Preparation method of biomimetic nanoscale enzyme fiber and application thereof in cell therapy

Abstract

The application belongs to the technical field of cell therapy, and particularly relates to a preparation method of a biomimetic nanoscale enzyme fiber and application thereof in cell therapy. The application provides a preparation method of a biomimetic nanoscale enzyme fiber, comprising: compounding and fiberizing an antioxidative metal nanoscale enzyme and a biodegradable polymer under a solvent condition to obtain the biomimetic nanoscale enzyme fiber; wherein the antioxidative metal nanoscale enzyme is loaded on the biomimetic nanoscale enzyme fiber. The biomimetic nanoscale enzyme fiber can be transplanted in situ in a damaged solid organ to regulate ROS homeostasis, can also synergize with a functional cell drug to enhance the effect of cell therapy, and effectively solves the problem that existing cell therapy drugs are prone to be blocked in the lung after intravenous injection or be removed by macrophages / phagocytes after transplantation.

Description

Technical Field

[0001] This application belongs to the field of cell therapy technology, and in particular relates to the preparation method of biomimetic nanoenzyme fibers and their application in cell therapy. Background Technology

[0002] Acute liver failure (ALF) is a life-threatening disease characterized by widespread hepatocellular necrosis and acute deterioration of liver function. However, currently, there is no satisfactory treatment other than liver transplantation. Excessive production and accumulation of reactive oxygen species (ROS) have been reported as an early pathological feature of ALF and are closely related to disease progression, caused by factors including induced oxidative stress, inflammatory storms, and massive hepatocellular necrosis. Therefore, early and effective elimination of ROS, prevention of irreversible hepatocellular damage, and replenishment of functional hepatocellular cells are crucial for the treatment of ALF.

[0003] While effective ROS clearance can alleviate the harsh local microenvironment, the extensive hepatocellular necrosis and loss of liver function in ALF still require functional hepatocytes for necessary compensation. However, limited donors, poor cell proliferation, and difficulties in maintaining hepatocyte function in vitro hinder its therapeutic application. Mesenchymal stem cells (MSCs) possess multi-lineage differentiation potential, self-renewal capacity, and antioxidant and anti-inflammatory functions, and can be easily isolated and expanded in vitro. Furthermore, MSCs exhibit low immune responses in clinical applications and do not raise ethical concerns. Studies have demonstrated that MSCs can be effectively induced to differentiate into functional hepatocyte-like cells (HLCs) through various cytokine phases. MSC-derived HLCs not only possess anti-inflammatory activity but also hepatocyte-specific functions. Therefore, MSC-derived HLCs show great potential as an alternative cell source for hepatocyte therapy. However, studies have shown that most implanted cells are obstructed in the lungs after intravenous injection or cleared by macrophages / phagocytes after intrasplenic transplantation. In addition, the liver microenvironment in ALF is a key consideration in cell therapy, as the inflammatory environment of cell implantation can cause necrosis and apoptosis of transplanted cells.

[0004] Therefore, there is an urgent need for a carrier or drug that can effectively eliminate ROS, has low side effects, and can be used for in situ transplantation of functional cells. Summary of the Invention

[0005] This application provides a method for preparing biomimetic nanoenzyme fibers and biomimetic nanoenzyme fibers, which can be transplanted in situ into damaged solid organs to regulate their ROS homeostasis. They can also synergistically enhance the effect of cell therapy with loaded functional cells, effectively solving the problem that existing cell therapies are easily blocked in the lungs after intravenous injection or cleared by macrophages / phagocytes after transplantation.

[0006] The first aspect of this application provides a method for preparing biomimetic nanoenzyme fibers, comprising:

[0007] Biomimetic nanoenzyme fibers are prepared by compounding and fiberizing antioxidant metal nanoenzymes and biodegradable polymers under solvent conditions; wherein the antioxidant metal nanoenzymes are loaded on the biomimetic nanoenzyme fibers.

[0008] In some embodiments, the antioxidant metal nanozyme is selected from one or more of copper-based nanozymes, gold nanozymes, silver nanozymes, and platinum nanozymes.

[0009] Preferably, the antioxidant metal nanozyme used in this application is a copper-based nanozyme (CuNZs), prepared according to the literature "Ultrasmall copper-based nanoparticles for reactive oxygen species scavenging and alleviation of inflammation-related diseases".

[0010] Although copper-based nanozymes (CuNZs) possess antioxidant capabilities, conventional intravenous injection of copper-based nanozymes often leads to suboptimal organ distribution, poor therapeutic effects, and side effects. Therefore, this application proposes to synthesize and fiberize copper-based nanozymes (CuNZs) with biodegradable polymers to prepare biomimetic antioxidant nanofibers that can be transplanted in situ and regulate ROS homeostasis, effectively addressing the shortcomings of suboptimal organ distribution of copper-based nanozymes.

[0011] In some embodiments, the biodegradable polymer is selected from one or more of polylactic acid-polyglycolic acid copolymer (PLGA), polycaprolactone (PCL), polylactic acid (PLA), and gelatin.

[0012] The solvent is selected from one or more of hexafluoroisopropanol (HFIP), dichloromethane, and tetrahydrofuran.

[0013] Preferably, the biodegradable polymer is polylactic acid-polyglycolic acid copolymer (PLGA); and the solvent is hexafluoroisopropanol (HFIP).

[0014] More specifically, in the polylactic acid-polyglycolic acid copolymer (PLGA), LA / GA = 50 / 50, and the maximum Mw of PLGA is 100,000. The polylactic acid-polyglycolic acid copolymer (PLGA) accounts for 10 wt / v% in the system.

[0015] In some embodiments, the fiberization is selected from electrospinning, 3D printing, or spin coating.

[0016] Preferably, the fiberization is performed by electrospinning; the voltage parameters for electrospinning are 13kV and the feed rate is 0.6mL / h. -1 .

[0017] Specifically, the biomimetic nanoenzyme fiber preparation method of this application includes: dispersing Cu NZs and polylactic acid-polyglycolic acid copolymer PLGA in hexafluoroisopropanol HFIP, and electrospinning them into nanofibers. The electrospun nanofibers are collected and placed under vacuum to remove residual solvent.

[0018] A second aspect of this application provides a biomimetic nanoenzyme fiber, comprising the biomimetic nanoenzyme fiber prepared by the aforementioned method.

[0019] The third aspect of this application discloses the biomimetic nanozyme fibers prepared by the aforementioned method or the application of the biomimetic nanozyme fibers in the preparation of in situ cell transplantation drugs.

[0020] The fourth aspect of this application provides an orthotopic cell transplantation drug, comprising:

[0021] Biomimetic nanoenzyme fibers and drug-loaded gels loaded with functional cells;

[0022] The biomimetic nanoenzyme fiber is the biomimetic nanoenzyme fiber prepared by the above preparation method.

