Ruthenium oxide nanoparticle composite for treating acute kidney injury, preparation method and application thereof

Abstract

The present application relates to the technical field of biological pharmacy, and particularly relates to a ruthenium oxide nanoparticle composite material for treating acute kidney injury, a preparation method and application, the ruthenium oxide nanoparticle composite material is coated by bovine serum albumin, and the surface is negatively charged.The ruthenium oxide nanoparticle composite material has hydrogen peroxidase and superoxide dismutase simulation activity, can effectively remove active oxygen, inhibit inflammatory reaction and reduce renal tubular cell apoptosis.The preparation method comprises the following steps: mixing RuCl3.3H2O solution and negatively charged BSA, then adding NaBH4 drop by drop for reduction, and obtaining the product through dialysis purification.Experiments show that the nanoparticle can significantly improve glomerular fluorescence intensity, reduce pericardial and ocular edema and other pathological phenotypes in a aristolochic acid-induced zebrafish acute kidney injury model, has good biocompatibility and treatment effect, and can be used for preparing a drug for treating acute kidney injury.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a ruthenium oxide nanoparticle composite material for treating acute kidney injury, its preparation method, and its application. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function, primarily manifested as decreased glomerular filtration rate, accumulation of nitrogenous waste, electrolyte disturbances, and systemic edema. The etiologies of AKI are complex and diverse, including ischemia-reperfusion injury, sepsis, and exposure to nephrotoxic drugs. Among these, drug-induced kidney injury is a common and preventable type. Aristolochic acids (AA), as typical nephrotoxic natural compounds, can induce characteristic lesions centered on damage to proximal renal tubular epithelial cells. Their toxic mechanisms involve multiple pathological processes, including DNA damage, oxidative stress, activated inflammatory responses, apoptosis, and interstitial fibrosis, making them ideal pathogenic factors for studying drug-induced AKI.

[0003] Currently, clinical treatment strategies for AA-induced acute kidney injury (AKI) mainly include supportive care, discontinuation of causative drugs, and symptomatic treatment, lacking specific interventions. Existing drug treatment research focuses on antioxidants (such as N-acetylcysteine), anti-inflammatory drugs (such as glucocorticoids), and cell protectants. However, these strategies mostly focus on single-target interventions (such as simple antioxidant or simple anti-inflammatory treatment), which are difficult to effectively block the complex pathological network mediated by multiple mechanisms such as oxidative stress, inflammatory response, and apoptosis in AA, resulting in limited treatment efficacy.

[0004] Therefore, there is an urgent need to develop a new drug that can treat acute kidney injury. Summary of the Invention

[0005] To address the above problems, this invention provides a ruthenium oxide nanoparticle composite material for treating acute kidney injury, its preparation method, and its application.

[0006] This invention is achieved through the following technical solution: A ruthenium oxide nanoparticle composite material is prepared by mixing a RuCl3·3H2O solution with a negatively charged bovine serum albumin solution; the mass ratio of RuCl3·3H2O to negatively charged bovine serum albumin is 1~5:1~9.

[0007] The ruthenium oxide nanoparticles are coated with bovine serum albumin and have a negatively charged surface; they possess catalase and superoxide dismutase-mimicking activities.

[0008] The preparation method of the ruthenium oxide nanoparticle composite material specifically includes the following steps: A RuCl3·3H2O solution was mixed with negatively charged bovine serum albumin to obtain a mixture; the volume ratio of the RuCl3·3H2O solution to the negatively charged bovine serum albumin was 1:1~3; the concentration of the RuCl3·3H2O solution was 1mg / mL~5mg / mL; and the concentration of the negatively charged bovine serum albumin was 1mg / mL~3mg / mL.

[0009] Add 1 mL to 5 mL of NaBH4 to the mixture to carry out the reaction, and obtain the reaction solution.

[0010] The entire reaction solution was dialyzed to obtain a ruthenium oxide nanoparticle composite material.

[0011] Preferably, the concentration of NaBH4 is 0.01 mol·L⁻¹. -1 ~0.1mol·L -1 .

[0012] Preferably, the molecular weight cutoff for dialysis is 3.5 kDa to 10 kDa.

[0013] Preferably, the dialysis time is 48h to 72h.

[0014] The application of the ruthenium oxide nanoparticle composite material in the preparation of a drug for treating aristolochic acid-induced acute kidney injury.

[0015] Preferably, the drug uses the ruthenium oxide nanoparticle composite material as the sole active ingredient.

