Hydrogel composite material for repairing acute kidney injury as well as preparation and application of hydrogel composite material

Abstract

The invention discloses a hydrogel composite material for repairing acute kidney injury as well as preparation and application thereof. The hydrogel composite material for repairing the acute kidney injury comprises peroxisome serving as an oxidative stress regulation center and self-assembly repairing polypeptide gel for encapsulating the peroxisome. According to the composite material, oxidative stress regulation is carried out through a peroxisome cascade catalytic system, a polypeptide precursor solution is initiated to be self-assembled and polymerized into gel through pH responsiveness, local drug delivery is carried out, and cell repair regulation and control of kidney injury are achieved. The problem that oxidative stress and tissue damage are difficult to solve at the same time in existing single drug treatment is solved, wound healing is accelerated by rapidly neutralizing free radicals, reducing tissue damage, activating cell proliferation and regenerating related signal channels, the composition can act on damaged tissue more accurately, the influence on healthy cells is reduced, and due to the synergistic effect mechanism, the composition has a good application prospect. More efficient and targeted kidney repair can be realized, so that remarkable advantages are shown in the repair treatment of the acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, in particular to a hydrogel composite material for repairing acute kidney injury, its preparation method and its application in preparing materials for regulating oxidative stress and repairing tissues of acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is a common clinical critical illness, and its incidence continues to rise worldwide. The pathophysiology of AKI is complex, involving multiple aspects such as renal tubular epithelial cell damage, microcirculatory disorders, inflammatory responses, and oxidative stress. Among them, oxidative stress is a core link in the occurrence and development of AKI, and its mechanism mainly involves the excessive production and imbalance of reactive oxygen species (ROS) clearance. When ROS levels exceed the body's antioxidant defense capacity, it can lead to lipid peroxidation, protein denaturation, and DNA damage, which in turn triggers 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 people and are positively correlated with the degree of renal function damage.

[0003] Currently, clinical treatment for AKI is still mainly supportive therapy, including fluid therapy, diuretic use, and renal replacement therapy. However, these methods can only temporarily maintain renal function and cannot effectively address the problem of tissue damage caused by oxidative stress. In recent years, the application of antioxidant therapy in AKI has received widespread attention, but traditional small molecule antioxidants such as N-acetylcysteine and vitamin C have defects such as low bioavailability, short half-life, and poor targeting, making it difficult to maintain effective therapeutic concentrations at the site of injury. In addition, these antioxidants are prone to nonspecific distribution in the body, which may lead to an imbalance in the redox homeostasis of normal tissues.

[0004] With the rapid development of biomaterials science, hydrogels, as a 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 sustained release of therapeutic molecules. However, the existing technology still lacks composite materials that can simultaneously meet the following requirements: (1) have ROS responsiveness, which can achieve intelligent release of antioxidants; (2) have good tissue adhesion, which can stably reside in the site of kidney damage; (3) have pro-regenerative function, which can promote the repair and regeneration of renal tubular epithelial cells; (4) have injectability, which is convenient for clinical minimally invasive application. Although studies have attempted to combine antioxidants (such as superoxide dismutase, glutathione, etc.) with hydrogels for tissue repair, these materials still have obvious shortcomings in kidney targeting, controllable drug release and tissue repair effects.

[0005] Therefore, it is of great practical significance to develop 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. Summary of the Invention

[0006] Due to the above-mentioned defects in the prior art, the present 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, the present invention provides a hydrogel composite material for acute kidney injury repair, a preparation method thereof, and its application in the preparation of acute kidney injury oxidative stress regulation and tissue repair materials, which overcomes the defect of the prior art that it cannot achieve a good balance between composite antioxidant function, tissue damage repair, targeted drug delivery to the injury site, and clinical-grade safety.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A hydrogel composite material for repairing acute kidney injury comprises peroxisomes serving as oxidative stress regulation centers and self-assembling repair polypeptide gel encapsulating the peroxisomes.

