A hydrogel composite for repairing acute kidney injury, preparation and application thereof

Abstract

The application discloses a kind of hydrogel composite material for acute kidney injury repair, its preparation and application, and the hydrogel composite material for acute kidney injury repair includes peroxisome as oxidative stress regulation center and self-assembled repair polypeptide gel encapsulating peroxisome.The above-mentioned composite material is carried out oxidative stress regulation and pH-responsive polypeptide precursor liquid self-assembly polymerization into glue by peroxisome cascade catalytic system, and local administration is realized to realize cell repair control to kidney injury.The application solves the problem that existing single drug treatment is difficult to simultaneously solve oxidative stress and tissue damage, by neutralizing free radicals quickly, reducing tissue damage, activating cell proliferation, regeneration-related signal pathway, accelerating wound healing, and can more accurately act on damaged tissue, reduce the influence on healthy cells, due to its synergistic mechanism, can realize more efficient and more targeted kidney repair, thereby showing significant advantages in the repair treatment of acute kidney injury.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, specifically to a hydrogel composite material for the repair of acute kidney injury, its preparation and application, and particularly to a hydrogel composite material for the repair of acute kidney injury, its preparation method and its application in the preparation of materials for regulating oxidative stress and repairing tissues in acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a common critical illness in clinical practice, and its incidence is on the rise globally. The pathophysiological mechanisms of AKI are complex, involving multiple aspects such as renal tubular epithelial cell damage, microcirculatory disturbances, inflammatory responses, and oxidative stress. Among these, oxidative stress is a core component in the development of AKI, and its mechanism mainly involves the excessive production and imbalance of reactive oxygen species (ROS). When ROS levels exceed the body's antioxidant defense capacity, it leads to lipid peroxidation, protein denaturation, and DNA damage, subsequently triggering apoptosis and necrosis of renal tubular epithelial cells. Studies have shown that ROS levels in AKI patients are 2-3 times higher than in healthy individuals, and are positively correlated with the degree of renal function impairment.

[0003] Currently, clinical treatment for AKI remains primarily supportive, including fluid resuscitation, diuretics, and renal replacement therapy. However, these methods only temporarily maintain kidney function and cannot effectively address tissue damage caused by oxidative stress. In recent years, the application of antioxidant therapy in AKI has received widespread attention. However, traditional small-molecule antioxidants such as N-acetylcysteine ​​and vitamin C suffer from low bioavailability, short half-life, and poor targeting, making it difficult to maintain effective therapeutic concentrations at the site of injury. Furthermore, these antioxidants tend to exhibit non-specific distribution in the body, potentially leading to an imbalance in redox homeostasis in normal tissues.

[0004] With the rapid development of biomaterials science, hydrogels, as a type of polymer material with a three-dimensional network structure, have shown unique advantages in the fields of tissue engineering and regenerative medicine. Hydrogel materials have the following characteristics: (1) excellent biocompatibility and degradability, and their degradation products are non-toxic to the body; (2) high water content (usually >90%) and good swelling properties, which can simulate the natural extracellular matrix environment; (3) adjustable mechanical properties and pore structure, which are conducive to cell migration and nutrient exchange; (4) can be used as a drug controlled-release carrier to achieve the continuous release of therapeutic molecules. However, there is still a lack of composite materials that can simultaneously meet the following requirements in the current technology: (1) have ROS responsive characteristics, which can realize the intelligent release of antioxidants; (2) have good tissue adhesion, which can stably reside in the kidney injury site; (3) have regeneration function, which can promote the repair and regeneration of renal tubular epithelial cells; (4) have injectability, which is convenient for clinical minimally invasive applications. Although some studies have attempted to combine antioxidants (such as superoxide dismutase and glutathione) with hydrogels for tissue repair, these materials still have significant shortcomings in terms of kidney targeting, controllable drug release, and tissue repair efficacy.

[0005] Therefore, developing a multifunctional hydrogel composite material for acute kidney injury (AKI) that combines ROS-responsive antioxidant controlled release, tissue damage repair, targeted drug delivery to the injury site, and clinical-grade safety is of great practical significance. Summary of the Invention

[0006] Due to the aforementioned deficiencies in existing technologies, this invention provides a multifunctional hydrogel composite material for acute kidney injury (AKI) that combines ROS-responsive antioxidant controlled release, tissue damage repair, targeted drug delivery to the injury site, and clinical-grade safety. Specifically, it is an acute kidney injury repair hydrogel composite material, its preparation method, and its application in the preparation of materials for regulating oxidative stress and repairing tissues in acute kidney injury. This invention overcomes the shortcomings of existing technologies in achieving a good balance between composite antioxidant function, tissue damage repair, targeted drug delivery to the injury site, and clinical-grade safety.

