A method for preparing an extracellular matrix nanofiber / growth factor preparation and its application in liver failure treatment

Abstract

The application belongs to the technical field of regenerative medicine and biomaterials, and discloses a preparation method of an extracellular matrix nanofiber / growth factor preparation and application of the preparation in liver failure treatment. The application discloses a biological preparation, which comprises extracellular matrix nanofibers and a drug modified on the surface of the extracellular matrix nanofibers, and the drug comprises a cell growth factor. The biological preparation provided by the application can effectively inhibit inflammatory cytokine storm, can be used for repair treatment after various liver injuries such as acute liver failure, liver fibrosis, sepsis-related liver dysfunction, has good growth factor slow-release performance, and can be administered through oral administration, intravenous injection or intraperitoneal injection, and has great clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of regenerative medicine and biomedical materials technology, specifically relating to a method for preparing an extracellular matrix nanofiber / growth factor preparation and its application in the treatment of liver failure. Background Technology

[0002] Liver injury is a common pathological condition characterized by hepatocyte degeneration and necrosis, as well as significant abnormalities in liver function indicators. The main pathogenesis of liver injury is closely related to oxidative stress and excessive inflammatory responses. In clinical practice, liver injury is often induced by alcohol abuse, drug overdose (such as acetaminophen), toxin exposure, or metabolic disorders. These factors directly attack hepatocytes, leading to mitochondrial dysfunction, excessive production of reactive oxygen species, and disruption of cell membrane integrity. Subsequently, resident immune cells in the liver (such as Kupffer cells) and recruited inflammatory cells (such as macrophages and neutrophils) are activated, releasing large amounts of pro-inflammatory cytokines and chemokines. These factors mediate the subsequent infiltration and activation of more immune cells, thereby amplifying the inflammatory damage to the liver through this cascade reaction.

[0003] To date, various treatment strategies aimed at reversing liver injury have been explored clinically, including the use of antioxidants (such as N-acetylcysteine), anti-inflammatory drugs, hepatoprotective and enzyme-lowering drugs (such as silymarin and glycyrrhizic acid preparations), and artificial liver support systems. However, due to the complexity of etiology, significant individual variability, and limited efficacy for severe liver injury, the treatment effect of liver injury remains significantly limited. To further improve treatment efficiency, researchers have attempted to develop nanoparticle-based therapeutic systems. These systems are typically designed to target and deliver antioxidant enzymes, hepatocyte growth factors, or gene silencing tools (such as siRNA). When administered in vivo, they can effectively accumulate in the liver and be internalized by hepatocytes or immune cells to regulate redox balance and inflammatory responses. However, because these nanoparticles are easily and rapidly cleared by non-parenchymal liver cells (such as Kupffer cells), or because long-term microenvironmental regulation is difficult to achieve after internalization by hepatocytes, liver tissue remains continuously exposed to inflammatory damage and oxidative stress inflammation. This poses a significant challenge to reversing hepatocyte necrosis, inhibiting fibrosis progression, and promoting liver regeneration. Therefore, there is an urgent need to develop a bioactive material or multifunctional preparation that can effectively inhibit hepatocyte death induced by various factors, regulate the local inflammatory microenvironment, and promote the proliferation of endogenous hepatocytes, so as to improve the treatment efficiency in various types of liver injury. Summary of the Invention

[0004] The first aspect of the present invention is to provide a biological agent.

[0005] The second objective of this invention is to provide a method for preparing the biological agent of the first aspect of this invention.

[0006] A third aspect of the present invention aims to provide the use of the biological agent of the first aspect of the present invention in the preparation of a medicament for the prevention and / or treatment of liver injury.

[0007] The fourth aspect of this invention is to provide a drug.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a biological agent comprising extracellular matrix nanofibers and a drug modified on the surface of the extracellular matrix nanofibers; The drug includes cell growth factors.

[0009] In some embodiments of the present invention, the extracellular matrix nanofibers are derived from human or animal tissues.

[0010] In some embodiments of the present invention, the umbilical cord, skin, uterus, testis, liver, heart, spleen, kidney, lung, bone, and smooth muscle tissue are derived from humans or animals; preferably, they are derived from human umbilical cord tissue.

[0011] In some embodiments of the present invention, the extracellular matrix nanofibers are prepared by a method comprising: decellularizing human or animal tissue to obtain decellularized tissue; digesting the tissue to obtain extracellular matrix nanofibers.

[0012] In some embodiments of the present invention, the decellularization process includes reacting human or animal tissue sequentially with pancreatic enzyme solution, Triton X-100 solution and sodium deoxycholate solution to obtain decellularized tissue.