[0009] The above-mentioned hydrogel composite material for repairing acute kidney injury has dual functional components, including peroxisomes as oxidative stress regulation centers and repair polypeptide gels. Among them, peroxisomes have cascade oxidoreductase activity (such as superoxide dismutase and catalase activity), which are used to provide ROS scavenging and homeostasis regulation capabilities for oxidative stress tissues. The self-assembled repair polypeptide gel has functions such as mediating cell adhesion and migration, regulating cell proliferation, etc., and is used to directional transport peroxisomes and promote tissue regeneration and repair.

[0010] The above-mentioned hydrogel composite material for repairing acute kidney injury can be injected in situ. It has excellent biocompatibility, good reactive oxygen species scavenging ability, strong inflammation regulation effect and excellent tissue repair potential. Through the composite functions of different components, it can respectively realize efficient loading of antioxidant components and promote tissue microenvironment of kidney repair, thereby realizing precise targeted treatment of the site of kidney damage.

[0011] Compared with the existing technology, the present invention solves the problem that existing single drug treatments (such as using only antioxidants or repair factors) are difficult to simultaneously solve the problem of oxidative stress and tissue damage. The material of the present invention can quickly neutralize free radicals, reduce tissue damage, activate signal pathways related to cell proliferation and regeneration, and accelerate wound healing. The composite material can act more accurately on damaged tissues and reduce the impact on healthy cells. Due to its synergistic mechanism, this material can achieve more efficient and targeted kidney repair, thereby showing significant advantages in the repair treatment of acute kidney injury.

[0012] As the preferred technical solution:

[0013] In the hydrogel composite material for repairing acute kidney injury as described above, the self-assembling repair polypeptide gel is formed by self-assembly of a polypeptide gel precursor solution by regulating pH.

[0014] In the hydrogel composite material for repairing acute kidney injury as described above, the oxidative stress regulation center is composed of a redox cascade catalytic system such as catalase and superoxide dismutase in the peroxisome component; the peroxisomes are obtained by density gradient centrifugation of liver tissue homogenate, specifically: adult healthy SD male mice are selected, fresh liver tissue is obtained after fasting for 18 hours, and crude peroxisomes (containing light mitochondria and lysosomes, etc.) are obtained after removing heavy mitochondria and cell debris in the cells by differential centrifugation; and the crude peroxisomes are further purified into purified peroxisomes by density gradient centrifugation.

[0015] The amino acid motif of the self-assembling repair polypeptide gel is selected from the functional motif of β-laminin. It has the functions of mediating cell adhesion and migration, regulating cell proliferation and restoring tissue function. It self-assembles into a gel by regulating pH in vitro, enabling in situ injection targeted drug delivery, and achieving low biological toxicity and immunogenicity. Specifically, two parts of the functional motif selected from β-laminin are combined to construct the YIGSRGD peptide. Based on the requirement of pH-regulated self-assembly into a gel, the hydrophobic amino acid sequence FF is added as the self-assembly motif in the polypeptide. Biotin is also added to the amino terminus of the polypeptide motif to improve its stability as a functional scaffold.

[0016] The present invention also provides a method for preparing the above-mentioned hydrogel composite material for repairing acute kidney injury, comprising the following steps:

[0017] S1: Purified peroxisomes were obtained by multiple differential centrifugation of rat liver tissue homogenate;

[0018] S2: dissolving the polypeptide in PBS and adjusting the pH to completely dissolve it to obtain a polypeptide gel precursor solution;

[0019] S3: The polypeptide gel precursor obtained in step S2 is self-assembled into a gel by adjusting the pH (the nanofibers undergo π-π stacking), and the purified peroxisomes obtained in step S1 are mixed with it before the gel is formed to obtain a hydrogel composite material for repairing acute kidney injury.

[0020] As the 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 a portion or multiple portions of a functional motif of β-laminin;

[0023] The pH regulator used for adjusting the pH is sodium carbonate, the mass ratio of the polypeptide to the pH regulator is 5-8:0.15-0.17, and the pH of the solution is 7.4-8 when the polypeptide is completely dissolved.

