Composite hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to composite hydrogel and a preparation method thereof, and the composite hydrogel comprises a miRNA delivery unit and a hydrogel slow-release unit coating the outer side of the miRNA delivery unit. According to the composite hydrogel provided by the invention, the miRNA delivery unit is wrapped by the hydrogel slow-release layer, so that the composite hydrogel has good stability, miRNA can be protected from being degraded by nuclease, and the half-life period of the miRNA in vivo is prolonged. Through wrapping of the hydrogel, local and continuous release of the medicine is achieved, the concentration of the medicine in the kidney lesion part is increased, and the treatment effect is enhanced. The composite hydrogel provided by the invention has a remarkable treatment effect on renal fibrosis, expression of related proteins and genes such as fibrosis can be reduced, pathological injury of kidney tissues is relieved, and meanwhile, the composite hydrogel has no obvious adverse effect on functions of other organs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a composite hydrogel and a preparation method thereof. Background Art

[0002] Chronic kidney disease (CKD) is a long-term disease in which the kidneys fail to function properly. Its prevalence is extremely high and poses a serious threat to human health. Diabetes and hypertension are the two main causes of CKD, which seriously affect the quality of life of patients. The progression of CKD is characterized by the loss of renal cells and their replacement by extracellular matrix (ECM), and renal fibrosis is a common pathological feature and final manifestation of CKD. Unfortunately, CKD is an extremely complex phenomenon controlled by the interaction between different cellular components and an intricate network of signaling pathways, which together lead to the loss of renal function and the replacement of renal parenchyma by scar tissue, making treatment very tricky. Therefore, effective prevention and management of renal fibrosis is crucial for the treatment of CKD. However, the pathogenesis of renal fibrosis has not been fully elucidated, and existing therapies can only slow down the progression of the disease but cannot cure the disease.

[0003] According to existing research, common drugs for treating renal fibrosis are renin-angiotensin system (RAS) blockers, SGLT2 inhibitors, GLP-1 receptor agonists, atrasentan (endothelin-1 blocker), tolvaptan (vasopressin receptor 2 antagonist) and non-steroidal antimineralocorticoids (Finerenone), etc. These drugs can improve the progression of the disease to varying degrees, but they also have significant side effects, and long-term use will produce drug resistance, causing a large economic burden on patients.

[0004] In view of this, how to improve the therapeutic effect on renal fibrosis while reducing toxic side effects is a technical problem that urgently needs to be solved. Summary of the invention

[0005] In order to solve the problem that the treatment methods for renal fibrosis in the prior art have large toxic and side effects, the present invention provides a composite hydrogel, which has a significant therapeutic effect on renal fibrosis and has no obvious adverse effects on the functions of other organs, thereby solving the problem that the treatment methods for renal fibrosis in the prior art have large toxic and side effects.

[0006] The technical solution adopted by the present invention to solve its technical problem is: A composite hydrogel comprises a miRNA delivery unit and a hydrogel sustained-release unit coated on the outside of the miRNA delivery unit.

[0007] Optionally, the miRNA delivery unit comprises miRNA, and cationic bovine serum albumin loaded with the miRNA.

[0008] Optionally, the miRNA is miR-21a-5p inhibitor.

[0009] Optionally, the material of the hydrogel sustained-release unit is F127.

[0010] Another object of the present invention is to provide a method for preparing the composite hydrogel as described above, comprising the following steps: S1: Mix the miRNA solution with the carrier and incubate at 1-10°C to obtain a mixed solution; S2: mixing the mixed solution with the hydrogel powder, and centrifuging to obtain a composite hydrogel.

[0011] Optionally, after incubating at 4° C. for 30 minutes in step S1, a mixed solution is obtained.

[0012] Optionally, the miRNA solution is prepared as follows: after centrifuging the miRNA, DEPC water is added and shaken to dissolve to obtain the miRNA solution.