[0013] In some embodiments of the present invention, the pancreatic enzyme solution is a 0.1%-0.6% aqueous pancreatic enzyme solution; further, it is a 0.1%-0.4% aqueous pancreatic enzyme solution; and even further, it is a 0.2%-0.3% aqueous pancreatic enzyme solution.

[0014] In some embodiments of the present invention, the Triton X-100 solution is a 1%-8% aqueous pancreatic enzyme solution; further, a 2%-6% aqueous pancreatic enzyme solution; and even further, a 3%-5% aqueous pancreatic enzyme solution.

[0015] In some embodiments of the present invention, the sodium deoxycholate solution is a 1%-8% sodium deoxycholate solution; further, it is a 2%-6% sodium deoxycholate solution; and even further, it is a 2%-4% sodium deoxycholate solution.

[0016] In some embodiments of the present invention, the reaction conditions of the human or animal tissue in the pancreatic enzyme aqueous solution are: stirring at 30-40 °C for 1-6 h; further, stirring at 34-38 °C for 2-5 h; and even further, stirring at 36-38 °C for 3-5 h.

[0017] In some embodiments of the present invention, the reaction conditions of the human or animal tissue in Triton X-100 solution are: stirring at 20-30 °C for 1-6 h; further, stirring at 25-30 °C for 2-5 h; and even further, stirring at 25-28 °C for 3-5 h.

[0018] In some embodiments of the present invention, the reaction conditions of the human or animal tissue in sodium deoxycholate solution are: stirring at 20-30 °C for 1-6 h; further, stirring at 25-30 °C for 2-5 h; and even further, stirring at 25-28 °C for 3-5 h.

[0019] In some embodiments of the present invention, the decellularized tissue is sterilized, dried, and pulverized before digestion.

[0020] In some embodiments of the present invention, conventional methods in the art can be used to achieve sterilization. This includes using peracetic acid solution to sterilize and disinfect the decellularized tissue.

[0021] In some embodiments of the present invention, drying can be carried out using conventional methods in the art, such as oven drying, vacuum drying, freeze drying, critical point drying, etc., with freeze drying being the preferred method.

[0022] In some embodiments of the present invention, conventional methods in the art can be used to pulverize decellularized tissues, such as grinding and vacuum crushing.

[0023] In some embodiments of the invention, the digestion includes digestion using a pepsin hydrochloric acid solution.

[0024] In some embodiments of the present invention, the concentration of the pepsin hydrochloric acid solution is 0.1-10 mg / mL; further, 0.5-5 mg / mL; and even further, 0.5-2 mg / mL.

[0025] In some embodiments of the present invention, the concentration of the decellularized tissue in the digestion system is 0.1-10 mg / mL; further, 0.5-5 mg / mL; and even further, 0.5-2 mg / mL.

[0026] In some embodiments of the present invention, the digestion time is 1-5 days; further, 1-4 days; and even further, 2-4 days.

[0027] In some embodiments of the present invention, after the digestion is completed, the pH value of the system is adjusted to neutral to obtain extracellular matrix nanofibers.

[0028] In some embodiments of the present invention, the growth factor may include at least one of hepatocyte growth factor (HGF), epidermal growth factor (EGF), heparin-bound EGF-like growth factor (HB-EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1), liver regeneration enhancer (ALR), and transforming growth factor-α (TGF-α); preferably hepatocyte growth factor (HGF) or epidermal growth factor (EGF).

[0029] In some embodiments of the present invention, the growth factor can be transferred to the extracellular matrix nanofibers through intermolecular interactions.

[0030] The biological agents provided by this invention can not only rebuild the organ microenvironment and promote cell proliferation, but also be used to control infection, improve organ function and inflammatory imbalance.

[0031] A second aspect of the present invention provides a method for preparing the biological agent of the first aspect of the present invention, comprising the following steps: Extracellular matrix nanofiber solution was mixed with growth factors and cross-linked to obtain a biological agent.

[0032] In some embodiments of the present invention, the crosslinking reaction includes physical crosslinking, chemical crosslinking, or enzymatic crosslinking.

[0033] In some embodiments of the present invention, the physical cross-linking includes ionic interactions, hydrogen bonding, hydrophobic interactions, electrostatic interactions, or a combination thereof; more preferably, the collagen in the extracellular matrix nanofiber solution undergoes self-assembly and fibrosis under near-neutral conditions induced by temperature, and forms a three-dimensional collagen fiber network through hydrogen bonding, hydrophobic interactions, electrostatic interactions, van der Waals forces, and physical entanglement between fibers; more preferably, the physical cross-linking is carried out at 35-39 °C and pH 7.0-7.6.