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

[0008] A hydrogel composite material for the repair of acute kidney injury includes a peroxisome as a regulatory center for oxidative stress and a self-assembled repair peptide gel encapsulating the peroxisome.

[0009] The aforementioned hydrogel composite material for acute kidney injury repair has dual functional components, including peroxisomes as the oxidative stress regulation center and a repair peptide gel. The peroxisomes have cascade oxidoreductase activity (such as superoxide dismutase and catalase activity), which is used to provide ROS scavenging and homeostasis regulation capabilities for oxidative stress tissues. The self-assembled repair peptide gel has functions such as mediating cell adhesion and migration and regulating cell proliferation, which is used to deliver peroxisomes in a targeted manner and promote tissue regeneration and repair.

[0010] The aforementioned hydrogel composite material for acute kidney injury repair can be injected in situ. It has excellent biocompatibility, good reactive oxygen species scavenging ability, strong inflammatory regulation effect and excellent tissue repair potential. Through the combined function of different components, it can achieve efficient loading of antioxidant components and tissue microenvironment that promotes kidney repair, thereby achieving precise targeted treatment of kidney injury sites.

[0011] Compared with existing technologies, this invention addresses the difficulty of simultaneously solving the problems of oxidative stress and tissue damage with existing single-drug treatments (such as using only antioxidants or repair factors). The material of this invention can rapidly neutralize free radicals, reduce tissue damage, activate signaling pathways related to cell proliferation and regeneration, accelerate wound healing, and the composite material can act more precisely on damaged tissues, reducing the impact on healthy cells. Due to its synergistic mechanism, this material can achieve more efficient and targeted kidney repair, thus showing significant advantages in the repair and treatment of acute kidney injury.

[0012] As a preferred technical solution:

[0013] The above-described hydrogel composite material for repairing acute kidney injury includes a self-assembled repair peptide gel formed by pH regulation of a peptide gel precursor solution.

[0014] The hydrogel composite material for acute kidney injury repair described above comprises an oxidative stress regulation center composed of a redox cascade catalytic system including catalase and superoxide dismutase in the peroxisomal component. The peroxisomals are obtained by density gradient centrifugation of liver tissue homogenate. Specifically, healthy adult male SD rats are selected, and fresh liver tissue is taken after fasting for 18 hours. The crude peroxisomals (containing light mitochondria and lysosomes) are obtained by removing heavy mitochondria and cell debris from the cells using differential centrifugation. The crude peroxisomals are further purified into purified peroxisomals using density gradient centrifugation.

[0015] The self-assembling repair peptide gel uses amino acid motifs selected from functional motifs of β-laminus, which mediate cell adhesion and migration, regulate cell proliferation, and restore tissue function. It achieves in-situ targeted drug delivery through in vitro pH-controlled self-assembly into a gel, while maintaining low biotoxicity and immunogenicity. Specifically, two parts selected from the functional motif of β-laminus are combined to construct the YIGSRGD peptide. Based on the requirement for pH-regulated self-assembly, a hydrophobic amino acid sequence FF is added as a self-assembly motif in the peptide, and biotin is added to the amino terminus of the peptide motif to improve its stability as a functional scaffold.

[0016] This invention also provides a method for preparing the hydrogel composite material for acute kidney injury repair as described above, comprising the following steps:

[0017] S1: Purified peroxisomes were obtained by repeatedly centrifuging rat liver tissue homogenate.

[0018] S2: Dissolve the peptide in PBS and adjust the pH to ensure complete dissolution, thus obtaining the peptide gel precursor solution;

[0019] S3: The polypeptide gel precursor solution obtained in step S2 is adjusted by pH to make it self-assemble into a gel (nanofibers stack ᴨ-ᴨ). Just before gel formation, the purified peroxisomes obtained in step S1 are mixed with it to obtain a hydrogel composite material for acute kidney injury repair.

[0020] As a preferred technical solution:

[0021] In the preparation method described above, in step S2, the concentration of the polypeptide in the polypeptide gel precursor solution is 5~8 mg / mL;

[0022] The amino acid motif of the polypeptide includes one or more parts of the functional motif of β-laminus.

[0023] The pH adjuster used to adjust the pH is sodium carbonate, and the mass ratio of the polypeptide to the pH adjuster is 5~8:0.15~0.17. When the polypeptide is completely dissolved, the pH of the solution is 7.4~8.

[0024] In the preparation method described above, in step S3, the mass fraction of the peroxidase in the hydrogel composite material for acute kidney injury repair is 0.01~0.5 mg / mL.

[0025] In the preparation method described above, in step S3, the pH adjuster used to adjust the pH is hydrochloric acid, the concentration of the hydrochloric acid is 0.05~0.5 mol / L, the pH when mixing peroxidase is 7.0~7.4, and the pH when self-assembling into a complete gel is 6.5~7.0.