[0023] In some embodiments, the functional cells are selected from one or more of functional hepatocyte-like cells, therapeutic stem cells, primary hepatocytes, and the hepatocyte line AML-12; the functional cells also include endothelial cells;

[0024] The drug-loaded gel is selected from one or more of decellularized matrix dECM hydrogel, PLGA-PEG-PLGA, natural polymer hydrogel and collagen hydrogel.

[0025] In some embodiments, the functional cells further include endothelial cells, namely, functional hepatocyte-like cells, therapeutic stem cells, primary hepatocytes, and hepatocyte line AML-12, combined with endothelial cells to obtain functional cells.

[0026] In some embodiments, the natural polymer hydrogel is a hydrogel such as hyaluronic acid.

[0027] Preferably, the functional cells are functional hepatocyte-like cells (HLCs), which are effectively differentiated from MSCs through a variety of cytokine-induced phases.

[0028] Specifically, HLCs possess liver functions similar to primary hepatocytes and exhibit anti-inflammatory and regenerative effects. Studies have shown that after hepatocyte differentiation, MSC-derived HLCs acquire hepatocyte functions, including ALB formation, cytochrome P450 enzyme activity, and urea secretion. HLCs can restore hepatic ammonia and purine metabolism, reduce transaminase and ammonia levels, and increase serum ALB levels, making them more effective than undifferentiated MSCs in treating liver diseases. HLCs also reduce the production of inflammatory factors IL-1β, IL-6, and TNF-α, alleviating inflammation in CCl4-induced ALF mice. Therefore, by inducing hADMSCs to differentiate into hepatic lines through cytokine therapy, HLCs for treating ALF can be obtained.

[0029] Preferably, the drug-loaded gel is a decellularized matrix (dECM) hydrogel, which provides an ideal 3D environment that is beneficial for enhancing the therapeutic effect of functional cells.

[0030] More preferably, the decellularized matrix hydrogel is a liver decellularized extracellular matrix (dECM) hydrogel. The liver decellularized extracellular matrix (dECM) hydrogel can provide a microenvironment similar to that of the natural liver for loaded functional cells, without adverse immune responses. However, this dECM hydrogel has relatively weak mechanical strength, and the harsh microenvironment at the injury site makes it difficult for the implanted functional cells to function effectively. This application discovers that combining biomimetic nanoenzyme fibers with the ability to regulate ROS homeostasis with a drug-loaded gel can effectively improve the therapeutic effect of functional cells in the drug-loaded gel.

[0031] The fifth aspect of this application discloses the use of the orthotopic cell transplantation drug in the preparation of drugs for treating liver diseases.

[0032] In some embodiments, the solid organ disease includes liver disease, skin trauma, or myocardial injury repair.

[0033] Preferably, the liver disease includes liver fibrosis or acute liver failure.

[0034] This application develops a biomimetic nanozyme fiber loaded with an antioxidant metal nanozyme and a biodegradable polymer. This biomimetic nanozyme fiber, after being combined with an oxidized metal nanozyme, exhibits significant ROS scavenging ability, good biocompatibility, and pro-angiogenic effects. Furthermore, the biomimetic nanozyme fiber of this application can also form a synergistic therapeutic platform with a drug-loaded gel for in-situ delivery of functional cells in solid organs, effectively treating solid organ diseases. This application discovers that the biomimetic nanozyme fiber can synergistically deliver functional cells in-situ in solid organs with a drug-loaded gel. On the one hand, the biomimetic nanozyme fiber provides mechanical support to the drug-loaded gel, enabling the latter to provide a 3D environment in the solid organ for the functional cells to exert their effects. On the other hand, the antioxidant metal nanozyme in the biomimetic nanozyme fiber can effectively scavenge ROS, alleviating the harsh local microenvironment, preventing functional cells from being interfered with by the oxidative microenvironment of the damaged organ, promoting the timely functioning of functional cells, and alleviating oxidative stress and inflammation in the early stages. The in-situ cell transplantation drug provided by this application has significant and efficient therapeutic effects on solid organ damage, manifested in reduced apoptosis and tissue necrosis, and increased cell proliferation and vascularization. Attached Figure Description

[0035] Figure 1 A schematic diagram of a composite material composed of Cu NZs@PLGA nanofibers and dECM hydrogel provided in this application embodiment, used for delivering hADMSCs-induced differentiated HLCs and treating CCl4-induced ALF. (A, B) Synthesis of Cu NZs@PLGA nanofibers, dECM hydrogel, and the composite material HLCs / Cu NZs@fiber / dECM. (C) Schematic diagram of the effects of HLCs / Cu NZs@fiber / dECM on the treatment of CCl4-induced ALF through ROS elimination, angiogenesis promotion, anti-inflammation, hepatocyte-related functions, and liver regeneration promotion;

[0036] Figure 2 Diameter distribution (A) and zeta potential (B) of Cu NZs prepared for the embodiments of this application;

[0037] Figure 3 Characterization of Cu NZs and Cu NZs@PLGA nanofibers provided in the embodiments of this application, and the effect of Cu NZs@PLGA nanofibers on H2O2, ·OH and O2· - The scavenging mechanism and ability of Cu NZs. (A) TEM image of Cu NZs (scale bar: 20 nm). (B) X-ray diffraction (XRD) pattern of Cu NZs. (C) SEM image (scale bar: 2 μm) and (D) elemental mapping image of Cu NZs@PLGA nanofibers (scale bar: 200 nm). (E) Scavenging of H2O2, ·OH and O2· by Cu NZs@PLGA nanofibers.- (F) Multiple enzyme activities of Cu NZs@PLGA nanofibers. (G) H2O2 detection mechanism. (H) UV-Vis absorption spectra of halogen products after 0-10 h of treatment with Cu NZs@PLGA nanofibers. (I) H2O2 scavenging rate of Cu NZs@PLGA nanofibers at 0, 2, 6, and 10 h. (J) ·OH detection mechanism. (K) UV-Vis absorption spectra of oxTMB after 0-12 h of treatment with Cu NZs@PLGA nanofibers. (L) ·OH scavenging rate of Cu NZs@PLGA nanofibers at 0, 2, 6, and 12 h. (M) O2· - Detection mechanism. UV-Vis absorption spectra of formazan after 0-12 h of treatment with (N)Cu NZs@PLGA nanofibers. O2·⁻¹ of (O)Cu NZs@PLGA nanofibers at 0, 2, 6, and 12 h. - Scavenging rate (in GO using 150 ng / mL of an equivalent Cu NZs concentration) -1 Cu NZs@PLGA nanofibers), all data are expressed as mean ± standard deviation (n = 3);

[0038] Figure 4 SEM(A) of PLGA nanofibers Figure 3 Diameter distribution of PLGA nanofibers (B) and Cu NZs@PLGA nanofibers (C);

[0039] Figure 5 for Figure 3 Results of the scavenging ability of Cu NZs for H2O2. (A) Color changes of Amplex Red and horseradish peroxidase (HRP) reaction mixture caused by 1 mM H2O2 Cu NZs (from left to right: 0, 10, 25, 50, 100, 150, 200 ng / mL) -1 (B) UV-Vis absorption spectra of halogenated products after incubation with different concentrations of Cu NZs. (C) Scavenging rate of H2O2 by different concentrations of Cu NZs, data are expressed as mean ± standard deviation (n = 3).