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

[0025] In the preparation method described above, in step S3, the pH regulator used to adjust the pH is hydrochloric acid, the concentration of the hydrochloric acid is 0.05-0.5 mol / L, the pH when the peroxisomes are mixed is 7.0-7.4, and the pH when the self-assembly is completely gelled is 6.5-7.0.

[0026] In addition, the present invention also provides the use of the above-mentioned hydrogel composite material for repairing acute kidney injury in the preparation of oxidative stress regulation and tissue repair materials for acute kidney injury, wherein the acute kidney injury includes ischemia-reperfusion type and drug-induced type. In practical applications, the polypeptide peroxisome composite gel of the present invention is completely self-assembled into a gel in vitro by regulating the pH, and then directedly delivered to the renal cortex by in situ injection, thereby achieving targeted drug delivery to the site of renal injury, while clearing ROS in renal tissue cells, it can also effectively regulate the inflammatory response of the tissue, maintain mitochondrial homeostasis in cells in the tissue, alleviate cell apoptosis, and promote endothelial cell proliferation and renal tubular regeneration and repair, thereby achieving the purpose of treatment.

[0027] The above technical solution is only a feasible technical solution of the present invention. The protection scope 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-functional design to achieve oxidative stress regulation and precise local drug delivery, quickly neutralizing free radicals, reducing tissue damage and activating 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 repair and treatment of acute kidney injury;

[0031] (3) The hydrogel composite material for repairing acute kidney injury of the present invention adopts a pH-responsive self-assembly gel system. The gel formation method is simple and rapid, no toxic cross-linking agent is added, and the drug is administered by in situ injection. It has low biological toxicity and immunogenicity, and can achieve targeted drug delivery without complex surgical operations, thereby reducing the impact on healthy tissues.

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

[0033] (5) The hydrogel composite material for repairing acute kidney injury of the present invention can regulate the remodeling of the extracellular matrix through a specific biochemical signaling pathway, enhance intercellular communication and signal transmission, help restore the integrity and function of damaged kidney tissue, further accelerate the repair process, and integrate oxidative stress regulation and tissue repair. It provides a new method for treating acute kidney injury, has important significance and promotion value, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention and its features, configurations, and advantages will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings. Like reference numerals indicate like parts throughout the drawings. The drawings are not necessarily drawn to scale, emphasis being placed on illustrating the subject matter of the present invention.

[0035] Figure 1 This is a schematic structural diagram of the hydrogel composite material for repairing acute kidney injury of the present invention;

[0036] Figure 2 The microscopic and macroscopic morphologies (transmission electron microscopy observation) of the hydrogel composite material for repairing acute kidney injury of the present invention, as well as its mechanical property test diagram, wherein a is a TEM image of peroxisomes, b is a TEM microstructure image of the self-assembled polypeptide hydrogel, c is a macroscopic vial gelation image, and d is the storage modulus (G') and loss modulus (G") curves in oscillation time sweep mode at 37°C;

[0037] Figure 3 Schematic diagram of the effect of peroxisome concentration on the anti-RONS ability of the composite gel, where a is the ABTS+ scavenging ability test and b is the H2O2 scavenging ability test;

[0038] Figure 4 Schematic diagram of the effect of polypeptide gel material on the migration and proliferation of endothelial cells (HUVECs);

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

[0040] Figure 6 Figure 1 shows the relevant legends of the C57 mouse acute kidney injury model after 3 days of treatment, where a is the H&E staining image of the renal cortex, b is the statistics of serum creatinine concentration, c is the statistics of serum urea nitrogen concentration, and d is the statistics of renal cortical necrosis;

[0041] Figure 7 Schematic diagram of the evaluation of oxidative stress and apoptosis in renal tissue of C57 mice after 3 days of treatment, where a is a biological projection electron microscopy image of mitochondria in renal cortical cells, and b is a fluorescence imaging of ROS staining in renal cortical tissue;

[0042] Figure 8 Schematic diagram of the evaluation of renal cortical angiogenesis in C57 mice after 3 days of treatment, where a is the VEGFD / CD31 protein immunofluorescence double-labeling test image, and b is the VEGFD / CD31 protein fluorescence intensity statistics. DETAILED DESCRIPTION

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

[0044] Unless otherwise specified in the following examples, each reagent can be a commercially available product that can be routinely obtained 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, a 1 mL syringe was used to perform in situ injection into the renal cortex for animal experimental treatment.