[0026] Furthermore, this invention also provides the application of the hydrogel composite material for acute kidney injury repair described above in the preparation of materials for regulating oxidative stress and repairing tissues in acute kidney injury, including ischemia-reperfusion type and drug-induced type. In practical applications, after the polypeptide peroxidase complex gel of this invention is completely self-assembled into a gel in vitro by adjusting the pH, it is then directionally delivered to the renal cortex via in situ injection. This achieves targeted drug delivery to the site of kidney injury, clearing ROS within renal tissue cells while effectively regulating the inflammatory response of the tissue, maintaining mitochondrial homeostasis within the tissue cells, alleviating cell apoptosis, and promoting endothelial cell proliferation and renal tubular regeneration and repair, thereby achieving the therapeutic purpose.

[0027] The above technical solution is only one feasible technical solution of the present invention. The scope of protection of the present invention is not limited thereto. Those skilled in the art can reasonably adjust the specific design according to actual needs.

[0028] The above invention has the following advantages or beneficial effects:

[0029] (1) The hydrogel composite material for acute kidney injury repair of the present invention adopts a dual-function design to achieve oxidative stress regulation and precise local drug delivery, rapidly neutralize free radicals, reduce tissue damage and activate cell regeneration signaling pathways;

[0030] (2) The hydrogel composite material for acute kidney injury repair of the present invention can precisely act on damaged tissues, reduce the impact on healthy cells, and achieve efficient and targeted kidney repair through a synergistic mechanism, overcoming the limitations of existing single drug treatments and showing significant advantages in the treatment of acute kidney injury repair.

[0031] (3) The hydrogel composite material for acute kidney injury repair of the present invention adopts a pH-responsive self-assembly gel system, which is simple and fast in gel formation, without the addition of toxic crosslinking agents, and is administered by in situ injection. It has the advantages of low biotoxicity and immunogenicity, and can achieve targeted drug delivery without complicated surgical operations, reducing the impact on healthy tissues.

[0032] (4) The hydrogel composite material for acute kidney injury repair of the present invention has simple raw materials, abundant peroxisomes, strong cascade oxidoreductase activity, and polypeptide motifs selected from intracellular structures. By simulating the extracellular matrix characteristics of natural kidney tissue, it provides suitable biophysical signals for cell adhesion and proliferation. It can utilize the peroxisome cascade catalytic system to rapidly neutralize a variety of free radicals and effectively alleviate oxidative stress. This redox regulation mechanism reduces inflammatory response and tissue damage.

[0033] (5) The hydrogel composite material for acute kidney injury repair of the present invention can regulate the remodeling of extracellular matrix through specific biochemical signaling pathways of polypeptide motifs, enhance intercellular communication and signal transduction, help restore the integrity and function of damaged kidney tissue, further accelerate the repair process, integrate oxidative stress regulation and tissue repair, provide a new method for the treatment of acute kidney injury, and has important significance and promotion value, with good application prospects. Attached Figure Description

[0034] The invention, its features, shape, and advantages will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings. Like reference numerals denote like parts throughout the drawings. The drawings are not drawn to scale; their focus is on illustrating the gist of the invention.

[0035] Figure 1 This is a schematic diagram of the structure of the hydrogel composite material for acute kidney injury repair according to the present invention;

[0036] Figure 2 The microscopic and macroscopic morphology (observed by transmission electron microscopy) of the hydrogel composite material for acute kidney injury repair of the present invention, as well as its mechanical property test diagram, are shown in the figure. In the figure, a is a TEM image of peroxisome, b is a TEM microstructure image of self-assembled polypeptide hydrogel, c is a macroscopic vial gelation image, and d is the storage modulus (G') and loss modulus (G") curves under the oscillation time scan mode at 37°C.

[0037] Figure 3 This is a schematic diagram showing the effect of peroxisome concentration on the anti-RONS ability of the composite gel, where a represents the ABTS+ scavenging ability detection and b represents the H2O2 scavenging ability detection.

[0038] Figure 4 This is a schematic diagram illustrating the effect of polypeptide gel materials on the migration and proliferation of endothelial cells (HUVECs).

[0039] Figure 5 This is a schematic diagram illustrating the protective effect of the hydrogel composite material for acute kidney injury repair of the present invention on oxidative stress in human renal proximal tubular epithelial cells. In the diagram, a shows the detection and observation of intracellular ROS by the DCFH-DA fluorescent probe after co-culturing with hydrogen peroxide / composite material; b shows the fluorescence intensity analysis of mitochondrial ROS; and c shows the cell viability detection after co-culturing with hydrogen peroxide / composite material.