[0040] Figure 6 for Figure 3 Results of the H2O2 scavenging ability of Cu NZs@PLGA nanofibers. After incubation in 1mM H2O2 for 0-10 h, the equivalent Cu NZs concentration was 0 ng / mL. -1 Up to 200 ng / mL -1 The H2O2 scavenging rate of Cu NZs@PLGA nanofibers varies over time. Data are expressed as mean ± standard deviation (n = 3).

[0041] Figure 7 for Figure 3 The appearance of O2 bubbles after removing H2O2 from Cu NZs@PLGA nanofibers is shown in the image. The nanofibers were incubated with 2 mL of H2O2 at 37°C for 4 hours. The Cu NZs@PLGA nanofibers (containing an equivalent Cu NZs concentration of 0 ng / mL) were used for this purpose. -1 Up to 200 ng / mL -1 The ability to generate oxygen;

[0042] Figure 8 This application illustrates the ability of CuNZs@PLGA nanofibers to scavenge intracellular ROS. (A) Schematic diagram of the in vitro ROS scavenging activity of CuNZs@PLGA nanofibers. (B, C) Flow cytometry analysis and fluorescence intensity quantification of AML12 cells treated with CuNZs@PLGA nanofibers after H2O2 incubation, using DCFH-DA staining. The CuNZs equivalent concentration was 0 ng / mL. -1 ~150ng mL -1 (D) Survival rate of AML12 cells (CCK-8). (E) Representative fluorescence image of AML12 cells (DCFH-DA staining) (scale bar: 50 μm). (F) Schematic diagram of Cu NZs@PLGA nanofibers promoting HUVEC migration. (G) HUVEC migration images after 24 h and 48 h of incubation with Cu NZs@PLGA nanofibers (scale bar: 400 μm). (H) Schematic diagram of Cu NZs@PLGA nanofibers promoting HUVEC proliferation. (I) EdU staining image of HUVECs after 48 h of incubation with Cu NZs@PLGA nanofibers (scale bar: 100 μm). All data are expressed as mean ± standard deviation (n = 3). *P<0.05,**P<0.01,***P<0.001,****P<0.0001 indicate comparison with the control group, ns indicates no significant difference compared with the control group. # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 indicates a difference of 150 ng / mL. -1 Comparison of Cu NZs@PLGA group (Cu NZs concentration 150 ng / mL) -1 );

[0043] Figure 9 for Figure 8 Biocompatibility evaluation of Cu NZs@PLGA nanofibers with AML 12 cells (A), hADMSCs (B) and HLCs (C). Data are expressed as mean ± standard deviation (n=3), and ns indicates no significant difference compared with the control group.

[0044] Figure 10 for Figure 8 Statistical analysis of migration and proliferation of HUVECs after treatment with CuNZs@PLGA nanofibers (including equivalent CuNZs concentration of 0 ng / mL) -1 Up to 150 ng / mL -1 Quantitative statistics on HUVEC migration after 24 hours (A) and 48 hours (B) of Cu NZs@PLGA nanofiber treatment. (C) Percentage of HUVEC proliferation (EdU staining positivity rate) after 48 hours of Cu NZs@PLGA nanofiber incubation. Data are expressed as mean ± standard deviation (n=3), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the control group. ## P<0.01 and 150 ng mL -1 Group comparison;

[0045] Figure 11 Characterization of HLCs induced by hADMSCs provided in this application embodiment. (A) Schematic diagram of liver differentiation timeline. (B) Bright-field images, PAS staining, and Oil red O staining of HLCs and hADMSCs (scale bar: 100 μm). (CE) Immunofluorescence staining of ALB, HNF4α, and ABCC2 in HLCs and hADMSCs (scale bar: 100 μm). (F) Liver gene expression levels of ALB, CYP3A4, and AFP (all data normalized to day 0 values) (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicates comparison with day 0). ## P<0.01 indicates comparison with day 7. (G) ALB and urea levels in HLC culture supernatant on day 7 and day 21 (all data normalized to day 7 values) (***P<0.001, ****P<0.0001 indicates comparison with day 7), all data are expressed as mean ± standard deviation (n=3);

[0046] Figure 12 for Figure 11 Graph showing the cellular morphological changes of hADMSCs differentiating into HLCs during the liver differentiation process from day 0 to day 21.

[0047] Figure 13 for Figure 11 The results of E-Cadherin and DPP4 immunofluorescence staining of HLCs and hADMSCs are shown in the figure.

[0048] Figure 14Anti-inflammatory effect of HLCs provided in this application (THP-1 cells). (A) Schematic diagram of HLC supernatant facilitating M2 polarization of macrophages. (B) Expression levels of IL 1β, NOS2, TGFβ1, IL 10 and MRC1 in THP-1 cells after culturing HLC supernatant for 24 or 48 h (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicates comparison with THP-1 (M0); ## P<0.01, ### P<0.001 indicates comparison with "HLCs supernatant 24 h"). (C) Schematic diagram of HLCs supernatant promoting macrophage phenotypic transformation under inflammatory conditions. (D) Expression levels of IL 1β, TNFA, TGFβ1, IL 10 and MRC 1 in LPS-stimulated THP-1 (*P<0.05,**P<0.01,***P<0.001,****P<0.0001 indicates comparison with THP-1(M0), ns indicates no significant difference compared with THP-1(M0)). ### P<0.001, #### P<0.0001 indicates a difference between 100 ng / mL and 100 ng / mL. -1 Compared to LPS, all data were normalized to THP-1(M0) and expressed as mean ± standard deviation (n=3).

[0049] Figure 15 The anti-inflammatory effect of HLCs provided in this application (RAW264.7 cells). (A) Schematic diagram of HLC supernatant promoting macrophage phenotypic transformation under inflammatory conditions. (B) Expression levels of IL 1β, TNFA, NOS2 and TGFβ1 in RAW264.7 cells stimulated by LPS (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicates no significant difference compared to the "RAW264.7" group, ns indicates no significant difference relative to RAW264.7). ### P<0.001, #### P<0.0001 indicates a difference between 100 ng / mL and 100 ng / mL. -1 Compared to the "LPS" group, all data were normalized to the values ​​of the "RAW264.7" group, and all data were expressed as mean ± standard deviation (n=3).