[0046] Example 1

[0047] A hydrogel composite material for repairing acute kidney injury, the structure of which is as follows Figure 1 As shown, it includes peroxisomes as oxidative stress regulation centers and self-assembling repair polypeptide gels that encapsulate peroxisomes.

[0048] The preparation process is as follows:

[0049] First, the purified peroxisomes and polypeptide solutions obtained by centrifugation are adjusted to obtain a hydrogel precursor solution, and then the pH is regulated to allow it to fully self-assemble into a gel. Finally, a hydrogel composite material for acute kidney injury repair is obtained, and then the in situ injection method is used for targeted drug delivery to the renal cortex of acute kidney injury.

[0050] The details are as follows:

[0051] 1) Peroxisome extraction: Using a peroxisome extraction kit, first homogenize SD rat liver tissue using a homogenizer, centrifuge at 1000×g, remove the floating lipid layer by suction, discard the precipitate, remove the nuclei and other debris; centrifuge at 2000×g, discard the lipid layer, discard the precipitate, and remove heavy mitochondria; centrifuge at 25,000×g for 20 minutes, and discard the supernatant to obtain a crude peroxisome fraction (CPF); suspend the CPF in a density gradient medium and centrifuge at high speed to obtain purified peroxisomes in a specific density solvent; observe its morphology using a transmission electron microscope. The experimental results are shown in Figure 2. Figure 2As shown in a;

[0052] 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 its resistance to enzymatic degradation. To further improve the assembly strength and impart pH-responsive self-assembly ability, a hydrophobic amino acid motif FF was introduced, which enables pH-regulated gelation 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) Successfully constructed a smart hydrogel system with controllable self-assembly properties;

[0053] 3) The peptide prepared above was dissolved in PBS and the pH was adjusted with 0.1 mol / L Na2CO3 solution to completely dissolve it (pH = 7.4 for complete dissolution; 2 μL of 0.1 mol / L Na2CO3 solution was added per 100 μL of precursor solution) to obtain a peptide gel precursor solution with a peptide concentration of 7 mg / mL.

[0054] 4) The polypeptide gel precursor solution was self-assembled into a gel by adjusting the pH using 0.1 mol / L HCl, and the purified peroxisomes were mixed with it just before gelation (pH = 7 when the purified peroxisomes were mixed, and the mass fraction of peroxisomes in the acute kidney injury repair hydrogel composite material was 0.05 mg / mL). HCl was continued to be added dropwise until the pH was 6.9 to allow the self-assembly to completely gel, thereby obtaining the acute kidney injury repair hydrogel composite material.

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

[0056] This invention offers the advantage of enabling the system to self-assemble into a gel within a narrow pH adjustment range (pH 7.4–7.0), a feature not previously reported in patents. Adding peroxisomes at varying pH levels results in changes in the final antioxidant properties, but the pH fluctuations are small and well within the pH range suitable for the human body, effectively preventing the loss of activity of bioactive substances due to environmental pH fluctuations. Furthermore, because 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 repairing acute kidney injury is basically the same as Example 1, except that the mass fraction of peroxisomes in the hydrogel composite material for repairing acute kidney injury is 0.1 mg / mL.

[0059] Example 3

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

[0061] Comparative Example 1

[0062] A hydrogel composite material is substantially the same as that of Example 1, except that peroxisomes are not extracted and peroxisomes are not mixed when the polypeptide gel precursor solution is gelled.

[0063] The composite materials prepared in Examples 1 to 3 and Comparative Example 1 were measured by rheometer. Figure 2 P0, P 0.05 、P 0.1 、P 0.5 Mechanical properties tests were performed on Example 1, Example 1, Example 2, and Example 3, respectively. The storage modulus (G') and loss modulus (G") of the hydrogel were measured using a Thermo Haake rheometer at a frequency variation of 0.01 to 10 Hz. The composite material was first dropped onto a parallel plate (20 mm diameter, 0.3 mm gap) at 37°C, and a dynamic frequency sweep of 0.01 to 10 Hz was performed. The experimental results are shown in FIG. Figure 2 As shown in Figure d, it can be seen that its storage modulus (G') and loss modulus (G") are relatively stable under the dynamic frequency change of 0.01~10 Hz, and G' is greater than G", showing a stable gel state.