[0013] The beneficial effects of the present invention are: The composite hydrogel provided by the present invention is wrapped with a miRNA delivery unit through a hydrogel sustained-release layer, so that the composite hydrogel has good stability, can protect miRNA from degradation by nucleases, and increase its half-life in the body. Through the encapsulation of the hydrogel, local and sustained release of the drug is achieved, the concentration of the drug in the kidney lesion site is increased, and the therapeutic effect is enhanced. The composite hydrogel provided by the present invention has a significant therapeutic effect on renal fibrosis, can down-regulate the expression of related proteins and genes such as fibrosis, reduce pathological damage to renal tissue, and has no obvious adverse effects on other organ functions. The material used in the present invention has good biocompatibility, reduces the toxic and side effects of the drug, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0015] Figure 1 The physicochemical characterization and biocompatibility test results of the hydrogel: A shows the state changes of the hydrogel caused by different temperatures; B shows the injectability test of the hydrogel; C shows the injection of the hydrogel into different parts of the mouse kidney; D shows whether the hydrogel is hemolytic; E shows the live-dead fluorescence test of the biocompatibility of the hydrogel; Figure 2The method for establishing the drug delivery system and model construction is shown in Figure 1. A is a schematic diagram of the combination of hydrogel-coated cBSA / miR-21a-5pinhibitor nanocomplexes; B is a schematic diagram of establishing the UUO model; C is a schematic diagram of the injection site; and D is a flowchart of drug delivery to mice. Figure 3 The results of HK-2 cell experiments: A is the Western blot results of different indicators; B is the fluorescence results of reactive oxygen species detection in cells of different groups; C is the fluorescence probe detection results of ferrous ions in cells of different groups; D is the fluorescence results of mitochondrial membrane potential detection in cells of different groups; Figure 4 The results of mouse kidney sampling and biochemical testing: A is the photos of intact and cut kidneys of mice in the 3-day group and the 7-day group; B is the quantitative analysis of serum creatinine and urea nitrogen in mice; C is the results of Western blot of different tissue indicators; D is the pathological section staining of mice in the 3-day group and the 7-day group; Figure 5 The results of Sirius red and immunohistochemical pathological section staining of the kidneys of the mouse models in the 3-day group and the 7-day group. DETAILED DESCRIPTION

[0016] The present invention is now further described in detail. The embodiments described below are exemplary and intended to be used to explain the present invention, but cannot be understood as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without creative work based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0017] In order to solve the problem of large toxic and side effects of the treatment methods for renal fibrosis in the prior art, the present invention provides a composite hydrogel, including a miRNA delivery unit, and a hydrogel sustained-release unit coated on the outside of the miRNA delivery unit.

[0018] MicroRNA (miRNA) is a class of small non-coding RNAs consisting of 18-24 nucleotide sequences that can bind to and inhibit mRNA and play an important role in regulating cellular physiological and pathological processes. MiRNA can inhibit its target through two different mechanisms, by cutting and degrading target mRNA or by inhibiting protein translation. At present, there are more than 1,000 known human microRNAs, and microRNA controls more than 50% of mammalian protein-coding genes. Therefore, imbalance in miRNA regulation plays an important role in the occurrence of renal fibrosis. A large number of studies have shown that abnormal expression of multiple miRNAs will lead to inflammation of cells inside the kidney, ECM degradation, and cell apoptosis, and accelerate the degree of renal fibrosis through different mechanisms of action. Therefore, the present invention searches for and explores the targets and rules of miRNAs, and regulates renal cell function through miRNAs gene therapy, which has great application prospects in improving renal fibrosis.

[0019] The composite hydrogel is an injectable hydrogel, which can achieve local and sustained release of miRNA. Compared with systemic delivery, the side effects of local delivery of any nucleic acid are much lower. Despite precise injection, miRNA is water-soluble and will be quickly degraded once it enters the body, without a sustained release effect. The present invention preferably encapsulates the miRNA delivery unit in a hydrogel, which can achieve local and sustained drug delivery, improve the therapeutic effect, and reduce toxic side effects.

[0020] In order to avoid dysfunction of other organs caused by traditional drug administration in the treatment of renal fibrosis, the present invention constructs a biomaterial that can wrap the drug and accurately locate the drug so that the drug can be continuously and slowly released at the damaged site. This is undoubtedly a once-and-for-all method that will not cause dysfunction of other organs and will not weaken the efficacy of the drug due to traditional drug administration.