[0040] Figure 6 The images show the results of treatment on the C57 mouse acute kidney injury model for 3 days. In the images, a is an H&E staining image of the renal cortex, b is a statistical analysis of serum creatinine concentration, c is a statistical analysis of serum urea nitrogen concentration, and d is a statistical analysis of the renal cortical necrosis score.

[0041] Figure 7 A schematic diagram of the assessment of oxidative stress and apoptosis in the kidney tissue of C57 mice after 3 days of treatment, where a is a biological projection electron microscope image of mitochondria in renal cortical cells and b is a fluorescence image of renal cortical tissue stained with ROS.

[0042] Figure 8 This is a schematic diagram of the assessment of renal cortical angiogenesis in C57 mice after 3 days of treatment. In the diagram, a is an image of the VEGFD / CD31 protein immunofluorescence double labeling assay, and b is a statistical analysis of the fluorescence intensity of the VEGFD / CD31 protein. Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0044] Unless otherwise specified in the following examples, all reagents can be commercially available products that are routinely obtainable by those skilled in the art. Process steps or preparation methods not mentioned in detail are all process steps or preparation methods well known to those skilled in the art.

[0045] In the examples, 1 mL syringes were used to inject the drug into the renal cortex in situ for animal experimental treatment.

[0046] Example 1

[0047] A hydrogel composite material for the repair of acute kidney injury, the structure of which is as follows: Figure 1 As shown, it includes a peroxisome as the oxidative stress regulation center and a self-assembled repair peptide gel encapsulating the peroxisome.

[0048] Its preparation process is as follows:

[0049] First, purified peroxidase and polypeptide solutions obtained by centrifugation were used to prepare a hydrogel precursor solution by pH adjustment. Then, the pH was adjusted to allow it to fully self-assemble into a gel, and finally, a hydrogel composite material for acute kidney injury repair was obtained. Subsequently, the drug was directed to the renal cortex of acute kidney injury by in situ injection.

[0050] Specifically as follows:

[0051] 1) Peroxisome extraction: Using a peroxisome extraction kit, SD rat liver tissue was first homogenized using a homogenizer. After centrifugation at 1000×g, the floating lipid layer was removed, the precipitate was discarded, and cell nuclei and other debris were removed. After centrifugation at 2000×g, the lipid layer was removed, the precipitate was discarded, and heavy mitochondria were removed. After centrifugation at 25,000×g for 20 minutes, the supernatant was discarded to obtain crude peroxisome fraction (CPF). The CPF was suspended in a density gradient medium and purified by high-speed centrifugation in a specific density solvent. The morphology was observed using a transmission electron microscope. The experimental results are shown below. Figure 2As shown in a;

[0052] 2) Design of peptide materials: A YIGSRGD functional peptide was constructed by coupling laminin YIGSR and RGD motifs, and biotin was covalently linked to the amino terminus to enhance its anti-enzymatic properties. To further improve the assembly strength and endow it with pH-responsive self-assembly ability, the hydrophobic amino acid motif FF was introduced, enabling it to achieve pH-regulated gelation transition through π-π stacking. The peptide was prepared by liquid-phase synthesis, and TEM characterization showed that it could form a three-dimensional network structure under specific pH conditions. Figure 2 (b) A smart hydrogel system with controllable self-assembly properties was successfully constructed.

[0053] 3) Dissolve the peptide obtained above in PBS and adjust the pH with 0.1 mol / L Na2CO3 solution to ensure complete dissolution (pH=7.4 when completely dissolved, add 2 μL of 0.1 mol / L Na2CO3 solution for every 100 μL of precursor solution) to obtain a peptide gel precursor solution with a peptide concentration of 7 mg / mL.

[0054] 4) The peptide gel precursor solution was self-assembled into a gel by adjusting the pH with 0.1 mol / L HCl. Just before gel formation, purified peroxidase was mixed with it (pH=7 when mixed with purified peroxidase, and the mass fraction of peroxidase in the hydrogel composite material for acute kidney injury repair was 0.05 mg / mL). HCl was added dropwise until pH=6.9 to allow complete self-assembly into a gel, thus obtaining the hydrogel composite material for acute kidney injury repair.

[0055] The gelation state of the composite material was observed using the vial inversion method. The experimental results are as follows: Figure 2 As shown in c.

[0056] This invention has the advantage of enabling the system to self-assemble into a gel within a small pH adjustment range (pH 7.4~7.0), a feature not reported in previous patents. Adding peroxidase at different pH levels alters the final antioxidant properties, with minimal pH changes occurring within the suitable pH range of the human body, effectively preventing the loss of bioactive substances due to environmental pH fluctuations. Furthermore, since the materials used are entirely derived from biological systems, their potential immunogenicity is significantly reduced, further enhancing the biocompatibility and safety of this technology.