[0034] In some embodiments of the present invention, the chemical crosslinking agent used in the chemical crosslinking includes at least one of EDC / NHS, glutaraldehyde, genipin, dialdehyde starch, proanthocyanidins, polyethylene glycol diacrylate, imine ester, maleimide, and ethylene glycol dimethacrylate.

[0035] In some embodiments of the present invention, during the chemical crosslinking, the concentration of the chemical crosslinking agent in the reaction system is 0.01-1000 mM.

[0036] In some embodiments of the present invention, the cross-linking agent used in the enzyme cross-linking includes at least one of transglutaminase (mTG or MTG), horseradish peroxidase (HRP), and coagulation factor XIII (FXIII).

[0037] In some embodiments of the present invention, during enzyme crosslinking, the concentration of the crosslinking agent in the reaction system is 0.01-100 U / mL.

[0038] In some embodiments of the present invention, in the system of the extracellular matrix nanofiber solution mixed with growth factors, the concentration of the extracellular matrix nanofibers is 0.01-100 mg / mL; further, 1-50 mg / mL; even further, 3-15, such as any value or a range formed by any combination of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg / mL.

[0039] In some embodiments of the present invention, in the system of the mixture of the extracellular matrix nanofiber solution and the growth factor, the concentration of the growth factor is 0.01-10000 ng / mL; further, 10-1000 ng / mL; and even further, 10-200 ng / mL.

[0040] In some embodiments of the present invention, in the system of the extracellular matrix nanofiber solution mixed with growth factors, the concentration of the hepatocyte growth factor is 10-200 ng / mL; further, 20-150 ng / mL; even further, 50-150 ng / mL, such as any value or a range formed by any two of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 ng / mL.

[0041] In some embodiments of the present invention, in the system of the extracellular matrix nanofiber solution mixed with growth factors, the concentration of the epidermal growth factor is 10-250 ng / mL; further, 50-200 ng / mL; even further, 70-150 ng / mL, such as any value or a range formed by any combination of 70, 80, 90, 100, 110, 120, 130, 140 or 150 ng / mL.

[0042] The biological agents provided by this invention have a simple preparation process, are widely available, have easily controllable experimental conditions, and are simple and easy to process and store, thus having certain clinical application value.

[0043] A third aspect of the invention provides the use of the biological agent of the first aspect of the invention in the preparation of a medicament for the prevention and / or treatment of liver injury.

[0044] In some embodiments of the present invention, the liver injury includes at least one of acute liver injury, acute liver failure, ischemia-reperfusion liver injury, sepsis-related liver dysfunction, alcoholic liver injury, non-alcoholic fatty liver disease, liver fibrosis, and cirrhosis with liver failure.

[0045] A fourth aspect of the present invention provides a medicament comprising the biological agent of the first aspect of the present invention.

[0046] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0047] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one selected from fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0048] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose, etc.; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin, or polyvinylpyrrolidone, etc.; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micronized silica gel, talc, or magnesium stearate; and the suspending agent is selected from micronized silica gel, beeswax, cellulose, or solid polyethylene glycol. The wetting agent is selected from glycerin, Tween-80, hydrogenated castor oil, or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, or vegetable oil, wherein the vegetable oil is selected from soybean oil, castor oil, peanut oil, blended oil, etc.; the surfactant is selected from sodium dodecylbenzenesulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan, or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin; the preservative is selected from at least one of methylparaben or propylparaben.

[0049] In some embodiments of the present invention, the dosage form of the drug includes a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0050] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of pills, powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0051] In some embodiments of the present invention, the gastrointestinal dosage forms include, but are not limited to, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratch tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, and oral patches.

[0052] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage form includes at least one of injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form, and cavity dosage form.

[0053] In some embodiments of the present invention, the injectable dosage forms include, but are not limited to, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, water injections, emulsions for injection, powder injections, injections, sterile powder injections, lyophilized powder injections, etc.

[0054] The beneficial effects of this invention are: The biological agent (i.e., extracellular matrix nanofiber / growth factor preparation) provided by this invention can effectively inhibit inflammatory cytokine storms and can be used for the repair treatment of various liver injuries, such as acute liver failure, liver fibrosis, and sepsis-related liver dysfunction. This biological agent has excellent growth factor sustained-release properties and can be administered orally, intravenously, or intraperitoneally, showing great promise for clinical application.