[0050] Figure 16Results of co-culturing AML 12 cells with hADMSCs or HLCs after H2O2 treatment according to embodiments of this application; (A, B) Bright-field microscopy images (A) and cell viability (B) of AML 12 cells co-cultured with hADMSCs or HLCs after 500 μM H2O2 treatment (scale bar: 500 μM). (C) Expression of antioxidant genes SOD2, KEAP1, and NQO1 in AML 12 cells co-cultured with hADMSCs or HLCs after 500 μM H2O2 treatment. All data are expressed as mean ± standard deviation (n = 3), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicates comparison with control, ns indicates no significant difference compared with control. # P<0.05 indicates a difference compared to the HLCs@dECM group;

[0051] Figure 17 Properties and functions of the HLCs / dECM hydrogels provided in the embodiments of this application. (A) Schematic diagram of dECM preparation and HLCs encapsulated in dECM hydrogel. (B) Rheological properties of dECM (storage modulus (G'), loss modulus (G″)). (C) Scanning electron microscope image of dECM hydrogel (scale bar: 50 μm). (D) Cell / Cu Scanning electron microscopy of NZs@fiber / dECM (yellow arrow: cells in dECM) (scale bar: 30 μm). (EH) Immunofluorescence staining of ALB, HNF4α, E-Cadherin, and ABCC2 in HLCs or hADMSCs cultured in dECM hydrogel for 3 days (scale bar: 100 μm). (I) Gene expression levels of ALB, CYP3A4, and CK18. (J) ALB and urea levels in the supernatant of HLCs cultured in 2D plates or dECM hydrogels (day 3 of culture). All data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicate comparison with HLCs cultured in 2D plates.

[0052] Figure 18 Properties of dECM and preparation of Cu NZs@fiber / dECM composite material provided in this embodiment. (A) Gelation properties of dECM hydrogel (scale bar: 1 cm). (B) Cu NZs@fiber / dECM composite material (scale bar: 1 cm). (C) Biocompatibility of dECM: Calcein-AM / PI staining images of HLCs cultured in dECM hydrogel on day 1 and day 3 (scale bar: 400 μm);

[0053] Figure 19This embodiment demonstrates the cell-protective effect of Cu NZs@PLGA nanofibers on HLCs embedded in dECM hydrogel. (A) Calcein-AM / PI staining images of HLCs in dECM. Blank: No pre-incubation with Cu NZs@PLGA nanofibers, no H2O2 treatment; Control: No pre-incubation with Cu NZs@PLGA nanofibers, 500 μM H2O2 treatment; Cu NZs@fiber: Pre-incubation with Cu NZs@PLGA nanofibers (isoconcentration 150 ng / mL). -1 Cu NZs), treated with 500 μM H2O2 (scale bar: 400 μM). (B) DCF fluorescence intensity of HLCs in dECM hydrogel (DCFH-DA staining, DCF fluorescence signal detected by microplate reader), all data were normalized to the blank group and expressed as mean ± standard deviation (n = 3). ***P<0.001, ****P<0.0001 indicates comparison with the control group;

[0054] Figure 20 The therapeutic effect of HLCs / Cu NZs@fiber / dECM provided in this embodiment on CCl4-induced ALF (treatment for 1 day). (A) Schematic diagram of the animal experimental treatment protocol for CCl4-induced ALF. (B) Liver HE staining (scale bar: 400 μm). (C) Liver TUNEL staining (scale bar: 200 μm). (D) Liver Ki67 staining (scale bar: 200 μm). (E) Quantitative analysis of necrotic areas (n=5). (FG) Quantitative analysis of TUNEL and Ki67 staining (n=3). (HJ) Serum ALB, AST, and ALT levels. All data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicates comparison with the control group, and ns indicates no significant difference compared with the control. # P<0.05, ## P<0.01, ### P<0.001 indicates a comparison with the HLCs / CuNZs@fiber / dECM group;

[0055] Figure 21 for Figure 20 Liver morphology images of each group one day after treatment. The white dashed lines represent the attachment areas of hADMSCs / Cu NZs@fiber / dECM and HLCs / Cu NZs@fiber / dECM, and the delineated areas show liver injury relief (scale bar: 0.5cm).

[0056] Figure 22The inflammation level of CCl4-induced ALF mice after treatment with HLCs / Cu NZs@fiber / dECM provided in this application embodiment. (AC) Staining of liver tissue with TNF-α, IL-6, and DHE (scale bar: 200 μm). (DF) Quantitative analysis of mean fluorescence intensity (MFI) of liver tissue with TNF-α (D), IL-6 (E), and DHE (F) staining. (GL) Expression levels of TNFA, IL-6, COX2, Pro-IL 1, INOS, and IL 10 mRNA. All data are expressed as mean ± standard deviation (n = 3), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate comparison with the control group, and ns indicates no significant difference compared with the control. # P<0.05, ## P<0.01, ### P<0.001 indicates a comparison with the HLCs / Cu NZs@fiber / dECM group;

[0057] Figure 23 The therapeutic effect of HLCs / Cu NZs@fiber / dECM provided in this application on CCl4-induced ALF (treatment for 3 days). (A) Liver morphology images of each group (scale bar: 0.5 cm). (B) Liver HE staining (scale bar: 400 μm). (C) Liver TUNEL staining (scale bar: 200 μm). (D) Liver Ki67 staining (scale bar: 200 μm). (EG) Serum AST, ALT, and ALB levels. (H) Changes in mouse body weight during treatment. All data are expressed as mean ± standard deviation (n = 5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate comparison with the control group. # P<0.05, ## P<0.01 indicates a significant difference compared to the HLCs / Cu NZs@fiber / dECM group, and ns indicates no significant difference compared to the blank group;

[0058] Figure 24The inflammation levels of CCl4-induced ALF mice treated with HLCs / Cu NZs@fiber / dECM provided in this application embodiment for 3 days. (A) DHE staining of liver tissue (scale bar: 50 μm). (B) IL-6 staining of liver tissue (scale bar: 200 μm). (C) TNF-α staining of liver tissue (scale bar: 200 μm). (DF) mRNA expression levels of TNFA, COX2 and NLRP3. All data are expressed as mean ± standard deviation (n = 3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate comparison with the control group, and ns indicates no significant difference compared with the control. # P<0.05, ## P<0.01 indicates a comparison with the HLCs / Cu NZs@fiber / dECM group;

