the Astragalus-Angelica composite carbon dot nanozyme according to claim 1, or the Astragalus-Angelica composite carbon dot nanozyme prepared by the preparation method according to any one of claims 2 to 8, in the preparation of products targeting the kidneys for the treatment of acute kidney injury.

10. The application according to claim 9, characterized in that, The acute kidney injury mentioned refers to acute kidney injury caused by ischemia-reperfusion.

11. A pharmaceutical composition for treating acute kidney injury, characterized in that, Include: (i) the Astragalus-Angelica composite carbon dot nanozyme according to claim 1, or the Astragalus-Angelica composite carbon dot nanozyme prepared by the preparation method according to any one of claims 2 to 8; and (ii) Pharmaceutically acceptable carriers or excipients.

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