[0055] The method for preparing extracellular matrix nanofiber / growth factor formulations provided by this invention is simple, uses widely available raw materials, and has easily controllable experimental conditions. Attached Figure Description

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Transmission electron microscopy (TEM) images of extracellular matrix nanofibers / growth factor formulations derived from human umbilical cord. From left to right, these are TEM images of the extracellular matrix, HGF / EGF, HGF / EGF particles uniformly distributed on the extracellular matrix nanofibers, and magnified TEM images of their local areas. The scale bars from left to right are 500 nm, 50 nm, 500 nm, and 200 nm.

[0057] Figure 2 Laser scanning confocal images of extracellular matrix nanofibers / growth factor formulations derived from human umbilical cord, from left to right: Cy5-dECM, FITC-HGF, Cy7-EGF, and Merge. Both growth factors show uniform adhesion on the surface of dECM nanofibers. Scale bar: 5 μm.

[0058] Figure 3The release results of HGF and EGF from human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations at different time points.

[0059] Figure 4 Results of in vitro hemolysis experiments on human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations at different concentrations.

[0060] Figure 5 In vivo fluorescence imaging (a) and statistical results (b) of mice injected with human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations at different time points. In the figures, express p <0.05, express p <0.01, express p <0.001.

[0061] Figure 6 The survival curves and mortality results of mice with acute liver failure in different treatment groups suggest that the extracellular matrix nanofiber / growth factor formulation in Example 1 can significantly improve the survival rate of mice with acute liver failure.

[0062] Figure 7 The figures show the serum ALT and AST levels in mice with acute liver failure from different treatment groups. In the figure, ns indicates no significant difference. express p <0.05, express p <0.01.

[0063] Figure 8 The figures show the detection results of IL-1, IL-6, and TNF-α in tissues of mice with acute liver failure in different treatment groups. In the figure, ns indicates no significant difference. express p <0.05, express p <0.01, express p <0.001. Detailed Implementation

[0064] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0065] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0066] With the approval of the Ethics Committee of Nanfang Hospital of Southern Medical University, this invention collects umbilical cord tissue that meets the inclusion criteria from the Department of Obstetrics and Gynecology of Nanfang Hospital after obtaining full knowledge and written informed consent from the pregnant woman and her family.

[0067] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0068] Example 1: Preparation of human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations This embodiment provides a method for preparing an extracellular matrix nanofiber / growth factor formulation derived from human umbilical cord, comprising the following steps: Human umbilical cord tissue was cut into 1 mm thick slices. 100 g of umbilical cord tissue was sequentially stirred in 1000 mL of 0.25% trypsin in PBS at 37 °C for 4 h, then in 4% Triton X-100 in PBS at 27 °C for 4 h, and finally in 3% sodium deoxycholate in PBS at 27 °C for 4 h to decellularize the tissue. Unless otherwise specified, all percentage concentrations of the solutions mentioned herein are weight-volume percentages (w / v). The decellularized tissue was sterilized by stirring in 1000 mL of 0.1% peracetic acid solution at 27 °C for 4 h. The resulting sample was then pre-frozen and freeze-dried. The pre-freezing temperature was [temperature missing]. The freeze-drying temperature was 80 °C, and the pre-freezing time was 12 h; the vacuum degree of the freeze-drying was 1 Pa, and the cold trap temperature was [missing information]. The extracellular matrix was freeze-dried at 40 °C for 24 h. The freeze-dried extracellular matrix was then placed in a grinding tank and ground into powder. The powder was then passed through a 100-mesh sieve and collected. 100 mg of extracellular matrix powder was added to 10 mL of 1 mg / mL pepsin hydrochloric acid solution and digested at 27 °C for 3 days. The digested extracellular matrix solution was neutralized to neutral. 1 μg of hepatocyte growth factor (HGF) and 1 μg of epidermal growth factor (EGF) were added to 10 mL of the neutralized extracellular matrix solution (i.e., extracellular matrix nanofiber solution). The mixture was sheared at 37 °C for 300 rpm for 3 h. Finally, it was centrifuged at 12000 rpm for 15 min and washed 3 times with PBS to obtain the extracellular matrix nanofiber / growth factor preparation.

[0069] Example 2 This embodiment characterizes the human umbilical cord-derived extracellular matrix nanofibers / growth factor formulation prepared in Example 1, as follows: The extracellular matrix nanofiber / growth factor formulation was characterized using transmission electron microscopy, and the results are as follows: Figure 1 As shown, under transmission electron microscopy, the extracellular matrix nanofiber / growth factor preparation derived from human umbilical cord exhibits a single dispersed fiber structure, with HGF / EGF particle size of approximately 4.141±1.040 nm. HGF / EGF is uniformly adhered to the extracellular matrix nanofiber.