[0059] Figure 25 The migration of CD31-positive endothelial cells in the host liver of the HLCs / Cu NZs@fiber / dECM treatment group provided in this application embodiment. (A) HLCs / Cu NZs@fiber / dECM attachment area on the liver 1 day after treatment, the attachment area is outlined by a black line (left: HLCs / Cu NZs@fiber / dECM attachment, right: HLCs / Cu NZs@fiber / dECM removal) (scale bar: 1cm). (B) HE staining and CD31 / HNF 4α immunofluorescence staining (red / CD31, green / HNF 4α, blue / DAPI) of liver tissue in the HLCs / Cu NZs@fiber / dECM transplantation area. The area outlined by the dashed line is the transplanted HLCs (scale bar: 100μm);

[0060] Figure 26 In vivo toxicity evaluation of HLCs / Cu NZs@fiber / dECM provided in the embodiments of this application in major organs, HE staining of heart, lung, spleen and kidney (scale bar: 100 μm);

[0061] Figure 27Transcriptome analysis of ALF mouse liver tissue was provided for the embodiments of this application. (A) Volcano plot of differentially expressed genes (≥2-fold difference. Red: upregulated genes; blue: downregulated genes). (B) Gene enrichment KEGG pathway analysis (showing the top 20 genes). (C) a) GSEA plot: drug metabolism cytochrome P450, enrichment score (ES) = 0.61718786, normalized enrichment score (NES) = 2.2412274; nominal p value < 0.001. b) GSEA plot: p53 signaling pathway (b), ES = -0.49999398, NES = -1.620217; nominal p value < 0.05. (D) Heatmap of differentially expressed genes related to oxidative stress, inflammation (a), antioxidation, angiogenesis (b), hepatocyte markers and liver regeneration (c);

[0062] Figure 28 for Figure 27 Heatmap of key genes in the cytochrome P450 pathway of drug metabolism in China;

[0063] Figure 29 for Figure 27 Heatmap of key genes in the p53 signaling pathway;

[0064] Figure 30 for Figure 27 Heatmap of genes upregulated by the Wnt signaling pathway. Detailed Implementation

[0065] This application discloses a method for preparing biomimetic nanoenzyme fibers and their application in cell therapy, which addresses the technical defects in the prior art where cell therapy drugs are easily blocked in the lungs after intravenous injection or cleared by macrophages / phagocytes after transplantation.

[0066] In the following examples, all reagents or drugs used are commercially available or self-made.

[0067] The primers used in the following examples are shown in Table 1.

[0068] Table 1

[0069]

[0070]

[0071] The test procedure of this application embodiment is as follows: Figure 1① Synthesize copper-based nanozymes Cu NZs, ② Prepare biomimetic nanozyme fibers (Cu NZs@PLGA nanofibers), ③ Combine Cu NZs@PLGA nanofibers with dECM hydrogels loaded with HLCs to form an in situ cell transplantation drug (HLCs / Cu NZs@fiber / dECM). This in situ cell transplantation drug is used to deliver HLCs differentiated by hADMSCs for the treatment of CCl4-induced ALF. Figure 1 Synthesis of (A,B)Cu NZs@PLGA nanofibers, dECM hydrogels and composite material HLCs / Cu NZs@fiber / dECM; Figure 1 (C) Schematic diagram of the effects of HLCs / Cu NZs@fiber / dECM on CCl4-induced ALF treatment through ROS elimination, angiogenesis promotion, anti-inflammation, hepatocyte-related function enhancement, and liver regeneration promotion.

[0072] Example 1

[0073] This embodiment provides the synthesis and characterization of copper-based nanozymes (CuNZs), specifically including:

[0074] CuCl2 (7447-39-4, Aladdin, China) was dispersed in 50 mL of deionized water to a final concentration of 10 mM. After stirring at 80 °C for 10 min, 50 mL of 100 mM ascorbic acid (50-81-7, AlfaAesar, USA) aqueous solution was slowly added to the CuCl2 solution. The pH of the mixed solution was adjusted to 8.5 ± 0.5 using 1 M NaOH (1310-73-2, Macklin, China), and stirred at 80 °C for 12 h. After the reaction was complete, the precipitate was removed by centrifugation, and the supernatant was further dialyzed against deionized water for 48 h using a 10 kDa dialysis tube (TX132, Viskase, China). The final Cu NZs were obtained by centrifugation. The morphology and size of the Cu NZs were characterized by transmission electron microscopy (TEM, FEI Tecnai G2 Spirit) at 120 kV. The crystal structure and oxidation state of Cu NZs were characterized using an X-ray diffractometer (Empyrean) at a voltage of 45 kV and a current of 40 mA.

[0075] The results are as follows Figure 2 and Figure 3 As shown, Cu NZs were first characterized by transmission electron microscopy, which revealed that they were uniformly distributed with an average particle size of approximately 4.0 nm. Figure 2 A, Figure 3 A). The zeta potential of Cu NZs is approximately -14.4 mV ( Figure 2B). According to the standard XRD pattern, Cu NZs is a compound of Cu₂O and Cu, indicating the successful synthesis of Cu NZs. Figure 3 B).

[0076] Example 2

[0077] This embodiment provides a method for preparing Cu NZs@PLGA nanofibers by electrospinning:

[0078] 1. Add Cu NZs (600 μg mL) -1 ) and 10 wt / v% poly(lactic acid-coglycolic acid) (PLGA, LA / GA = 50 / 50, Mw ~ 100,000, Shenzhen Polymtek Biomaterial) were uniformly dispersed in hexafluoroisopropanol (HFIP, 99.8%, Aladdin, China) at a feed rate of 0.6 mL / h under 13 kV voltage. -1 Electrospinning was used to prepare nanofibers. The electrospun nanofibers were collected and placed in a vacuum at room temperature overnight to remove residual solvent. Ordinary PLGA nanofibers without CuNZs were prepared using the same process. The morphology of the nanofibers was characterized and analyzed by energy-dispersive X-ray spectroscopy (EDS) at 20 kV.

[0079] 2. Characterization of Cu NZs and Cu NZs@PLGA nanofibers, the results are as follows: Figure 3 and Figure 4 As shown, SEM imaging revealed that Cu NZs did not significantly alter the smooth surface and long fiber morphology of the PLGA nanofibers, with an average diameter of 382.5 ± 74.5 nm. Figure 3 C, Figure 4 EDS elemental mapping imaging revealed that C, O, and Cu were uniformly distributed in the PLGA nanofibers. Figure 3 D), further confirming the successful loading of Cu NZs in nanofibers.