[0021] The composite hydrogel provided by the present invention is wrapped with a miRNA delivery unit through a hydrogel sustained-release layer, so that the composite hydrogel has good stability, can protect miRNA from degradation by nucleases, and increase its half-life in the body. Through the encapsulation of the hydrogel, local and sustained release of the drug is achieved, the concentration of the drug in the kidney lesion site is increased, and the therapeutic effect is enhanced. The composite hydrogel provided by the present invention has a significant therapeutic effect on renal fibrosis, can down-regulate the expression of related proteins and genes such as fibrosis, reduce pathological damage to renal tissue, and has no obvious adverse effects on other organ functions. The material used in the present invention has good biocompatibility, reduces the toxic and side effects of the drug, and has good application prospects.

[0022] Currently, the two most commonly used miRNA delivery systems include viral and non-viral vectors. Although viral vectors have high transfection efficiency, the safety of viral vectors remains a controversial issue. At the same time, non-viral vectors, such as liposomes and cationic polymers, have defects such as easy aggregation, high toxicity, and poor stability, which also limit their widespread use.

[0023] In order to take into account the stability and safety of the therapeutic effect, the miRNA delivery unit of the present invention preferably includes miRNA and cationic bovine serum albumin (cBSA) loaded with miRNA. The cationic bovine serum albumin (cBSA) has low cost, abundant sources, low immunogenicity, and can be directly electrostatically adsorbed with negatively charged hydrophilic miRNA to form a nanocomplex (cBSA / miRNA), thereby improving stability while ensuring safety.

[0024] The preferred miRNA of the present invention is miR-21a-5 inhibitor, which is an inhibitor of miRNA overexpression in counteracting renal fibrosis. The inhibitor is combined with cBSA and loaded onto the hydrogel to obtain a hydrogel-encapsulated cBSA / miRNA nanocomplex sustained-release system.

[0025] Compared with systemic delivery, the side effects of local delivery of any nucleic acid are much lower, but cBSA / miRNA is water-soluble. Once they enter the body, they will be quickly degraded and do not have a sustained release effect. Therefore, the present invention uses hydrogel polymer materials to load cBSA / miRNA, and utilizes the characteristics of hydrogels having three-dimensional network structures, good biocompatibility and adjustable physicochemical properties to achieve the loading and sustained release of cBSA / miRNA. The present invention can achieve local and continuous delivery of cBSA / miRNA nanocomplexes by encapsulating cBSA / miRNA nanocomplexes in hydrogels, thereby improving therapeutic effects and reducing toxic side effects.

[0026] In order to ensure the mechanical properties of the composite hydrogel, the preferred material of the hydrogel sustained-release unit of the present invention is F127.

[0027] The composite hydrogel provided by the present invention is injectable, degradable, and has good biocompatibility. It is loaded with electrostatically adsorbed cBSA / microRNA nanocomplexes. The injectable hydrogel is combined with the cBSA / microRNA nanocomplexes to realize a hybrid platform system. The sustained release of therapeutic drugs is achieved through local administration at fixed points. The hydrogel delivery system has the effects of inhibiting ferroptosis and anti-fibrosis, and can locally alleviate or reverse the effect of renal fibrosis. The present invention provides a new idea for treating renal fibrosis, avoids the mental stress and economic burden brought to patients by long-term treatment, and has good application prospects.

[0028] Another object of the present invention is to provide a method for preparing the composite hydrogel as described above, the preparation method comprising the following steps: S1: Mix the miRNA solution with the carrier and incubate at 1-10°C to obtain a mixed solution; S2: The mixed solution is mixed with the hydrogel powder and centrifuged to obtain a composite hydrogel.

[0029] The preparation method of the composite hydrogel provided by the present invention has a simple process and is easy to operate; the prepared composite hydrogel is wrapped with a hydrogel sustained-release layer to encapsulate the miRNA delivery unit, so that the composite hydrogel has good stability, can protect miRNA from degradation by nucleases, and increase its half-life in the body. Through the encapsulation of the hydrogel, the local and sustained release of the drug is achieved, the concentration of the drug in the renal lesion site is increased, and the therapeutic effect is enhanced. The composite hydrogel provided by the present invention has a significant therapeutic effect on renal fibrosis, can downregulate the expression of related proteins and genes such as fibrosis, reduce pathological damage to renal tissue, and has no obvious adverse effects on the functions of other organs. The material used in the present invention has good biocompatibility, reduces the toxic and side effects of the drug, and has good application prospects.

[0030] Furthermore, in the preferred embodiment of the present invention, step S1 is incubated at 4° C. for 30 minutes to obtain a mixed solution.