[0070] Based on the preparation method in Example 1, extracellular matrix nanofibers were further covalently labeled with NHS-Cy5-Ester fluorescent dye. Simultaneously, HGF and EGF were labeled using FITC and NHS-Cy7-Ester, respectively. For each desired dispersion (concentration identical to the original formulation, e.g., 10 mg / mL), 10 μL of fluorescent dye (pre-prepared stock solution using the supplier's recommended solvent, purchased from Xi'an Ruixi Biotechnology Co., Ltd., catalog numbers R-FF-0025; RH-5101; RH-7352) was added, followed by reaction at 4 ℃ in the dark for 12 h (overnight). After the reaction, the reaction solution was transferred to a 3500 Da molecular weight cutoff dialysis bag and dialyzed with PBS at 4 ℃ to remove unreacted free fluorescent molecules (the dialysate was changed periodically until no obvious fluorescence signal was observed in the external solution). After dialysis, the sample in the bag was collected and stored at 4 ℃ in the dark for later use, yielding a fluorescently labeled extracellular matrix nanofiber / growth factor formulation. The distribution of fluorescent markers in this extracellular matrix nanofiber / growth factor formulation was detected using laser scanning confocal microscopy. The results are as follows: Figure 2 As shown, the two growth factors are uniformly adhered to the extracellular matrix nanofibers.

[0071] Example 3 This example is used to investigate the encapsulation efficiency, drug release, and safety of the human umbilical cord-derived extracellular matrix nanofiber / growth factor formulation prepared in Example 1, as detailed below: (1) Encapsulation efficiency of HGF and EGF In preparing the human umbilical cord-derived extracellular matrix nanofibers / growth factor formulation in Example 1, after centrifugation, the amount of HGF / EGF in the supernatant was measured using an ELISA kit (Elabscience, catalog number E-EL-H0084; E-EL-H0059), and the encapsulation efficiency of HGF / EGF was calculated. The encapsulation efficiency was calculated using the formula: Encapsulation efficiency EE (%) = (1 - Amount of drug in supernatant / Total amount of drug added) × 100%. Wherein, "Amount of drug in supernatant" was obtained by multiplying the supernatant concentration measured by ELISA by the supernatant volume; "Total amount of drug added" was the total amount of HGF / EGF added to the system. The results showed that the encapsulation efficiencies of HGF and EGF were 22.70 ± 0.36% and 27.23 ± 1.61%, respectively.

[0072] (2) In vitro drug release To evaluate the release behavior of HGF and EGF under in vitro conditions, PBS was used to simulate a physiological environment. One mL of human umbilical cord-derived extracellular matrix nanofibers / growth factor formulation was placed in a 3500 Da molecular weight cutoff dialysis bag (pre-hydrated according to the instructions), sealed, and placed in a centrifuge tube containing 50 mL of 0.1 M PBS (pH 7.4). Dialysis was performed at room temperature with gentle shaking (100-150 rpm). At preset time points (0.5, 3, 6, 12, 24, 48, 72 h, etc.), 1 mL samples were taken from the dialysate, and an equal volume of fresh PBS was immediately added to maintain the flow conditions. The concentrations of HGF and EGF in the collected dialysate were determined using an ELISA kit, and the cumulative release was corrected based on the sample volume and the cumulative replenishment volume to calculate the cumulative release percentage at each time point. In vitro drug release experiments showed that both hepatocyte growth factor (HGF) and epidermal growth factor (EGF) exhibited sustained release characteristics. Figure 3 ).

[0073] (3) Safety Hemolysis tests were conducted using extracellular matrix nanofiber / growth factor preparations at concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL. The specific experimental procedure was as follows: Fresh anticoagulated rabbit blood was washed three times with physiological saline to prepare a 2% (v / v) erythrocyte suspension. The experiment consisted of a positive control (deionized water), a negative control (physiological saline), and an extracellular matrix nanofiber / growth factor preparation group, with final concentrations of 0.5, 1, 2, 4, 5, 10, 15, and 20 mg / mL (prepared with physiological saline), respectively. 200 μL of each concentration of sample or control solution was added to a 1.5 mL centrifuge tube, followed by 200 μL of 2% erythrocyte suspension. After gentle mixing, the mixture was incubated at 37°C for 60 min. After incubation, centrifuge at 1000 rpm for 5 min, and transfer 100 μL of supernatant to a 96-well plate. Measure the absorbance (OD value) at 540 nm using a microplate reader. Calculate the hemolysis rate for each group using the formula: Hemolysis rate (%) = (OD experimental group - OD negative control) / (OD positive control - OD negative control) × 100%. Each group has 3 replicates, and the experiment is repeated 3 times. Result interpretation criteria: Hemolysis rate <5% is considered no hemolysis or mild hemolysis, meeting the blood compatibility requirements of the biomaterial; hemolysis rate between 5% and 25% indicates moderate hemolysis; hemolysis rate greater than 25% indicates severe hemolysis, and the material does not have acceptable blood compatibility.