[0057] Example 2

[0058] A method for preparing a hydrogel composite material for acute kidney injury repair is basically the same as that in Example 1, except that the mass fraction of peroxidase in the hydrogel composite material for acute kidney injury repair is 0.1 mg / mL.

[0059] Example 3

[0060] A method for preparing a hydrogel composite material for acute kidney injury repair is basically the same as that in Example 1, except that the mass fraction of peroxidase in the hydrogel composite material for acute kidney injury repair is 0.5 mg / mL.

[0061] Comparative Example 1

[0062] A hydrogel composite material is basically the same as that in Example 1, except that peroxidase is not extracted and peroxidase is not mixed when the polypeptide gel precursor solution is gelled.

[0063] The composite materials prepared in Examples 1-3 and Comparative Example 1 were subjected to rheological analysis using a rheometer. Figure 2 d in P0, P 0.05 P 0.1 P 0.5 Mechanical properties were tested for Examples 1, 2, and 3 respectively. The storage modulus (G') and loss modulus (G") of the hydrogel were measured using a Thermo Haake rheometer at frequencies ranging from 0.01 to 10 Hz. The composite material was first dropped onto a parallel plate (20 mm in diameter, 0.3 mm gap) at 37°C, and a dynamic frequency scan of 0.01–10 Hz was performed. The experimental results are as follows: Figure 2 As shown in d, it can be seen that its storage modulus (G') and loss modulus (G") are relatively stable under dynamic frequency changes of 0.01~10 Hz, and G' is greater than G", showing a stable gel state.

[0064] The composite materials (P) prepared in Examples 1-3 0.05 P 0.1 P 0.5 Antioxidant performance was evaluated for examples 1, 2, and 3 respectively.

[0065] 1) ABTS + Free radical scavenging: 38.41 mg of ABTS was dissolved in 10 mL of deionized water to prepare 7 mM ABTS reagent; 13.37 mg of potassium persulfate was dissolved in 10 mL of deionized water to prepare 4.95 mM potassium persulfate reagent; 2 mL of the above ABTS reagent and 2 mL of potassium persulfate reagent were incubated in the dark at room temperature for 16 h to obtain ABTS. + The working solution was diluted to 5% ABTS with PBS (0.1 M pH=7.4). + Working solution: Prepare 200 μL hydrogel with 1 mL 5% ABTS +After incubating the working solution for 1 hour, 100 μL was added to a 96-well plate, and the absorbance at 734 nm was measured using a microplate reader.

[0066] ABTS + Free radical scavenging rate = [1-(A s -A1) / A0]*100%, where A s A1 is the absorbance of the sample alone (PBS instead of ABTS). + (Working solution), A0 is the absorbance of the control (water instead of the test sample). Three parallel experiments were conducted; see [link to results]. Figure 3 a(P) 0.05 P 0.1 P 0.5 (Corresponding to Example 1, Example 2, and Example 3, respectively).

[0067] 2) H2O2 removal: Prepare a 1 mM H2O2 solution by dissolving 1 g TiOSO4 in 100 mL of 2 M H2SO4 and storing in the dark. Add 200 μL of hydrogel to 5 mL of H2O2 solution, shake in the dark (25℃, 1 h), and use an equal volume of PBS as a control group. Centrifuge (12000 rpm, 5 min) and collect the supernatant. Take 0.5 mL of the supernatant to be tested, add 0.5 mL of titanium sulfate solution, mix well, and let stand at room temperature for 10 minutes. Measure the absorbance at 405 nm.

[0068] H2O2 scavenging rate (%) = [(C0 - C) / C0] × 100% (C0: concentration of control group, C: concentration of sample group). Three parallel experiments were conducted; detailed results can be found in [link to relevant data]. Figure 3 b(P) 0.05 P 0.1 P 0.5 (Corresponding to Example 1, Example 2, and Example 3, respectively).

[0069] Peroxidases provide antioxidant properties in the composite material, with ABTS at a concentration of 0.05 mg / mL. + The free radical and H2O2 scavenging rates were 88.97% and 27.53%, respectively. Higher antioxidant activity could be obtained by increasing the concentration to 0.1 mg / mL. (ABTS) + The free radical and H2O2 scavenging rates were 91.30% and 44.70%, respectively, and there was no significant increase in antioxidant performance after increasing the concentration to 0.5 mg / mL. ABTS + The free radical scavenging rate and the H2O2 scavenging rate (%) were 93.17% and 44.71%, respectively.

[0070] The cellular antioxidant effects of the composite materials prepared in Examples 1-3 were evaluated using an H2O2-induced oxidative stress model of human renal proximal tubular epithelial cells (HK-2). A blank group (without H2O2 and composite materials), a control group (without composite materials), and a peroxisome-only group (P only, P) were established. 0.1 Specifically, the only groups are: a peroxidase concentration of 0.1 mg / mL, a peptide gel only group, and peptide composite groups with peroxidase concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.5 mg / mL (corresponding to Examples 1, 2, and 3, respectively).