[0080] Example 3

[0081] This embodiment provides Cu NZs and Cu NZs@PLGA nanofibers for their effects on H2O2, ·OH, and O2· - Its clearing capabilities specifically include:

[0082] 1. Test the ability to remove H2O2:

[0083] Different concentrations of Cu NZs were placed in phosphate buffer solution, from 0 to 200 ng / mL. -1The reagents were incubated with 1 mM H₂O₂ (CS7730, G-Clone, China) at 37 °C for 2 hours. The remaining H₂O₂ was detected by Amplex Red (Meilunbio, China) and HRP (Meilunbio, China). Amplex Red reacted with H₂O₂ to generate a halogenated reagent with an absorption peak at 571 nm. Quantification was performed using a UV-2600 UV-Vis spectrophotometer (SHIMADZU, China). A Cu- and NZs-free phosphate buffer solution served as a control.

[0084] Contains varying amounts of Cu and NZs (0-200 ng / mL) -1 Cu NZs@PLGA nanofibers were immersed in phosphate buffer solution and incubated with 1 mM H2O2 at 37°C for 0-10 h. Residual H2O2 was measured in each group at 2, 4, 6, 8, and 10 h. PLGA nanofibers without Cu NZs were used as a control. The H2O2 scavenging rate was calculated using the formula: Scavenging rate (%) = (Ac - Ai) / Ac × 100%. Ac is the absorbance of the control group, and Ai is the absorbance of the sample.

[0085] 2. Detection of the ·OH scavenging ability of Cu NZs@PLGA nanofibers:

[0086] The ·OH scavenging rate at different time points was monitored using a typical 3,3',5,5'-tetramethylbenzidine (TMB) (54827-17-7, Aladdin, China) colorimetric method. Cu NZs@PLGA nanofibers were pre-incubated in acetate / sodium acetate buffer (HAc / NaAc, 0.5 M, pH 4.5) (Aladdin, China), with a final Cu NZs concentration of 150 ng / mL. -1 The cells were incubated for 0, 2, 6, and 12 hours, respectively. The supernatant was collected and incubated in the dark for 7 minutes with a working solution containing 1 mM FeSO4 (7782-63-0, Sangon, China), 1 mM H2O2, and 250 μM TMB. HAc / NaAc buffer without nanofiber pretreatment was used as a control. The ·OH-oxidized TMB (oxTMB) was blue, and its absorbance at 652 nm was measured using a UV-Vis spectrophotometer. Based on this, the ·OH scavenging rate was calculated using the same formula as for H2O2 scavenging.

[0087] 3. Cu NZs@PLGA nanofibers remove O2· - Ability test:

[0088] The O2· of CuNZs@PLGA nanofibers was studied using the nitrotetrazole blue (NBT) colorimetric method. -Scavenging activity was assessed. CuNZs@PLGA nanofibers were immersed in Tris-HCl buffer (10 mM, pH 7.4) (Biosharp, China) to achieve a final CuNZs concentration of 150 ng / mL. -1 After pre-incubation for 0, 2, 6, and 12 hours, the supernatant was collected and thoroughly mixed with working solutions containing 6.5 mM L-methionine (C11385354, Macklin, China), 37.5 μM NBT (C11167116, Macklin, China), and 10 μM riboflavin (C11507808, Macklin, China), respectively. The mixture was then irradiated with UV light using a SCIENTZ03-II UV irradiator (Scientz, China) at a wavelength of 254 nm (60 W, 150 mm). UV irradiation caused riboflavin and L-methionine to generate O2· - The NBT was further converted to formazan. After illumination, the absorbance of the mixed solution at 560 nm was measured using a UV-Vis spectrophotometer. - The removal rate is calculated according to the H2O2 removal rate formula.

[0089] Cu NZs and Cu NZs@PLGA nanofibers resist H2O2, ·OH and O2· - The removal efficiency test results are as follows: Figure 3 and Figure 5-7 As shown, with increasing Cu NZs concentration, the UV absorption intensity at 571 nm gradually decreases and the colorimetric color fades, indicating that the scavenging efficiency of Cu NZs for H2O2 is concentration-dependent. Figure 5 It is worth noting that adding an ultra-low concentration (200 ng / mL) -1 Cu NZs can eliminate up to 83.4% of H2O2 ( Figure 5 C). When Cu NZs are encapsulated in PLGA nanofibers, the scavenging efficiency of H2O2 is unaffected and exhibits time- and concentration-dependent characteristics. Figure 6 Contains 150 ng / mL -1 and 200ng mL -1 Cu NZs@PLGA nanofibers with equivalent Cu NZs concentrations showed H2O2 removal rates of 68.1% and 75.0% after 10 h, respectively. Figure 3 GI, Figure 6 Furthermore, during the removal of H2O2, bubbles appeared on the pipe wall, indicating that H2O2 was converted into O2 by Cu NZs. Figure 7 In addition, with a Cu NZs equivalent concentration of 150 ng / mL -1Taking Cu NZs@PLGA nanofibers as an example, the effects of Cu NZs@PLGA nanofibers on ·OH and O2· were measured. - CuNZs@PLGA nanofibers can eliminate ·OH generated by the Fenton reaction. With prolonged incubation time, the UV absorption peak of oxTMB gradually decreases, indicating that CuNZs in CuNZs@PLGA nanofibers can effectively eliminate Fe. 2+ The ·OH generated by H2O2. In particular, CuNZs@PLGA nanofibers showed a near 100% ·OH removal efficiency after 12 hours of incubation. Figure 3 JL). Furthermore, the decrease in UV absorbance of the converted NBT at 560 nm also indicates that the CuNZs@PLGA nanofibers successfully eliminated O2· - At 12h, O2· - The clearance rate was approximately 48.5%. Figure 3 In summary, these results strongly demonstrate that Cu NZs@PLGA nanofibers can effectively eliminate H2O2, ·OH, and O2· - .

[0090] Example 4

[0091] This embodiment provides experiments on the ability of Cu NZs@PLGA nanofibers to scavenge intracellular ROS and their effects on the migration and proliferation of HUVECs, including:

[0092] 1. Cell culture:

[0093] hADMSCs were purchased from Cellera Stem Cell Technology Co., Ltd., China. hADMSCs were cultured in serum-free medium containing 5% serum substitute (G03010, Saliai, China). hADMSCs between passages 5 and 9 were used for experiments. AML12 cells were purchased from a nationally certified cell culture bank (China) and cultured with 10% fetal bovine serum (FBS) (Gibco, USA), 1× insulin-transferrin-selenium supplement (ITS) (ITSS-10201, Cyagen, China) and 40 ng / mL. -1Dexamethasone (D4902, Sigma-Aldrich, USA) was used in DMEM / F-12 medium (11330-032, Gibco, USA). Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM / F-12 medium containing 10% FBS and 1% penicillin antibiotics (Gibco, USA). Human monocytes THP-1 were cultured in RPMI 1640 medium (Gibco, USA), 10% FBS, 1% penicillin antibiotics, and 50 μM β-mercaptoethanol (Sigma-Aldrich, USA). RAW264.7 cells were cultured in DMEM medium (Gibco, USA), 10% heat-inactivated FBS, and 1% penicillin antibiotics. All cells were cultured in an incubator at 37°C and 5% CO2.