[0074] The results showed that the human umbilical cord-derived extracellular matrix nanofiber / growth factor formulation did not induce hemolytic reactions at any of the tested concentrations. Figure 4 ).

[0075] Example 4 This embodiment investigates the role of human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations in hepatocyte injury, as detailed below: Human umbilical cord-derived extracellular matrix nanofibers / growth factor preparations were synthesized according to Example 1 and added to the hepatocyte APAP injury model culture system. Specifically, HL-7702 cells in logarithmic growth phase were seeded in 96-well plates at a cell density of 5 × 10⁶ cells / well. 3Cells were incubated at 37 °C for 12 h, and then an acute cell injury model was constructed. 100 μL of complete culture medium containing 10 mM APAP was added to each well. A control group was incubated with 100 μL of complete culture medium. Incubation continued for 24 h. Then, different treatment groups were added, with 5 wells per group. After discarding the supernatant, 100 μL of complete culture medium containing 10 μL PBS, 1 mg / mL ECM, 0.5 mg / mL extracellular matrix nanofibers / growth factor preparation, 1 mg / mL extracellular matrix nanofibers / growth factor preparation, and 2 mg / mL extracellular matrix nanofibers / growth factor preparation were added respectively. Incubation continued for 24 h. Finally, cell viability was assessed using CCK-8 assay. The old culture medium was discarded, and 100 μL of pre-prepared serum-free culture medium containing 10% CCK-8 solution was added. Cells were transferred to an incubator and incubated for another 1 h. The multi-sensor microplate reader was preheated to 37 °C, and the cells were incubated at 450 °C. The absorbance of the 96-well plate was measured at nm, the OD value was recorded, and the cell viability was calculated according to the formula.

[0076] The test results showed that the proliferation rate of hepatocytes increased by 31.6% at 48 h and by 81.4% at 72 h, indicating that the human umbilical cord-derived extracellular matrix nanofiber / growth factor preparation prepared in Example 1 has a significant effect on promoting hepatocyte proliferation and maintaining function.

[0077] Example 5 This embodiment uses an in vivo imaging system (IVIS) to examine the in vivo distribution of the human umbilical cord-derived extracellular matrix nanofibers / growth factor formulation prepared in Example 1, as detailed below: Based on the preparation method in Example 1, extracellular matrix nanofibers were covalently labeled with NHS-Cy7 fluorescent dye to obtain the labeled extracellular matrix nanofiber / growth factor formulation. 200 μL of the 10 mg / mL extracellular matrix nanofiber / growth factor formulation was injected intraperitoneally into C57 mice, while free NHS-Cy7 fluorescent dye was simultaneously injected as a control group. IVIS imaging was performed at different time points (0.5, 6, 12, 24, 48, 72, and 96 h).

[0078] IVIS imaging showed that human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations were effectively accumulated in the liver. Unlike free NHS-Cy7, which is completely metabolized within 24 hours, human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations exhibited stronger liver accumulation capacity, and their fluorescence signals could be detected up to 96 hours after administration. Figure 5 ).

[0079] Example 6 This embodiment was used to investigate the therapeutic effect of the human umbilical cord-derived extracellular matrix nanofibers / growth factor formulation prepared in Example 1 on acute liver failure in mice, as detailed below: Establishment of an acute liver failure mouse model: Eight-week-old female C57BL / 6 mice (weighing 16-18 g) were housed in a laminar flow chamber in a light-dark cycle environment for 12 h. All feed, water, bedding, and cages were autoclaved. An acute liver failure (ALF) model was established by intraperitoneal injection of 600 mg / kg acetaminophen (APAP). All mice were randomly divided into 5 groups (n=20 per group): Group 1 consisted of untreated female mice as a negative control; 2 h after modeling, Groups 2 through 5 were intravenously injected with 200 μL of 0.9% (w / v) physiological saline (negative control), 3 mg of human umbilical cord-derived extracellular matrix nanofibers, 200 μL of 1 μg / mL HGF / EGF, and 3 mg of human umbilical cord-derived extracellular matrix nanofibers / growth factor preparation, respectively. The animals were further divided into two subgroups: the survival of the first 8 mice in each group was observed every 0.5 h; the other 12 mice in each group were sacrificed at 1, 6, 24 and 48 h after injection, and serum and sections of major organs were collected and fixed in 10% neutral buffered formalin. Their liver and kidney functions were compared with those of healthy control mice.