[0071] HK-2 cells were loaded at 8×10 3 Seeded at a density of [number] cells / well in 96-well plates and incubated in MEM complete medium for 24 h. Afterward, the control group received only 100 µL of medium, while the H2O2 group received 100 µL of medium containing 500 µM H2O2 for 1 h. Simultaneously, the material group was treated with 100 µL of medium containing 500 µM H2O2 and 25 µL of material for 1 h. Subsequently, all groups were stained in the dark for 20 min with DCFH-DA solution (10 µM). Fluorescence images were observed and recorded using a fluorescence microscope, and the results are as follows: Figure 4 As shown in figure a. The fluorescence intensity was analyzed and statistically analyzed using ImageJ software, and the results are as follows. Figure 4 As shown in b. The treated cells were added to a culture medium containing 10% CCK-8 solution via medium exchange. After incubation for 2 hours, the OD value of each well at 450 nm was measured using a microplate reader to assess cell viability. The results are shown in Figure b. Figure 4 As shown in c.

[0072] Experimental results showed a significant decrease in green fluorescence intensity in the peroxidase group, confirming the material's excellent antioxidant properties. In the control group, a strong green fluorescence signal was observed, indicating the large-scale generation of intracellular reactive oxygen species (ROS) under H2O2 stimulation. Notably, the treatment group using only the peptide gel also exhibited a high green fluorescence signal. In cell viability testing, cell viability in the control group significantly decreased to below 50%, while the composite material treatment group showed a significant protective effect on cell viability. Subsequent testing was conducted using a peroxidase concentration of 0.05 mg / mL. These results strongly support the composite material's significant protective effect against oxidative stress-damaged epithelial cells in an acute kidney injury model.

[0073] To evaluate the effects of the hydrogel composite material of this invention on endothelial cell proliferation and migration, we conducted cellular-level experiments using human umbilical vein endothelial cells (HUVECs). The groups were designated as a control group, a peptide hydrogel-only group, and a peroxisome peptide hydrogel composite material group.

[0074] 1) Cell proliferation promotion: 100 μL of hydrogel was prepared for each group, and 1 mL of complete culture medium was added. The mixture was incubated at 37℃ for 24 h to obtain the hydrogel extract. The cell suspension was then diluted to a cell density of 3 × 10⁻⁶ cells / mL. 3 After achieving a cell / mL ratio, cells were seeded into 96-well plates, with 3 wells per group. The cells were then incubated at 37°C in a 5% CO2 incubator for 24 h. Once cells had adhered well, the culture medium was aspirated from each well, and 100 μL of the above hydrogel extraction solution was added to each well of the experimental groups. Cells were incubated for 24 h, 48 h, and 72 h. Cell proliferation was assessed using the CCK-8 assay kit; the results are shown below. Figure 5 a (P in the diagram) 0.1 only, Pipetide7only 、 Pipetide 7+P 0.05 Pipetide 7+P 0.1 Pipetide 7+P 0.5 The results correspond to the peroxidase-only group with a peroxidase concentration of 0.1 mg / mL, the peptide-only gel group with a peptide concentration of 7 mg / mL, and Examples 1, 2, and 3, respectively. It can be seen that the hydrogel composite material containing peptides has the effect of promoting endothelial cell proliferation, and the composite material has no cytotoxicity.

[0075] 1) Cell migration promotion: Using a Transwell chamber with a pore size of 3.0 μm, suitable for a 24-well plate, 100 μL of hydrogel was seeded in the lower chamber, and 500 μL of complete culture medium was added. Endothelial cells were digested with trypsin, centrifuged, and resuspended in serum-free culture medium. Cells were counted using a cell counting chamber, and the cell density was adjusted to 1 × 10⁶ cells / well. 5 Cells / mL: 100 μL of cell suspension was added to the upper chamber of a Transwell chamber. The cells were incubated at 37°C with 5% CO2 for 24 hours to induce cell migration. The Transwell chamber was removed, the upper chamber was washed with PBS, and the chamber was fixed in 4% paraformaldehyde for 15 minutes. The cells were stained with crystal violet for 10 minutes, followed by washing three times with PBS to remove excess dye. The migrated cells in the lower chamber were observed under a microscope. Results are shown below. Figure 5 As shown in b (in the figure, control, Pipetide7, Pipetide7+P) 0.1The groups were a blank control group, a peptide-only gel group with a peptide concentration of 7 mg / mL, and Example 2. Three fields of view were randomly selected, and the number of cells in each field was counted. The number of migrating cells was also statistically analyzed. The results are as follows: Figure 5 As shown in c (in the figure, control, Pipetide7, Pipetide7+P) 0.1 The groups were a blank control group, a peptide-only gel group with a peptide concentration of 7 mg / mL, and Example 2, respectively. The experimental results showed that the peptide hydrogel composite material promoted endothelial cell migration, and the addition of peroxisomes had no significant effect on its migration ability.