[0016] Preferably, the concentration of the ruthenium oxide nanoparticle composite material is 25 μg / mL to 100 μg / mL.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a ruthenium oxide nanoparticle composite material for treating acute kidney injury (AKI), the surface of which is coated with bovine serum albumin (BSA) and exhibits a negative charge. The particles are synthesized via a biotemplate method, and the resulting BSA-RuO2 NPs possess both catalase (CAT) and superoxide dismutase (SOD) mimicry activities. Due to the presence of CAT-mimicry activity, this nanomaterial can simultaneously generate a large amount of oxygen during the scavenging of reactive oxygen species (ROS), thereby synergistically regulating the inflammatory response and achieving efficient ROS scavenging and anti-inflammatory effects. Furthermore, an aristolochic acid (AA)-induced AKI model was constructed using zebrafish, and the therapeutic effect of BSA-RuO2 NPs was evaluated using real-time fluorescence imaging and phenotypic analysis. The results show that the ruthenium oxide nanoparticle composite material prepared in this invention can significantly repair AKI, demonstrating good therapeutic potential. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The images show glomerular fluorescence after treatment with different concentrations of AA according to the present invention.

[0020] Figure 2 Fluorescence micrograph of BSA-RuO2NPs nanoparticles used to rescue AA-induced kidney injury.

[0021] Figure 3 Whole-body bright-field photograph of BSA-RuO2NPs nanoparticles used to rescue AA-induced kidney injury. Detailed Implementation

[0022] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0024] The inventive concept of this invention is as follows: The core concept of this invention lies in designing and constructing a multifunctional nanoenzyme material, namely, a ruthenium oxide nanoparticle composite material, for targeted treatment of aristolochic acid-induced acute kidney injury. First, ruthenium oxide nanoparticles (BSA-RuO2NPs) coated with negatively charged bovine serum albumin (BSA) are synthesized using a biotemplate method, giving them both catalase (CAT) and superoxide dismutase (SOD) mimicry enzyme activities. Due to its CAT-mimicking activity, this nanomaterial can generate a large amount of oxygen during the scavenging of reactive oxygen species (ROS), thereby more effectively regulating the inflammatory response and achieving efficient ROS scavenging and anti-inflammatory effects.

[0025] Aristolochic acids (AA) are a classic pathogenic factor that induces kidney injury. Its toxic mechanism involves multiple pathological links: (1) DNA adduct formation: After AA is bioactivated in the body, it generates aristolochic acid-DNA adduct, which leads to multi-target gene mutations (such as TP53) and triggers cell damage and apoptosis. This is the core pathogenic mechanism of AAN; (2) Oxidative stress and mitochondrial damage: AA significantly increases intracellular ROS production, causing lipid peroxidation, loss of mitochondrial membrane potential and energy metabolism disorders; (3) Activation of inflammatory response: AA can upregulate inflammatory factors such as TNF-α, IL-1β, and IL-6, activate the NF-κB signaling pathway, and induce immune cell infiltration; (4) Epithelial cell apoptosis / necrosis and renal tubular dysfunction; (5) Interstitial fibrosis formation: AA can continuously activate the TGF-β / Smad pathway, promote epithelial-interstitial transition (EMT) and excessive deposition of extracellular matrix, causing progressive renal interstitial fibrosis. AA-induced kidney injury models have well-defined targets, high reproducibility, and stable models, and are widely used to study the pathogenesis of acute kidney injury (AKI) and for drug screening. Based on the compatibility between the multiple pathogenic mechanisms of AA and the multiple enzyme activities of this nanoparticle, this invention selects AA-induced acute kidney injury as the therapeutic research subject.

[0026] To directly evaluate the therapeutic effect of ruthenium oxide nanoparticle composite materials, zebrafish were selected as a model animal in this invention. Zebrafish have advantages such as high egg production, rapid reproductive cycle, transparent embryos, synchronous embryonic development, and rapid development speed. Combined with glomerular-specific marker fluorescent transgenic lines... Tg ( Pod:mcherry This allows for real-time observation of the degree of kidney damage and the repair process. AA-induced kidney injury in a zebrafish model manifests as decreased glomerular fluorescence intensity and systemic phenotypes such as pericardial edema, ocular edema, and cerebral edema. These indicators can be used to accurately quantify the degree of kidney damage and treatment efficacy.

[0027] Therefore, this invention utilizes zebrafish to construct an AA-induced AKI model, and precisely quantifies the degree of kidney injury through real-time fluorescence imaging and phenotypic analysis. Finally, the therapeutic effect of BSA-RuO2NPs is systematically evaluated in this in vivo model. By analyzing key indicators such as glomerular fluorescence recovery and edema reduction, its role in repairing damage by scavenging ROS, reducing inflammation, and protecting renal cells is verified, thus providing a new material and strategy for nanozyme therapy of acute kidney injury.