[0031] The miRNA solution of the present invention is preferably prepared according to the following method: after the miRNA is centrifuged, DEPC water is added and shaken to dissolve to obtain the miRNA solution.

[0032] Specifically, the miRNA solution can be prepared according to the following method: S101: Place the tube containing miR-21a-5p inhibitor in a desktop centrifuge and centrifuge at 4000 rpm for 2 minutes; S102: Slowly open the cap of the miR-21a-5p inhibitor tube and add enough DEPC water; S103: Cover the tube and shake to dissolve.

[0033] The present invention preferably has a usage ratio of miR-21a-5p inhibitor to DEPC water of (0.66-0.8) μg: (0.25-1) mL; and preferably has a mass ratio of miR-21a-5p inhibitor to carrier cBSA of (0.5-5): (3-45).

[0034] Preferably, the ratio of the hydrogel to the mixed solution in this step is (0.2-0.3) g: 1 mL.

[0035] The composite hydrogel provided by the present invention can be used for intervention and treatment of renal fibrosis. The method has good treatment effect and significant improvement effect.

[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0037] Example 1 This embodiment provides a method for preparing a composite hydrogel, comprising the following steps: 1. Preparation of miR-21a-5p inhibitor solution: 1) Place the tube containing 0.66 μg of miR-21a-5p inhibitor in a desktop centrifuge and centrifuge at 4000 rpm for 2 minutes; 2) Slowly open the cap of the miR-21a-5p inhibitor tube and add 0.5 mL of DEPC water; 3) Cover the tube and shake to dissolve.

[0038] 2. Preparation of cBSA / miR-21a-5p inhibitor nanocomplex: 1) Pipette 5.28 μL of cBSA solution (concentration of 2 mg / mL) and add it to the prepared miR-21a-5p inhibitor solution (concentration of 1.32 μg / mL). The mass ratio of cBSA to miR-21a-5p inhibitor is 10.56:1.32 to obtain a mixture. 2) The mixture of cBSA and miR-21a-5p inhibitor was incubated at 4°C for 30 minutes to allow the miR-21a-5p inhibitor and cBSA to undergo an electrostatic adsorption reaction and bind tightly to obtain a cBSA / miR-21a-5p inhibitor nanocomplex.

[0039] 3. Loading cBSA / miR-21a-5p inhibitor nanocomplex onto F127: 0.25 g of F127 powder was added to 1 mL of cBSA / miR-21a-5p inhibitor solution, and the mixture was shaken and centrifuged several times at 4°C to obtain F127 loaded with cBSA / miR-21a-5p inhibitor, i.e., composite hydrogel, which was stored at 4°C.

[0040] Example 2 This embodiment provides a method for preparing a composite hydrogel, comprising the following steps: 1. Preparation of miR-21a-5p inhibitor solution: 1) Place the tube containing 0.66 μg of miR-21a-5p inhibitor in a desktop centrifuge and centrifuge at 4000 rpm for 2 minutes; 2) Slowly open the cap of the miR-21a-5p inhibitor tube and add 1 mL of DEPC water; 3) Cover the tube and shake to dissolve.

[0041] 2. Preparation of cBSA / miR-21a-5p inhibitor nanocomplex: 1) Pipette 1.98 μL of cBSA solution (concentration of 2 mg / mL) and add it to the prepared miR-21a-5p inhibitor solution (concentration of 0.66 μg / mL). The mass ratio of cBSA to miR-21a-5p inhibitor is 3.96:0.66 to obtain a mixture. 2) The mixture of cBSA and miR-21a-5p inhibitor was incubated at 4°C for 30 minutes to allow the miR-21a-5p inhibitor and cBSA to undergo an electrostatic adsorption reaction and bind tightly to obtain a cBSA / miR-21a-5p inhibitor nanocomplex.

[0042] 3. Loading cBSA / miR-21a-5p inhibitor nanocomplex onto F127: 0.2 g of F127 powder was added to 1 mL of cBSA / miR-21a-5p inhibitor solution, and the mixture was shaken and centrifuged several times at 4°C to obtain F127 loaded with cBSA / miR-21a-5p inhibitor, i.e., composite hydrogel, which was stored at 4°C.