[0076] A clinical ischemia-reperfusion acute kidney injury model (IRI-AKI) was established for animal experimental validation. First, C57 mice (6 weeks old) were anesthetized with isopentane, and bilateral or unilateral renal arteries were exposed and clamped for 20 minutes. The clamps were then released to restore blood flow and achieve reperfusion. Kidney damage was observed 72 hours later. This model simulates the pathological process of acute kidney injury by inducing renal tubular epithelial cell damage, inflammatory response, and oxidative stress through the dual effects of ischemia and reperfusion. A sham surgery group, a model group (AKI modeling), and a treat group (AKI modeling + composite material therapy) were established.

[0077] Three groups of mouse kidney tissue specimens were collected and fixed overnight in 4% paraformaldehyde at 4℃, then fixed in paraffin. Sections (4 μm) were prepared and stained with hematoxylin and eosin (H&E) for observation of the renal cortex. The experimental results are as follows: Figure 6 As shown in Figure a (black triangle: complete renal cortical tubular structure; red triangle: complete renal cortical tubular structure), the condition of the renal cortex was observed. In the sham-operated group, the kidney structure was normal, with obvious brush borders within the tubules. In the AKI model group, kidney necrosis was severe, with renal epithelial cell shedding, brush border disruption, and nephron atrophy. In the composite gel treatment group, kidney necrosis was rare, and the brush borders of the tubules were relatively intact. Creatinine (SCr) in mouse serum samples was detected using a fully automated biochemical analyzer. Figure 6 b) and blood urea nitrogen (BUN) Figure 6 c) The level, as a traditional marker of renal function, showed a high level in the model group, indicating renal function impairment, while the treatment group showed a significant decrease, verifying the restorative effect of the composite material on renal function, and correlated with the renal necrosis score ( Figure 6 d) Corresponding.

[0078] This indicates that the hydrogel composite material prepared in this invention exhibits protective effects on renal tissue and recovery of renal function in mice with acute kidney injury due to ischemia-reperfusion injury.

[0079] To evaluate the effects of the hydrogel composite material of this invention on antioxidant and apoptosis signal regulation in mice with acute kidney injury, kidney tissue from the experimental mice was used for further related experimental verification.

[0080] 1) Protective role of mitochondrial homeostasis

[0081] In acute kidney injury (AKI), ischemia-reperfusion injury leads to impaired mitochondrial function, a major source of reactive oxygen species (ROS) production, which further triggers inflammatory responses. Bio-transmission electron microscopy (Bio-TEM) was used to observe mitochondria in renal cortical epithelial cells from different groups to evaluate the protective effect of the composite material on mitochondrial homeostasis in mouse proximal tubule cells. Fresh kidney tissue (n=3) was fixed overnight in 2.5% glutaraldehyde at 4℃, prepared into ultrathin sections, and observed under Bio-transmission electron microscopy. The results are as follows: Figure 7 As shown in Figure a, the mitochondrial structure in the Shame group remained intact, while AKI caused extensive damage to mitochondria in proximal tubule cells, specifically manifested as numerous swollen mitochondria with lost cristae, partial mitochondrial membrane rupture, and release of matrix material into the cytoplasm. In contrast, the composite material treatment in the Treat group significantly reduced the number of damaged mitochondria in the proximal tubule cells of AKI mice.

[0082] 2) ROS scavenging effect in kidney tissue

[0083] The large amount of ROS generated during ischemia-reperfusion is a major cause of kidney injury, further triggering downstream inflammatory responses. ROS staining was performed on kidney tissue to assess the ability of the composite material to clear ROS from the tissue. Fresh mouse kidney tissue was harvested, flash-frozen in liquid nitrogen, and 8 μm frozen sections were prepared. These sections were stained with ROS fluorescent probe working solution (e.g., DHE, 5 μM), incubated at 37°C in the dark for 30 minutes, and observed under a confocal fluorescence microscope. The results are as follows: Figure 7 b shows that the Shame group exhibited some endogenous ROS with weak red fluorescence intensity, while AKI induced the production of a large amount of ROS in the kidney tissue, showing strong red fluorescence intensity. In the Treat group, the composite material effectively cleared excess ROS from the tissue, resulting in a significant decrease in red fluorescence intensity compared to the AKI group. Figure 7 c).