[0064] The composite materials (P 0.05 、P 0.1 、P 0.5 Corresponding to Example 1, Example 2, and Example 3, the antioxidant performance evaluation was performed:

[0065] 1) ABTS + Free radical scavenging: Weigh 38.41 mg ABTS and dissolve it in 10 mL deionized water to prepare 7 mM ABTS reagent. Weigh 13.37 mg potassium persulfate and dissolve it in 10 mL deionized water to prepare 4.95 mM potassium persulfate reagent. Take 2 mL of the above ABTS reagent and 2 mL of potassium persulfate reagent and incubate them at room temperature in the dark for 16 h to prepare ABTS. + Working solution: dilute to 5% ABTS in PBS (0.1 M pH = 7.4) + Working solution: prepare 200 μL of hydrogel and 1 mL of 5% ABTS +The working solution was incubated for 1 h, 100 μL was taken into a 96-well plate, and its absorbance at 734 nm was detected using a microplate reader.

[0066] ABTS + Free radical scavenging rate = [1-(A s -A1) / A0]*100%, where A s is the sample absorbance, 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 sample to be tested). Three sets of parallel experiments were conducted. The specific results are shown in 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 of TiOSO₄ in 100 mL of 2 M H₂SO₄ and store in the dark. Add 200 μL of the hydrogel to 5 mL of the H₂O₂ solution and shake in the dark (25°C, 1 h). For the control, replace the sample with an equal volume of PBS. Centrifuge (12,000 rpm, 5 min) and remove the supernatant. Add 0.5 mL of the supernatant to be tested to 0.5 mL of the titanium sulfate solution, mix thoroughly, and incubate at room temperature for 10 minutes. Measure the absorbance at 405 nm.

[0068] H2O2 clearance rate (%) = [(C0 - C) / C0] × 100% (C0: control group concentration, C: sample group concentration) Three parallel experiments were performed, and the specific results are shown in Figure 3 b(P 0.05 、P 0.1 、P 0.5 Corresponding to Example 1, Example 2, and Example 3 respectively).

[0069] Peroxisomes provide antioxidant properties in the composite material, and their ABTS at a concentration of 0.05 mg / mL + The free radical and H2O2 scavenging rates (%) were 88.97% and 27.53%, respectively. By increasing its concentration to 0.1 mg / mL, higher antioxidant activity could be obtained. + The free radical and H2O2 scavenging rates (%) were 91.30% and 44.70%, respectively. After increasing to 0.5 mg / mL, the antioxidant activity did not increase significantly. + The free radical and H2O2 scavenging rates (%) were 93.17% and 44.71%, respectively.

[0070] The cellular antioxidant effects of the composite materials prepared in Examples 1 to 3 were evaluated by the H2O2-induced human renal proximal tubular epithelial cell (HK-2) oxidative stress model, and a blank group (without H2O2 and composite materials), a control group (without composite materials), and a peroxisome-only group (P only, P 0.1 only (specifically, the peroxisome concentration was 0.1 mg / mL), only polypeptide gel group (Pipetide only), and polypeptide composite material groups with peroxisome concentrations of 0.05 mg / mL, 0.1 mg / mL and 0.5 mg / mL (corresponding to Example 1, Example 2, and Example 3, respectively).