[0043] Example 3 This embodiment provides a method for preparing a composite hydrogel, comprising the following steps: 1. Preparation of miR-21a-5p inhibitor solution: 1) Place the tube containing 0.66 μg of miR-21a-5p inhibitor in a desktop centrifuge and centrifuge at 4000 rpm for 2 minutes; 2) Slowly open the cap of the miR-21a-5p inhibitor tube and add 0.25 mL of DEPC water; 3) Cover the tube and shake to dissolve.

[0044] 2. Preparation of cBSA / miR-21a-5p inhibitor nanocomplex: 1) Pipette 21.12 μL of cBSA solution (concentration of 2 mg / mL) and add it to the prepared miR-21a-5p inhibitor solution (concentration of 2.64 μg / mL). The mass ratio of cBSA to miR-21a-5p inhibitor is 42.24:2.64 to obtain a mixture. 2) The mixture of cBSA and miR-21a-5p inhibitor was incubated at 4°C for 30 minutes to allow the miR-21a-5p inhibitor and cBSA to undergo an electrostatic adsorption reaction and bind tightly to obtain a cBSA / miR-21a-5p inhibitor nanocomplex.

[0045] 3. Loading cBSA / miR-21a-5p inhibitor nanocomplex onto F127: 0.3 g of F127 powder was added to 1 mL of cBSA / miR-21a-5p inhibitor solution, and the mixture was shaken and centrifuged several times at 4°C to obtain F127 loaded with cBSA / miR-21a-5p inhibitor, i.e., composite hydrogel, which was stored at 4°C.

[0046] The following specific test examples demonstrate the mechanism research and application of the composite hydrogel in intervening in renal fibrosis, and prove the beneficial effects of the present invention.

[0047] In the application provided by the present invention, renal fibrosis is manifested as glomerular atrophy, necrosis and shedding of renal tubular epithelial cells, renal tubular dilatation, distal renal tubular dilatation and disappearance of the space within the glomerular capsule.

[0048] In the above applications, renal fibrosis is a common pathological pathway for multiple chronic kidney diseases to develop into end-stage renal failure. It is caused by abnormal tissue repair caused by repeated or long-term damage. This dynamic process involves an impaired balance between the synthesis and degradation of extracellular matrix (ECM) proteins, which can lead to significant impairment of kidney function or even loss of its function.

[0049] In the above application, the hydrogel-coated cBSA / miR-21a-5p inhibitor nanoparticle sustained-release system, i.e., the composite hydrogel, takes the composite hydrogel prepared in Example 1 as an example, the dosage is 300nM / 10g body weight, the subjects are C57 mice, the administration route is renal capsule injection, and the treatment method is efficient, safe and reasonable.

[0050] The present invention provides a method for constructing a renal fibrosis disease drug verification model. The model construction method comprises: injecting renal fibrosis disease drugs into mice after renal fibrosis disease modeling, and the method is safe and effective for the model animals without obvious side effects and complications.

[0051] The method for constructing the above-mentioned disease-related drug validation model specifically includes the following steps: 1) Select mice for renal fibrosis disease modeling; 2) While establishing the model, injecting the renal fibrosis disease drug into the mice through the renal capsule; The above-mentioned method for constructing a drug validation model for renal fibrosis diseases includes HE staining, Masson staining, Sirius red staining, immunohistochemistry staining, real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and biochemical index detection.

[0052] The present invention creates a renal fibrosis model by performing unilateral ureteral ligation (UUO) on C57 mice, and the microRNA chip technology and RT-qPCR quantitative analysis show that the expression of miRNA-21 is significantly upregulated. Exogenous miR-21a-5p inhibitor is selected, and western blot, RT-qPCR, HE staining, transmission electron microscopy, immunofluorescence and other techniques are used for identification and analysis, and miRNA-21 is determined to be the therapeutic target of the injury model; a renal fibrosis disease drug is prepared by hydrogel-coated cBSA / miR-21a-5p inhibitor nanoparticle sustained-release system to prevent, protect and improve symptoms such as renal injury in infected mice, thereby playing a protective role against renal injury caused by other reasons, and a new treatment method is proposed.

[0053] Therefore, the present invention provides a novel microRNA-loaded injectable hydrogel for studying the mechanism of intervention in renal fibrosis and its application.

[0054] The hydrogel-coated cBSA / miR-21a-5p inhibitor nanocomplex sustained-release system can improve renal fibrosis and other injuries. It is safe and effective for model animals without obvious side effects and complications.