[0094] 2. Determination of the ability of Cu NZs@PLGA nanofibers to scavenge ROS in AML12 cells:

[0095] AML12 cells were distributed at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells / well in 48-well plates and treated with different concentrations of CuNZs@PLGA nanofibers (equivalent CuNZs concentrations from 0 ng / mL). -1 Up to 200 ng / mL -1 Cells were pre-incubated overnight, then stimulated with cell culture medium containing 500 μM H2O2 for 24 hours. Cell viability was then assessed using CCK-8 assay, and intracellular ROS were labeled using 2',7'-dichlorofluorescein diacetic acid (DCFH-DA, S0033S, Beyotime, China) according to the manufacturer's instructions. AML12 cells were incubated with 10 μM DCFH-DA staining solution in the dark at 37°C for 30 minutes. After removing the staining solution, cells were fixed with 4% paraformaldehyde (PFA, Biosharp, China) and treated with 0.2 μg / mL... -1 4,6-Diamidinyl-2-phenylindole (DAPI) was stained at room temperature for 15 min, and fluorescence imaging was performed using an inverted fluorescence microscope (Nikon, Japan). The average fluorescence intensity was quantified using a flow cytometer (Beckman, CytoFLEX, USA).

[0096] 3. Determination of the biocompatibility of Cu NZs@PLGA nanofibers:

[0097] AML12 cells, hADMSCs, and HLCs were respectively loaded at 1×10⁶ cells per well. 4 Cells were seeded at a density in 96-well plates. Cu NZs@PLGA nanofibers (equivalent Cu NZs concentration from 0 ng / mL) were then seeded. -1 Up to 200 ng / mL -1Cells were incubated for 24 hours, and cell viability was assessed using a CCK-8 assay. Cell viability was quantified by measuring absorbance at 450 nm using a BioTek microplate reader.

[0098] 4. Experiment to determine the effect of Cu NZs@PLGA nanofibers on the migration and proliferation of HUVECs:

[0099] The effect of CuNZs@PLGA nanofibers on the migration of HUVECs was investigated by scratch assay. HUVECs were distributed at a density of 5 × 10⁻⁶ nanofibers per pore. 4 Cells were seeded at a density of [insert density here] in 24-well plates and cultured overnight. Cells were then cultured in serum-free medium for 24 hours, and a straight line was drawn at the bottom of each well using the tip of a 1 mL sterile pipette. Cells were gently washed twice with phosphate-buffered saline to remove cell debris, and then seeded with nanofibers containing 1% FBS and Cu NZs@PLGA nanofibers (equivalent Cu NZs concentration from 0 ng / mL). -1 Up to 200 ng / mL -1 Cells were cultured in complete culture medium. Images of the scratched areas were taken at 24 and 48 hours, and measurements and statistics were performed using ImageJ software (NIH, USA). Cell migration statistics formula: Cell migration (%) = ((S0 - S...)) / (S0 - S0 ... t ) / S0)×100%, where S0 is the initial scratch area, S t The area of ​​the scratched area after 24 or 48 hours.

[0100] The EdU kit (K1076, APExBIO, USA) was used to evaluate the effect of Cu NZs@PLGA nanofibers on the proliferation of HUVECs. Cell treatment was the same as in the scratch assay, except that the cell seeding density per well was lower, at 1 × 10⁻⁶. 4 After treatment with Cu NZs@PLGA nanofibers for 48 h, HUVECs were stained with an EdU kit and imaged using an inverted fluorescence microscope. The proliferation of EdU-positive cells was measured using ImageJ, with HUVEC proliferation rate (%) = EdU-positive cells / total cells × 100%. EdU-positive cells were quantified in three randomly selected regions within each well.

[0101] The results are as follows Figures 8-10 As shown, cells were inoculated with Cu NZs@PLGA nanofibers (containing an equivalent Cu NZs concentration of 0 ng / mL). -1 Up to 200 ng / mL -1 After 24 hours of incubation, CCK-8 assays showed that cell viability ranged from 95.0% to 107.9% compared to the untreated control group, indicating that CuNZs@PLGA nanofibers possess good biocompatibility. Figure 9Inspired by the excellent ROS scavenging ability of Cu NZs@PLGA nanofibers, this embodiment examines the scavenging and cell-protective capabilities of Cu NZs@PLGA nanofibers against intracellular ROS. Intracellular ROS generation after DCFH-DA staining was analyzed by flow cytometry. When the Cu NZs equivalent concentration in Cu NZs@PLGA nanofibers reached 150 ng / mL... -1 At that time, the DCF fluorescence intensity after Cu NZs@PLGA nanofiber treatment decreased by 1.8 times compared with the control group. The results indicate that intracellular ROS can be significantly eliminated. Figure 8 BC), consistent with DCFH-DA staining images ( Figure 8 E). Similarly, the CCK-8 test ( Figure 8 D) It was also confirmed that, compared with the control group (cell viability 53.0%), Cu NZs@PLGA nanofibers at an equivalent Cu NZs concentration of 100 ng / mL -1 and 150 ng mL -1 At the same time, it significantly improved the cell survival rate after H2O2 treatment, reaching 82.2% and 88.3%, respectively. Therefore, CuNZs@PLGA nanofibers can significantly alleviate cell damage caused by oxidative stress, thereby promoting the survival of hepatocytes under high ROS conditions. In subsequent experiments, the equivalent of 150 ng / mL -1 Cu NZs@PLGA nanofibers were used as the optimal concentration.

[0102] In this embodiment, scratch assays and EdU staining imaging were used to further determine the effects of CuNZs@PLGA nanofibers on the migration and proliferation of HUVECs. Figure 8 As shown in Figure FG, compared to the untreated control group, more cells migrated towards the center of the scratch after incubation with Cu NZs@PLGA nanofibers in a concentration-dependent manner. Specifically, at a Cu NZs equivalent concentration of 150 ng / mL... -1 After 24 hours of treatment with Cu NZs@PLGA nanofibers, the scratched areas were almost completely covered by cells. After 48 hours of treatment with Cu NZs@PLGA nanofibers, the scratched areas in all concentration groups were completely recovered. Figure 8 FG). EdU staining imaging showed that after 48 h of treatment with CuNZs@PLGA nanofibers, the number of EdU-positive cells increased ( Figure 8 HI). Compared with the untreated control group, 150 ng / mL -1 The proliferation rate of HUVECs increased by 21.1% in the Cu NZs@PLGA nanofiber concentration group. Figure 10The pro-angiogenic effect of Cu NZs@PLGA nanofibers may accelerate the integration and functionalization of transplanted HLCs with the host liver, thereby promoting liver regeneration. These results indicate that Cu NZs@PLGA nanofibers have a pro-angiogenic effect, thus promoting liver regeneration.