[0071] HK-2 cells were cultured at 8×10 3 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated in MEM complete medium for 24 hours. Afterwards, the control group only replaced 100 μL of medium, and the H2O2 group replaced 100 μL of medium containing 500 μM H2O2 for 1 hour. At the same time, the material group was treated with 100 μL of medium containing 500 μM H2O2 and 25 μL of material for 1 hour. Subsequently, different groups were stained with DCFH-DA solution (10 μM) in the dark for 20 minutes. Fluorescence images were observed and recorded using a fluorescence microscope. The results are shown in Figure 2. Figure 4 The fluorescence intensity was analyzed and statistically analyzed using imageJ software. The results are shown in Figure 4 d. The treated cells were prepared with CCK-8 solution to prepare a culture medium containing 10% CCK-8 solution and added in the form of liquid replacement. After incubation for 2 hours, the OD value of each well at a wavelength of 450nm was measured using a microplate reader to detect the cell viability. The results are shown in Figure 4 As shown in e.

[0072] The experimental results showed that the green fluorescence intensity in the peroxisome group was significantly reduced, which confirmed that the material has excellent antioxidant properties. In the control group, we observed a strong green fluorescence signal, indicating that intracellular reactive oxygen species (ROS) were generated in large quantities under the stimulation of H2O2. It is worth noting that the group treated with only the peptide gel also showed a high green fluorescence signal. In the cell viability test, the cell viability of the control group dropped significantly to below 50%, while the composite material treatment group showed a significant cell viability protection effect. The peroxisome concentration of 0.05 mg / mL was subsequently selected for testing. These test results strongly support the composite material's significant protective effect on tissue epithelial cells damaged by oxidative stress in the acute kidney injury model.

[0073] To evaluate the effects of the hydrogel composite material of the present invention on endothelial cell proliferation and migration, we used human umbilical vein endothelial cells (HUVEC) for cellular level experimental verification. These groups were set up as a blank group (control), a peptide hydrogel-only group, and a peroxisomal peptide hydrogel composite 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 cells were incubated at 37°C for 24 h to prepare the hydrogel extract. The cell suspension was diluted to a cell density of 3×10 3 After the cells were plated at 400 μg / mL, the cells were plated in 96-well plates, with 3 wells per group. The cells were then cultured in a 37°C, 5% CO2 cell culture incubator for 24 h. After the cells were well attached, the culture medium of each well was aspirated, and 100 μL of the above hydrogel extract was added to each well of the experimental group and incubated for 24 h, 48 h, and 72 h. The cell proliferation was detected using the CCK-8 kit. The specific results are shown in the table. Figure 5 a (P 0.1 only、Pipetide7only 、 Pipetide7+P 0.05 、Pipetide7+P 0.1 、Pipetide7+P 0.5 These correspond to the peroxisome-only group with a peroxisome concentration of 0.1 mg / mL, the polypeptide gel-only group with a polypeptide concentration of 7 mg / mL, Example 1, Example 2, and Example 3, respectively. It can be seen that the hydrogel composite material containing the polypeptide has the effect of promoting endothelial cell proliferation and has no cytotoxicity.

[0075] 1) Cell migration promotion: A Transwell chamber with a pore size of 3.0 μm and adapted to a 24-well plate was used. 100 μL of hydrogel was inoculated into the lower chamber and 500 μL of complete culture medium was added. Endothelial cells were trypsinized, centrifuged, and resuspended in serum-free culture medium. Cell counts were performed using a cell counter and the cell density was adjusted to 1×10 5 cells / mL, add 100 μL of cell suspension to the upper chamber of the Transwell chamber. Incubate in a 37°C, 5% CO2 incubator for 24 hours to allow cell migration. Remove the Transwell chamber, wash the upper chamber with PBS, and immerse the chamber in 4% paraformaldehyde for 15 minutes. Stain with crystal violet for 10 minutes, then wash three times with PBS to remove excess dye. Observe the migrated cells in the lower chamber under a microscope. The results are as follows. Figure 5 b (control, Pipetide7, Pipetide7+P 0.1Three visual fields were randomly selected, the number of cells in each visual field was counted, and the number of migrated cells was statistically analyzed. The results are shown in Figure 2. Figure 5 c (control, Pipetide7, Pipetide7+P 0.1 The experimental results showed that the polypeptide hydrogel composite material has the function of promoting endothelial cell migration, and the addition of peroxisomes has no significant effect on its migration ability.