[0055] The method for constructing a renal fibrosis disease drug validation model is as follows: injecting renal fibrosis disease drugs into the renal capsule of the modeling side kidney of mice after renal fibrosis modeling.

[0056] Its treatment includes the following steps: 1. Establish the UUO model of C57 mice to cause renal fibrosis. 2. Drugs prepared by exogenous hydrogel-coated cBSA / miR-21a-5p inhibitor nanocomplex sustained-release system.

[0057] The drug prepared by the hydrogel-coated cBSA / miR-21a-5p inhibitor nanocomplex sustained-release system was injected into the model kidney of the injured animal through the renal capsule.

[0058] 3. Analysis of each indicator: HE staining was used to detect the damage to the kidneys of the mouse model; toxicity experiments were used to detect whether the drugs had any functional damage to other organs; Masson staining was used to detect the fibrosis damage to the kidneys of the mouse model; immunohistochemical staining was used to detect the damage to the ferroptosis-related indicators, fibrosis-related indicators, and inflammation-related indicators of the mice; Western blot was used to detect the expression levels of ferroptosis-, fibrosis-, and inflammation-related proteins in the mice; biochemical indicators were used to detect the degree of damage to the kidney function of the mice (such as the detection of aspartate aminotransferase and alanine aminotransferase in the blood). The hydrogel-coated cBSA / miR-21a-5p inhibitor nanocomplex can partially reverse the above phenomenon and intervene and treat renal fibrosis.

[0059] In summary, the present invention discloses a novel microRNA-loaded injectable hydrogel for intervention in renal fibrosis, its application and model construction method, and the drug for treating renal fibrosis is prepared by F127-coated cBSA / miR-21a-5p inhibitor nanocomposite sustained-release system; the method for verifying the construction of a renal fibrosis inhibitor model includes: injecting drugs that inhibit renal fibrosis into the capsule of the mouse model kidney after modeling normal mice. The present invention found that the hydrogel-coated cBSA / miR-21a-5p inhibitor nanocomposite sustained-release system can be used to prepare anti-renal fibrosis drugs, thereby improving kidney damage, preventing and protecting chronic kidney diseases caused by inflammation and other causes. The treatment sensitivity of this method is high and the improvement effect is significant. The present invention has good application value and promotion prospects.

[0060] Experimental Example 1 Physicochemical Characterization and Biocompatibility Testing of Hydrogel 1. Test Method Two bottles of F127 hydrogel with a concentration of 25% w / v were prepared, placed at different temperatures (4°C and 37°C), and the state of F127 was observed; F127 was drawn into a syringe to observe whether it was injectable; the administration site was determined by injecting the dyed hydrogel into different parts of the mouse kidney (renal parenchyma and renal capsule); the rupture of red blood cells in the mouse was observed to determine whether the hydrogel was hemolytic; live-dead fluorescence was used to observe the coexistence of hydrogel and cells.

[0061] 2. Test Results The physicochemical characterization and biocompatibility test results of the hydrogel are shown in Figure 1 As shown: Figure 1 A shows the states of 25% w / v F127 hydrogel at 4°C and 37°C respectively. The left side is liquid at low temperature (4°C) and solid at body temperature (37°C), indicating that the hydrogel can be sucked into the syringe at low temperature and can be accurately injected at body temperature.

[0062] Figure 1 B is the injection diagram of the hydrogel in aqueous solution and on a glass slide, indicating that it has good injectability.

[0063] Figure 1 C shows injections at different locations in the kidney, with the left side showing an untreated mouse kidney, the middle showing injections at the renal parenchyma, and the right side showing injections at the renal capsule. Injections at the renal parenchyma will cause the hydrogel to spread and cause some damage, while injections at the renal capsule will retain the hydrogel at the administration site and will not cause damage to the kidney, so the administration site was determined to be the renal capsule.

[0064] Figure 1 D shows mouse red blood cells in PBS solution, water, F127 hydrogel, and F127 / miR-21a-5pinhibitor, respectively. Only the second one (water) showed hemolysis, indicating that the hydrogel will not damage mouse red blood cells and is safe.

[0065] Figure 1 E is the live-dead fluorescence image of HK-2 cells co-cultured with hydrogel. It was observed that the hydrogel can coexist with the cells and does not inhibit cell growth, indicating that the hydrogel has good biocompatibility.