[0028] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0029] The pharmaceuticals or reagents used in this invention are all commercially available products.

[0030] Example 1 The synthesis of ruthenium oxide nanoparticle composite materials using bovine serum albumin (BSA) modification of RuO2 is described in the following specific method: Add RuCl3·3H2O solution and negatively charged bovine serum albumin (BSA) to a 50 mL beaker to obtain a mixture; wherein the volume ratio of RuCl3·3H2O solution to negatively charged bovine serum albumin is 1:1; the concentration of RuCl3·3H2O solution is 1 mg / mL; the concentration of negatively charged bovine serum albumin is 1 mg / mL.

[0031] 1 mL of 0.01 mol·L⁻¹ -1 Fresh NaBH4 was added dropwise to the mixture for reaction, and the mixture was stirred until the solution turned into a dark green reaction solution. The entire reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed in double-distilled water for 48 h to obtain ruthenium oxide nanoparticle composite material, denoted as BSA-RuO2NPs.

[0032] Subsequent experiments verified the resuscitation effect of different concentrations of BSA-RUO2NPs solution on kidney injury.

[0033] Example 2 The synthesis of ruthenium oxide nanoparticle composite materials using bovine serum albumin (BSA) modification of RuO2 is described in the following specific method: Add RuCl3·3H2O solution and negatively charged bovine serum albumin (BSA) to a 50 mL beaker to obtain a mixture; wherein the volume ratio of RuCl3·3H2O solution to negatively charged bovine serum albumin is 1:2; the concentration of RuCl3·3H2O solution is 3 mg / mL; and the concentration of negatively charged bovine serum albumin is 2 mg / mL.

[0034] 2 mL of 0.05 mol·L⁻¹ -1Fresh NaBH4 was added dropwise to the mixture for reaction, and the mixture was stirred until the solution turned into a dark green reaction solution. All the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 7 kDa and dialyzed in double-distilled water for 56 h to obtain ruthenium oxide nanoparticle composite material, denoted as BSA-RuO2NPs.

[0035] Subsequent experiments verified the resuscitation effect of different concentrations of BSA-RUO2NPs solution on kidney injury.

[0036] Example 3 The synthesis of ruthenium oxide nanoparticle composite materials using bovine serum albumin (BSA) modification of RuO2 is described in the following specific method: Add RuCl3·3H2O solution and negatively charged bovine serum albumin (BSA) to a 50 mL beaker to obtain a mixture; wherein the volume ratio of RuCl3·3H2O solution to negatively charged bovine serum albumin is 1:3; the concentration of RuCl3·3H2O solution is 5 mg / mL; and the concentration of negatively charged bovine serum albumin is 3 mg / mL.

[0037] 5 mL of 0.1 mol·L⁻¹ -1 Fresh NaBH4 was added dropwise to the mixture for reaction, and the mixture was stirred until the solution turned into a dark green reaction solution. All the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed in double-distilled water for 72 h to obtain ruthenium oxide nanoparticle composite material, denoted as BSA-RuO2NPs.

[0038] Subsequent experiments verified the resuscitation effect of different concentrations of BSA-RUO2NPs solution on kidney injury.

[0039] Experimental Example 1 1. Construction of an AA-induced drug-induced acute kidney injury (AKI) model in zebrafish Retrieved 3 days post-fertilization (3dpf), with normal development and glomerular cells specifically labeled with red fluorescence. Tg ( Pod: mcherry Zebrafish juveniles were immersed in AA solutions at concentrations of 1.6 mg / L and 1.8 mg / L for 72 hours. Changes in glomerular fluorescence intensity were observed using fluorescence microscopy. The results are as follows: Figure 1 As shown, the glomerular fluorescence intensity of zebrafish juveniles significantly decreased after soaking in 1.6 mg / L and 1.8 mg / L solutions, indicating that the zebrafish acute kidney injury (AKI) model was successfully established and can be used for subsequent rescue experiments.

[0040] 2. Comparison of the therapeutic effects of BSA-RUO2NPs on acute kidney injury Based on the successful establishment of a zebrafish acute kidney injury model, this invention further evaluated the therapeutic effect of BSA-RUO2NPs.