[0076] A clinical model of acute kidney injury (IRI-AKI) was established for animal experimental validation. First, C57 mice (6 weeks old) were anesthetized with isopentane, and either the bilateral or unilateral renal artery was exposed and clamped for 20 minutes. The clamp was then released to restore blood flow and achieve reperfusion. Renal injury 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. Sham group (i.e., shame), model group (AKI modeling), and treat group (AKI modeling plus composite material treatment) (i.e., treat) were established.

[0077] Kidney tissue specimens from three groups of mice were collected and fixed in 4% paraformaldehyde at 4°C overnight, then fixed in paraffin. Sections (4 μm) were made and stained with hematoxylin-eosin (H&E) to observe the renal cortex. The experimental results are shown in Figure 2. Figure 6 As shown in a (in the figure, black triangle: complete renal cortical lumen structure; red triangle: complete renal cortical lumen structure), the renal cortex was observed. The kidney structure of the sham operation group was normal, with obvious brush borders in the tubules. The renal necrosis in the AKI model group was severe, with renal epithelial cells falling off, brush borders destroyed, and nephrons atrophied. The kidneys of the mice in the composite gel treatment group rarely showed necrosis, and the brush borders of the tubules were relatively intact. The creatinine (SCr) in the serum samples of mice was detected using an automatic biochemical analyzer ( Figure 6 b) and urea nitrogen (BUN) ( Figure 6 c) level, which is a traditional renal function marker, showed a higher level in the model group, indicating renal function damage, while the treatment group showed a significant decrease, verifying the restorative effect of the composite material on renal function and was consistent with the renal necrosis score results ( Figure 6 d) Correspondence.

[0078] It shows that the hydrogel composite material prepared by the present invention exhibits protective effects on renal tissue and recovery of renal function in mice with acute renal injury caused by ischemia-reperfusion.

[0079] To evaluate the effects of the hydrogel composite material of the present invention on anti-oxidation and apoptosis signal regulation in mice with acute kidney injury, renal tissues of experimental mice were taken for further relevant experimental verification.

[0080] 1) Protective effect on mitochondrial homeostasis

[0081] In AKI, ischemia-reperfusion injury leads to impaired mitochondrial function, which is the main source of ROS production and further triggers an inflammatory response. Bio-TEM was used to observe mitochondria in renal cortical epithelial cells in different groups to evaluate the protective effect of the composite material on mitochondrial homeostasis in mouse proximal tubule cells. Fresh renal tissue (n=3) was fixed in 2.5% glutaraldehyde at 4°C overnight, prepared into ultrathin sections, and observed using bio-TEM. The results are shown in Figure 2. Figure 7 (a) While the mitochondrial structure of the shame group was intact, AKI caused extensive mitochondrial damage in proximal tubular cells, as evidenced by the observation of numerous swollen mitochondria with loss of cristae, partial mitochondrial membrane rupture, and release of matrix material into the cytoplasm. In the treat group, treatment with the composite material significantly reduced the number of damaged mitochondria in the proximal tubular cells of AKI mice.

[0082] 2) Clearing of ROS in renal tissue

[0083] The large amount of ROS generated by ischemia-reperfusion is the main cause of renal injury, which further triggers downstream inflammatory responses. ROS staining was performed on renal tissue to evaluate the ability of the composite material to clear ROS in the tissue. Fresh mouse kidney tissue was taken, quickly frozen in liquid nitrogen, and 8 μm frozen sections were prepared. ROS fluorescent probe working solution (such as DHE, 5 μM) was used for staining, incubated at 37°C in the dark for 30 minutes, and observed under a confocal fluorescence microscope. The results are shown in Figure 2. Figure 7 b shows that the Shame group showed a certain amount of endogenous ROS, with a weak red fluorescence intensity. AKI caused the production of a large amount of ROS in the renal tissue, showing a strong red fluorescence intensity. The composite material in the Treat group could effectively remove the excessive ROS in the tissue, and the red fluorescence intensity decreased significantly compared with the AKI group ( Figure 7 c).