[0066] Experimental Example 2: Drug delivery system establishment and model building method 1. Test Method The composite hydrogel was prepared according to the method of Example 1 to prepare a drug delivery system.

[0067] 2. Test Results Drug delivery system establishment and model building methods such as Figure 2 As shown: Figure 2 A is a schematic diagram of the combination of hydrogel-coated cBSA / miR-21a-5p inhibitor nanocomplex. Since cBSA is positively charged, it can electrostatically adsorb the negatively charged miR-21a-5p inhibitor to form a nanocomplex. The hydrogel and the nanocomplex are mixed to form a drug delivery system.

[0068] Figure 2 B is a diagram of a unilateral ureteral ligation in mice, indicating that successful surgery will lead to renal fibrosis in mice.

[0069] Figure 2 C is a diagram of the drug administration site. After the ligation was completed, the drug administration group was immediately injected into the renal capsule.

[0070] Figure 2 D is a flowchart of drug administration for mice, which illustrates the entire animal drug administration and sample collection time period.

[0071] Test Example 3, HK-2 Cell Experiment Results 1. Test Method Western blot was used to detect the quantitative changes of cell proteins through a series of routine steps including gel preparation, sample loading, electrophoresis, membrane transfer, blocking, overnight incubation with primary antibodies, incubation with secondary antibodies, and development, and finally imaging was performed using a chemiluminescence development system. Immunofluorescence was used to detect the status of cells through cell culture, confocal dishes, drug treatment, addition of fluorescent reagents, washing, and addition of culture medium, and observation under a fluorescence microscope.

[0072] 2. Test Results The results of HK-2 cell experiments are as follows Figure 3 As shown: Figure 3 A is the expression diagram of Western blot results of different indicators, which shows that TGF-β can indeed damage cells, affecting the increase of cell ferroptosis indicators, inflammatory indicators, and fibrosis indicators, and miR-21a-5p inhibitor can improve the above phenomenon.

[0073] Figure 3 B is the fluorescence result of intracellular reactive oxygen species detection in different groups. The green fluorescence in the TGF-β group increased significantly, indicating that TGF-β would lead to increased generation of reactive oxygen species (ROS) in HK-2 cells, while miR-21a-5p inhibitor could alleviate the cellular oxidative damage caused by TGF-β.

[0074] Figure 3 C is the result of the fluorescent probe detection of intracellular ferrous ions in different groups. The red fluorescence in the TGF-β group increased significantly, indicating that TGF-β can cause the accumulation of ferrous ions in HK-2 cells, and miR-21a-5p inhibitor can reduce the accumulation of ferrous ions caused by TGF-β, thereby reducing HK-2 cell damage.

[0075] Figure 3D is the fluorescence result of mitochondrial membrane potential detection in cells of different groups. In the CON group cells, the mitochondrial membrane potential is higher, so JC-1 aggregates in the mitochondrial matrix to form polymers that produce red light. When the cells are stimulated by TGF-β, their mitochondrial membrane potential decreases, and JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is a monomer that produces green fluorescence, indicating that TGF-β destroys the integrity of the mitochondrial membrane and impairs mitochondrial function. The miR-21a-5p inhibitor can alleviate the damage to mitochondria in HK-2 cells.

[0076] Test Example 4: Mouse kidney sampling and biochemical test results 1. Test Method When sampling, the mouse kidneys were photographed to observe the modeling and treatment conditions; the mouse serum was collected, and the serum creatinine and urea nitrogen levels were tested according to the instructions of the relevant kits to evaluate renal function damage; Western blot was used to detect the quantitative changes in renal tissue protein; the tissue sections were stained with hematoxylin and eosin to observe the kidney morphology, and the tissue sections were processed using a Masson trichrome staining kit to observe the degree of fibrosis.

[0077] 2. Test Results Mouse kidney sampling and biochemical test results Figure 4 As shown: Figure 4 A is a comparison of the intact and cut parts of the mouse model kidneys in the 3-day group and the 7-day group with different treatments and the untreated kidneys. In particular, the UUO model kidneys in the 7-day group were significantly larger than the normal kidneys, indicating that fibrosis was severe, and the improvement was very significant with F127 / cBSA / miR-21a-5p inhibitor.