[0080] The results are as follows Figures 6-8 As shown.

[0081] The results showed that in a mouse model of acute liver failure treated with human umbilical cord-derived extracellular matrix nanofibers / growth factor preparations, the survival rate was significantly better than other treatment groups. The survival rates were ranked as follows: PBS control group (100%) > treatment group (71.4%) > HGF / EGF group (42.9%) > ECM nanofiber group (28.6%) > APAP model group (0%). Figure 6 Serum ALT and AST levels (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., catalog numbers: C009-1-1; C010-2-1) showed that the levels of these indicators were the lowest among all groups in the human umbilical cord-derived extracellular matrix nanofiber / growth factor formulation group. Both indicators showed an increase within the first 6 hours but returned to normal within 48 hours. Figure 7In the APAP model group, the levels of pro-inflammatory cytokines (including IL-1, IL-6, and TNF-α; Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog numbers: ml106733, ml063159, ml002095) in liver tissue were significantly increased. In contrast, the levels of pro-inflammatory cytokines (IL-1, IL-6, and TNF-α) in the human umbilical cord-derived extracellular matrix nanofiber / growth factor preparation treatment group showed a brief increase at the beginning of treatment (within 6 hours) followed by a sustained decrease. Figure 8 These results clearly demonstrate that human umbilical cord-derived extracellular matrix nanofibers / growth factor formulations can effectively inhibit inflammatory cytokine storms, thereby significantly improving the survival rate of mice with acute liver failure.

[0082] Example 7 To further illustrate the effect of different extracellular matrix concentrations on the biological properties of the resulting formulation, this example modifies the extracellular matrix concentration based on Example 1 and compares the therapeutic effects of the resulting formulation.

[0083] The difference between this embodiment and Example 1 is that: after digestion and neutralization, the extracellular matrix solution was diluted with PBS to adjust the final concentration of the extracellular matrix in the preparation to 3 mg / mL, and then growth factors were loaded; the remaining preparation conditions were the same as in Example 1.

[0084] The therapeutic effects of the human umbilical cord-derived extracellular matrix nanofibers / growth factor preparations obtained in Example 1 and this Example were verified in a mouse model of acute liver failure. The experimental procedure was the same as in Example 6.

[0085] The results showed that the survival rate of mice treated with the formulation obtained in Example 1 was 71.4%, while the survival rate of mice treated with the formulation obtained in this example was 52.8%. These results indicate that, compared with this example, the human umbilical cord-derived extracellular matrix nanofiber / growth factor formulation obtained in Example 1 has superior efficacy in the treatment of acute liver failure.

[0086] Example 8 To further illustrate the effect of different decellularization conditions on the biological properties of the resulting formulation, this example modifies the decellularization treatment conditions based on Example 1 and compares the therapeutic effects of the resulting formulation.

[0087] The difference between this embodiment and Example 1 is that the decellularization conditions are changed. Specifically, human umbilical cord tissue sections are placed in 0.1 M PBS solution and stirred at 4°C and 120 rpm for 48 hours, with the solution changed 3-5 times during this period; then the tissue is placed in 0.02% trypsin solution and 0.05% EDTA solution and stirred at 37°C and 120 rpm for 2 hours; after rinsing the tissue with deionized water again, it is placed in 0.1% peracetic acid solution for sterilization and then freeze-dried; the remaining preparation conditions are the same as in Example 1.

[0088] The therapeutic effects of the human umbilical cord-derived extracellular matrix nanofibers / growth factor preparations obtained in Example 1 and this Example were verified in a mouse model of acute liver failure. The experimental procedure was the same as in Example 6.

[0089] The results showed that the survival rate of mice treated with the formulation obtained in Example 1 was 71.4%, while the survival rate of mice treated with the formulation obtained in this example was 38.6%. These results indicate that, compared with Example 3, the decellularization preparation conditions described in Example 1 are more conducive to obtaining a human umbilical cord-derived extracellular matrix nanofiber / growth factor formulation with therapeutic effects for acute liver failure.