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

[0085] Oxidative stress caused by AKI often leads to apoptosis of tissue cells. We used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method to detect apoptotic cells in AKI-injured kidneys. Paraffin sections of renal tissue were prepared and incubated with proteinase K for 20 minutes at room temperature. TUNEL labeling was then performed according to the kit instructions. Confocal fluorescence microscopy was used for observation ( 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 number of apoptotic cells in the Treat group was the least, indicating that the hydrogel composite had the best anti-apoptotic effect.

[0086] 4) Promoting angiogenesis in the renal cortex

[0087] The proliferation and migration of endothelial cells play a key role in the regeneration of renal tissue angiogenesis. As an important part of the acute injury repair process, angiogenesis significantly accelerates the repair and functional reconstruction of damaged tissue 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 animal models. The co-expression of VEGFD (vascular endothelial growth factor D), a key factor in angiogenesis, and CD31 (platelet-endothelial cell adhesion molecule), a marker of vascular endothelial cells, was detected using immunofluorescence double-labeling experimental technology. Double-label co-localization analysis aims to reveal the spatial distribution relationship between VEGFD and CD31 in the process of angiogenesis and their potential functional associations. The results are shown in Figure 2. Figure 8 As shown in a, the results showed that the hydrogel composite material was able to promote the secretion of multiple cytokines that are related to repair and have angiogenesis and anti-inflammatory properties.

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

[0089] Those skilled in the art should understand that they can implement variations by combining the prior art with the above embodiments, which will not be described in detail here. Such variations do not affect the essence of the present invention and will not be described in detail here.

[0090] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-mentioned disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which 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 that do not depart from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A hydrogel composite material for repairing acute kidney injury, characterized in that: It includes peroxisomes as oxidative stress regulation centers and self-assembling repair polypeptide gels that encapsulate peroxisomes.

2. The hydrogel composite material for repairing acute kidney injury according to claim 1, characterized in that The self-assembling repair polypeptide gel is formed by self-assembly of a polypeptide gel precursor solution by regulating pH.

3. The hydrogel composite material for repairing acute kidney injury according to claim 2, characterized in that The oxidative stress regulation center is composed of a peroxisome component redox cascade catalytic system; The amino acid motif of the self-assembling repair polypeptide gel is selected from the functional motif in β-laminin.

4. The method for preparing the hydrogel composite material for repairing acute kidney injury according to any one of claims 1 to 3, wherein: The following steps are involved: S1: Purified peroxisomes were obtained by multiple differential centrifugation of rat liver tissue homogenate; S2: dissolving the polypeptide in PBS and adjusting the pH to completely dissolve it to obtain a polypeptide gel precursor solution; S3: The polypeptide gel precursor solution obtained in step S2 is self-assembled into a gel by adjusting the pH, and the purified peroxisomes obtained in step S1 are mixed with the precursor solution before gelation to obtain a hydrogel composite material for repairing acute kidney injury.

5. The preparation method according to claim 4, characterized in that In step S2, the concentration of the polypeptide in the polypeptide gel precursor solution is 5-8 mg / mL; The amino acid motif of the polypeptide includes a portion or multiple portions of a functional motif of β-laminin; The pH regulator used for adjusting the pH is sodium carbonate, the mass ratio of the polypeptide to the pH regulator is 5-8:0.15-0.17, and the pH of the solution is 7.4-8 when the polypeptide is completely dissolved.

6. The preparation method according to claim 4, characterized in that In step S3, the mass fraction of the peroxisome in the hydrogel composite material for repairing acute kidney injury is 0.01-0.5 mg / mL.

7. The preparation method according to claim 4, characterized in that In step S3, the pH regulator used to adjust the pH is hydrochloric acid, the concentration of the hydrochloric acid is 0.05-0.5 mol / L, the pH when the peroxisomes are mixed is 7.0-7.4, and the pH when the self-assembly is completely gelled is 6.5-7.

0.

8. Use of the acute kidney injury repair hydrogel composite material according to any one of claims 1 to 3 in the preparation of acute kidney injury oxidative stress regulation and tissue repair materials, characterized in that: The acute kidney injury includes ischemia-reperfusion type and drug-induced type.

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