[0078] Figure 4 B. Quantitative analysis of serum creatinine and urea nitrogen in mice showed that UUO induced severe renal damage, while miR-21a-5p inhibitor, cBSA / miR-21a-5p inhibitor, and F127 / cBSA / miR-21a-5p inhibitor improved the renal damage induced by UUO to varying degrees, among which F127 / cBSA / miR-21a-5p inhibitor had the most obvious effect.

[0079] Figure 4 C is the expression diagram of Western blot results of different indicators, indicating that UUO-induced renal fibrosis affects the increase of tissue ferroptosis indicators, inflammatory indicators, and fibrosis indicators, while the treatment effect of F127 / cBSA / miR-21a-5p inhibitor is obvious.

[0080] Figure 4D is the HE and Masson pathological section staining of mice in the 3-day group and the 7-day group. In the HE staining, glomerular atrophy, distal tubular dilatation and disappearance of the interspaces in the glomerular capsule were observed in the UUO group, UUO+F127 group and UUO+ inhibitor NC group, which was particularly obvious in the 7-day group. The condition improved significantly after treatment with F127 / cBSA / miR-21a-5p inhibitor. The blue color in Masson staining represents the degree of fibrosis, which intuitively shows that the treatment effect of F127 / cBSA / miR-21a-5p inhibitor is significant.

[0081] Test Example 5: Sirius red and immunohistochemical pathological section staining results of the kidneys of the mouse models in the 3-day group and the 7-day group 1. Test Method Collagen fibers were observed with Sirius red through sectioning, dewaxing, conventional staining and dehydration, adding Sirius red, rinsing, staining cell nuclei with hematoxylin, rinsing, sealing with neutral resin, and observation under a microscope. Immunohistochemical staining was used to determine antigens (peptides and proteins) in tissue cells through sectioning, dewaxing, conventional staining and dehydration, sodium citrate antigen retrieval, primary antibody incubation overnight, secondary antibody incubation, DAB color development, hematoxylin counterstaining, ammonia anti-blueing, rehydration, neutral resin sealing, and observation under a microscope.

[0082] 2. Test Results The results of Sirius red and immunohistochemical pathological section staining of the kidneys of the mouse models in the 3-day group and the 7-day group are as follows Figure 5 As shown: In the Sirius red staining results, red represents the degree of fibrosis. The degree of fibrosis in the 3-day group was not obvious, while in the 7-day group, the UUO group, UUO+F127 group, and UUO+ inhibitor NC group, obvious fibrosis appeared. There were fewer red areas in the F127 / cBSA / miR-21a-5p inhibitor group, indicating that F127 / cBSA / miR-21a-5p inhibitor had an inhibitory effect on renal fibrosis. In the immunohistochemical staining of TGF-β, FTH-1, and IL-6, brown represents damage. Obviously, miR-21a-5p inhibitor, cBSA / miR-21a-5p inhibitor, and F127 / cBSA / miR-21a-5p inhibitor improved the damage to varying degrees, among which F127 / cBSA / miR-21a-5p inhibitor had the best improvement effect.

[0083] Based on the above ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above description. The technical scope of the present invention is not limited to the contents of the specification, and its technical scope must be determined according to the scope of the claims.

Claims

1. A composite hydrogel, characterized in that: It comprises a miRNA delivery unit and a hydrogel sustained-release unit coated on the outside of the miRNA delivery unit.

2. The composite hydrogel according to claim 1, characterized in that The miRNA delivery unit includes miRNA and cationic bovine serum albumin loaded with the miRNA.

3. The composite hydrogel according to claim 2, characterized in that The miRNA is miR-21a-5p inhibitor.

4. The composite hydrogel according to any one of claims 1 to 3, characterized in that The material of the hydrogel sustained-release unit is F127.

5. A method for preparing the composite hydrogel according to any one of claims 1 to 4, characterized in that: The steps include: S1: Mix the miRNA solution with the carrier and incubate at 1-10°C to obtain a mixed solution; S2: mixing the mixed solution with the hydrogel powder, and centrifuging to obtain a composite hydrogel.

6. The method for preparing the composite hydrogel according to claim 5, characterized in that: Step S1: After incubation at 4°C for 30 minutes, a mixed solution is obtained.

7. The method for preparing the composite hydrogel according to claim 5, characterized in that: The miRNA solution is prepared as follows: after centrifuging the miRNA, DEPC water is added and shaken to dissolve, thereby obtaining the miRNA solution.

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