[0084] 3) Regulatory role of renal tissue cell apoptosis

[0085] Oxidative stress induced by AKI typically leads to apoptosis of tissue cells. We used a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay to detect apoptotic cells in AKI-damaged kidneys. Paraffin sections of kidney tissue were prepared, incubated with proteinase K at room temperature for 20 minutes, and then TUNEL labeled according to the kit instructions. Observation was performed using confocal fluorescence microscopy. Figure 7d), quantitative analysis was performed by counting the number of positive cells in three different fields of view in each renal cortex ( Figure 7 e). Compared with the AKI group, the Treat group had the fewest apoptotic cells, indicating that the hydrogel composite material had the best anti-apoptotic effect.

[0086] 4) Renal cortex promotes angiogenesis

[0087] Endothelial cell proliferation and migration play a crucial role in renal angiogenesis, and angiogenesis, as an important step in the acute injury repair process, significantly accelerates the repair and functional reconstruction of damaged tissues by improving local blood supply and maintaining tissue homeostasis. Based on the significant effect of the composite material in promoting endothelial cell proliferation and migration, its potential ability to promote angiogenesis was further evaluated in an animal model. Immunofluorescence double-labeling assays were used to detect the co-expression of the key angiogenesis factor VEGFD (vascular endothelial growth factor D) and the vascular endothelial cell marker CD31 (platelet-endothelial cell adhesion molecule). Double-label co-localization analysis was used to reveal the spatial distribution relationship of VEGFD and CD31 in the angiogenesis process and their potential functional associations. Results are as follows: Figure 8 As shown in figure a, the results indicate that the hydrogel composite material can promote the secretion of a variety of cytokines, which are related to repair and have angiogenesis and anti-inflammatory properties.

[0088] The above results indicate that the hydrogel composite material has excellent in vitro and in vivo antioxidant capacity, good endothelial cell proliferation regulation performance, tissue repair capacity, high biocompatibility and easy operation, and has excellent antioxidant and repair-promoting effects on acute kidney injury, which has important clinical application value.

[0089] Those skilled in the art should understand that variations can be implemented by combining existing technology with the above embodiments, which will not be elaborated here. Such variations do not affect the substantive content of the present invention, and will not be elaborated here.

[0090] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. A hydrogel composite material for the repair of acute kidney injury, characterized in that: This includes peroxisomes, which serve as regulatory centers for oxidative stress, and self-assembled repair peptide gels that encapsulate peroxisomes. Its preparation process is as follows: First, purified peroxidase and polypeptide solution obtained by centrifugation were used to prepare hydrogel precursor solution by pH adjustment. Then, the pH was adjusted to allow it to fully self-assemble into a gel, and finally, a hydrogel composite material for acute kidney injury repair was obtained. Subsequently, the drug was directed to the renal cortex of acute kidney injury by in situ injection. Specifically as follows: 1) Extraction of peroxisomes: Using a peroxisome extraction kit, the liver tissue of SD rats was first homogenized using a homogenizer, centrifuged at 1000×g, the floating lipid layer was removed by aspiration, the precipitate was discarded, and cell nuclei and other debris were removed. Centrifuge at 2000×g to remove the lipid layer, precipitate, and heavy mitochondria; centrifuge at 25,000×g for 20 minutes, aspirate the supernatant to obtain crude peroxisomal fraction (CPF); suspend the CPF in a density gradient medium and centrifuge at high speed in a specific density solvent to obtain purified peroxisomes. 2) Design of peptide materials: YIGSRGD functional peptide was constructed by coupling laminin YIGSR and RGD motifs, and biotin was covalently linked to the amino terminus to enhance the anti-enzymatic properties. To further improve the assembly strength and endow it with pH-responsive self-assembly ability, the hydrophobic amino acid motif FF was introduced, which can achieve pH-regulated gelation transition through π-π stacking. The peptide was prepared by liquid phase synthesis. 3) Dissolve the peptide obtained above in PBS and adjust the pH with 0.1 mol / L Na2CO3 solution to ensure complete dissolution. The pH when completely dissolved is 7.

4. Add 2 μL of 0.1 mol / L Na2CO3 solution to every 100 μL of precursor solution to obtain a peptide gel precursor solution with a peptide concentration of 7 mg / mL. 4) The peptide gel precursor solution was self-assembled into a gel by adjusting the pH with 0.1 mol / L HCl. Just before gel formation, purified peroxidase was mixed with it. The pH of the purified peroxidase mixture was 7. The mass fraction of peroxidase in the hydrogel composite material for acute kidney injury repair was 0.05, 0.1, and 0.5 mg / mL. HCl was added dropwise until the pH reached 6.9 to allow complete self-assembly into a gel, thus obtaining the hydrogel composite material for acute kidney injury repair.

2. The application of the hydrogel composite material for acute kidney injury repair as described in claim 1 in the preparation of materials for regulating oxidative stress and repairing tissues in acute kidney injury, characterized in that, The acute kidney injury includes ischemia-reperfusion type and drug-induced type.

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