[0041] Zebrafish juveniles were treated with aristolochic acid (AA) solutions of the aforementioned concentrations (1.6 mg / L and 1.8 mg / L) and 100 μg / mL BSA-RUO2NPs for 72 hours. A normal control group, an AA-damaged model group, and a control group treated solely with BSA-RUO2NPs were also included. After treatment, under the same imaging parameters, fluorescence microscopy of the kidney region and whole-body bright-field photography were performed on each group of zebrafish juveniles. Figure 2 and Figure 3 As shown in the image. By comparing the differences in renal injury and edema phenotypes among the groups, the ameliorative effect of BSA-RUO2NPs on acute kidney injury was evaluated, and representative images were used to demonstrate the treatment effect. Specific methods and results are as follows:

[0042] First, zebrafish juveniles were immersed in aristolochic acid (AA) solutions at concentrations of 1.6 mg / L and 1.8 mg / L for 72 hours to induce stable kidney damage. These concentrations consistently induced significant edema phenotypes, including in the pericardial cavity and periorbital region.

[0043] To clearly evaluate the rescue effect of BSA-RUO2NPs, the following experimental groups were established: Normal control group (PTU fish fluid); 1.6 mg / L AA model group (1.6 mg / L AA); 1.8 mg / L AA model group (1.8 mg / L AA); Treatment group: 1.6 mg / L AA + 100 μg / mL BSA-RUO2NPs; Treatment group: 1.8 mg / L AA + 100 μg / mL BSA-RUO2NPs; BSA-RUO2NPs as a standalone control group (100 μg / mL).

[0044] After treatment, the juvenile fish were fixed in a lateral position, and high-resolution images of the eye area were systematically acquired under uniform high-magnification bright-field microscope parameters. The focus was on observing and quantifying the translucent gap between the eyeball and surrounding tissues caused by edema.

[0045] The results are as follows Figure 3As shown, compared with the AA model group, BSA-RUO2NPs treatment significantly reduced ocular swelling and effectively reversed abnormal fluid accumulation around the eyeball. Furthermore, at this concentration, treatment with nanoparticles alone did not cause any toxic phenotypes, indicating that BSA-RUO2NPs have significant therapeutic effects on acute kidney injury and good biocompatibility.

[0046] It should be noted that the present invention used BSA-RUO2NPs at concentrations of 25 μg / mL and 50 μg / mL to conduct the same rescue experiment as in Experimental Example 1. The results showed that BSA-RUO2NPs at concentrations of 25 μg / mL and 50 μg / mL could also significantly reduce ocular swelling, effectively reverse abnormal fluid accumulation around the eyeball, and did not cause any toxic phenotypes.

[0047] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A ruthenium oxide nanoparticle composite material for treating acute kidney injury, characterized in that, The ruthenium oxide nanoparticle composite material is prepared by mixing RuCl3·3H2O solution with negatively charged bovine serum albumin solution; the mass ratio of RuCl3·3H2O to negatively charged bovine serum albumin is 1~5:1~9; The ruthenium oxide nanoparticles are coated with bovine serum albumin and have a negative surface charge.

2. The method for preparing the ruthenium oxide nanoparticle composite material according to claim 1, characterized in that, Specifically, the following steps are included: A RuCl3·3H2O solution was mixed with negatively charged bovine serum albumin to obtain a mixture; the volume ratio of the RuCl3·3H2O solution to the negatively charged bovine serum albumin was 1:1~3; the concentration of the RuCl3·3H2O solution was 1 mg / mL~5 mg / mL; and the concentration of the negatively charged bovine serum albumin was 1 mg / mL~3 mg / mL. Add 1 mL to 5 mL of NaBH4 to the mixture to carry out the reaction, and obtain the reaction solution; The entire reaction solution was dialyzed to obtain a ruthenium oxide nanoparticle composite material.

3. The preparation method according to claim 2, characterized in that, The concentration of NaBH4 is 0.01 mol·L⁻¹. -1 ~0.1mol·L -1 .

4. The preparation method according to claim 2, characterized in that, The molecular weight cutoff for the dialysis is 3.5 kDa to 10 kDa.

5. The preparation method according to claim 2, characterized in that, The dialysis time is 48h~72h.

6. The use of the ruthenium oxide nanoparticle composite material according to claim 1 in the preparation of a medicament for treating aristolochic acid-induced acute kidney injury.

7. The application according to claim 6, characterized in that, The drug uses the aforementioned ruthenium oxide nanoparticle composite material as its sole active ingredient.

8. The application according to claim 7, characterized in that, The concentration of the ruthenium oxide nanoparticle composite material is 25 μg / mL to 100 μg / mL.

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