[0090] Example 9 To further illustrate the effect of different growth factor dosages on the biological properties of the resulting formulation, this embodiment modifies the dosage of growth factors based on Example 1 and compares the therapeutic effects of the resulting formulation.

[0091] The difference between this embodiment and Example 1 is that 0.2 μg of hepatocyte growth factor (HGF) and 0.5 μg of epidermal growth factor (EGF) were added to 10 mL of neutralized extracellular matrix solution at 10 mg / mL, and the mixture was sheared at 4 °C to prepare an extracellular matrix nanofiber / growth factor formulation; the remaining preparation conditions were the same as in Example 1.

[0092] The therapeutic effects of the human umbilical cord-derived extracellular matrix nanofibers / growth factor preparations obtained in Examples 1 and 6 were verified in a mouse model of acute liver failure. The experimental procedure was the same as in Example 6.

[0093] The results showed that the survival rate of mice treated with the formulation obtained in Example 1 was 71.4%, while the survival rate of mice treated with the formulation obtained in this example was 45.6%. These results indicate that, compared with this example, the human umbilical cord-derived extracellular matrix nanofiber / growth factor formulation obtained in Example 1 has superior efficacy in the treatment of acute liver failure.

[0094] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A biological agent comprising extracellular matrix nanofibers and a drug modified on the surface of the extracellular matrix nanofibers; The drug includes cell growth factors.

2. The biological agent according to claim 1, characterized in that, The extracellular matrix nanofibers are derived from human or animal tissues; Preferably, the tissues are derived from human or animal umbilical cord, skin, uterus, testis, liver, heart, spleen, kidney, lung, bone, or smooth muscle tissue.

3. The biological agent according to claim 2, characterized in that, The extracellular matrix nanofibers are prepared by the following method: decellularizing human or animal tissue to obtain decellularized tissue; digesting the tissue to obtain extracellular matrix nanofibers. Preferably, the decellularization process involves reacting human or animal tissue sequentially with pancreatic enzyme solution, Triton X-100 solution, and sodium deoxycholate solution to obtain decellularized tissue; Preferably, the digestion includes digestion using a pepsin hydrochloric acid solution.

4. The biological agent according to any one of claims 1-3, characterized in that, The cytokines include at least one of hepatocyte growth factor, epidermal growth factor, heparin-bound EGF-like growth factor, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, liver regeneration enhancer, and transforming growth factor-α.

5. A method for preparing the biological agent according to any one of claims 1-4, comprising the following steps: Extracellular matrix nanofiber solution was mixed with growth factors and cross-linked to obtain a biological agent.

6. The preparation method according to claim 5, characterized in that, The cross-linking reaction includes physical cross-linking, chemical cross-linking, or enzymatic cross-linking; Preferably, the physical cross-linking is achieved through ionic interactions, hydrogen bonding, hydrophobic interactions, electrostatic interactions, or a combination thereof; more preferably, the collagen in the extracellular matrix nanofiber solution undergoes self-assembly and fibrosis under near-neutral conditions induced by temperature, and forms a three-dimensional collagen fiber network through hydrogen bonds, hydrophobic interactions, electrostatic interactions, van der Waals forces, and physical entanglement between fibers; more preferably, the physical cross-linking is carried out at 35-39 °C and pH 7.0-7.

6. Preferably, the chemical crosslinking agent used in the chemical crosslinking includes at least one of EDC / NHS, glutaraldehyde, genipin, dialdehyde starch, proanthocyanidins, polyethylene glycol diacrylate, imine ester, maleimide, and ethylene glycol dimethacrylate. Preferably, the cross-linking agent used in the enzyme cross-linking process includes at least one of transglutaminase, horseradish peroxidase, and coagulation factor XIII.

7. The preparation method according to claim 5 or 6, characterized in that, In the system of mixing the extracellular matrix nanofiber solution with growth factors, the concentration of the extracellular matrix nanofibers is 0.01-100 mg / mL; And / or, in the system of the extracellular matrix nanofiber solution mixed with growth factors, the concentration of the growth factors is 0.01-10000 ng / mL.

8. Use of the biological agent according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of liver injury.

9. The application according to claim 8, characterized in that, The liver injury includes at least one of the following: acute liver injury, acute liver failure, ischemia-reperfusion liver injury, sepsis-related liver dysfunction, alcoholic liver injury, non-alcoholic fatty liver disease, liver fibrosis, and cirrhosis with liver failure.

10. A drug comprising the biological agent according to any one of claims 1-4.

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