[0103] Example 5

[0104] This embodiment provides HLCs differentiated from hADMSCs in vitro and their characterization, specifically including:

[0105] 1. Inducing hADMSCs to differentiate into hepatocyte lineages using cytokines. First, in a solution containing 20 ng / mL... -1 Epidermal growth factor (EGF, PeproTech, USA) and 10 ng / mL -1 Cells were cultured for two days in low-glucose DMEM (Gibco, USA) serum-free medium containing basic fibroblast growth factor (bFGF, PeproTech, USA). Subsequently, the cells were cultured in 20 ng / mL of DMEM containing basic fibroblast growth factor (bFGF, PeproTech, USA). -1 Hepatocyte growth factor (HGF, PeproTech, USA), 10 ng / mL -1 bFGF and 0.61 mg mL -1 Differentiation was induced for one week in a low-glucose DMEM medium containing nicotinamide (Sigma-Aldrich, USA). Afterwards, differentiation was carried out in a medium containing 20 ng / mL... -1 Continue culturing for two weeks in low-glucose DMEM medium containing oncostatin M (OSM, PeproTech, USA), 1 mM dexamethasone, and 1 × ITS. Change the medium twice a week.

[0106] like Figure 11 A is a schematic diagram of the timeline for hADMSCs-induced liver differentiation. Albumin (ALB) and urea production in the culture supernatant were measured on days 7 and 21 using an ALB assay kit (CH0101002, Maccura, China) and a urea assay kit (CH0101051, Maccura, China), respectively. Cells were fixed in 4% PFA for 15 minutes, and accumulated lipids and glycogen were stained with Oil Red O (Sigma-Aldrich, USA) and periodic acid-Schiff (PAS, Sigma-Aldrich, USA), respectively, according to the manufacturer's instructions.

[0107] 2. Real-time quantitative PCR and immunofluorescence staining were used to detect whether hADMSCs were successfully induced to differentiate into HLCs, and to detect the characteristics of HLCs, specifically including:

[0108] Real-time quantitative PCR: Total RNA was extracted from cells using TRIzon reagent (CW0580S, Cwbiotech, China) and reverse transcribed using the HiFiScript cDNA Synthesis Kit (CW2569M, Cwbiotech, China). Real-time quantitative PCR was performed using UltraSYBR Mixture (CW2601, Cwbiotech, China) and QuantStudio. TM 5. Applied Biosystems were used for detection. All data were normalized to ACTB or GAPDH CT values, and relative expression levels were calculated using the delta-deltaCt method.

[0109] Immunofluorescence staining: Cells were fixed with 4% PFA at room temperature for 15 min, incubated with 5% goat serum for 1 h, and then incubated overnight with primary antibody at 4℃. Rabbit anti-human primary antibody dilutions were as follows: ALB (16475-1-AP, Proteintech, USA) (1:500), E-cadherin (710161, Invitrogen, USA) (1:200), DPP4 (PAB32046, China) (1:200), HNF4α (61189, Active Motif, USA) (1:200), ABCC2 (PAB35870, China) (1:200). Cells were incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:500) at room temperature in the dark for 2 h, and washed twice with phosphate buffer. 0.2 μg / mL -1 Cell nuclei were stained with DAPI and incubated at room temperature for 15 minutes. Finally, imaging was performed using an inverted fluorescence microscope.

[0110] 3. Determine the anti-inflammatory ability of HLCs, specifically including:

[0111] THP-1 cells were spaced at 8 × 10⁸ cells per well. 5 Inoculate at a density of 100 ng / mL into 6-well plates. -1 THP-1 cells (M0) were activated by treatment with 12-myristate-13-acetate (PMA) (Sigma-Aldrich, USA) medium for 48 h. The PMA-containing medium was then removed. THP-1 cells (M0) were cultured with fresh medium and HLC supernatant (1:1) for 24 or 48 h.

[0112] Lipopolysaccharide (LPS, 100 ng / mL) -1THP-1 (M0) and Raw264.7 cells were stimulated for 24 h at (Sigma-Aldrich, USA) to induce M1 polarization. Then, the lipopolysaccharide-containing medium was removed. Polarized THP-1 and Raw264.7 cells were treated with fresh medium and HLC supernatant (1:1) for 24 h. Cells were collected, and the expression of IL-1β, TNFA, NOS2, TGFβ1 (pro-inflammatory cytokine), IL-10 (anti-inflammatory cytokine), and MRC1 (marker of M2 macrophages) in THP-1 and Raw264.7 cells was detected by qPCR to assess macrophage polarization status.

[0113] 4. Characteristic results of HLCs after hADMSCs-induced differentiation are as follows: Figure 11-13 This embodiment uses a two-step differentiation protocol based on cytokines to induce liver differentiation of stem cells. Figure 11 A). After differentiation, the spindle-shaped hADMSCs become polygonal in shape. Figure 11 B, Figure 12 Compared to hADMSCs, induced HLCs showed significant glycogen production and lipid accumulation as indicated by PAS staining and Oil Red O staining, respectively. Figure 11 B). In addition, hepatocyte-associated proteins such as ALB, HNF4α, ABCC2, E-cadherin, and DPP4 are also highly expressed in HLCs. Figure 11 CE, Figure 13 From the beginning to the end of cell differentiation, the mRNA expression of hepatocyte-specific genes was tracked. The mRNA expression levels of ALB and CYP3A4 genes in HLCs on day 21 were more than 10.0 times that on day 0. Figure 11 F). Conversely, compared to day 7, the level of the immature hepatocyte marker AFP was significantly lower on day 21 (F). Figure 11 F). Furthermore, 21 days after differentiation, the secretion levels of ALB and urea in the supernatant of HLC culture medium were 5.3 times and 2.1 times higher, respectively, than on day 7 of differentiation. Figure 11 (G). In summary, these results indicate that hADMSCs were successfully induced into HLCs with typical hepatocyte morphology and related functions, which is beneficial for improving liver biological function in ALF.

[0114] 5. Results of the inflammatory regulatory effects of HLCs are as follows: Figure 14-15This example investigated the effect of HLC supernatant on macrophage polarization. First, HLC supernatant was incubated with THP-1 (M0) for 24 or 48 h. Macrophage gene expression was analyzed by qPCR to assess macrophage polarization. Compared to THP-1 (M0), with prolonged treatment time, the expression of pro-inflammatory genes IL-1β, NOS2, and TGFβ1 in THP-1 treated with HLC supernatant was significantly reduced, while the expression of anti-inflammatory genes IL-10 and MRC1 increased by 3.3-fold and 33.9-fold, respectively, at 48 h. Figure 14 (AB). This indicates that HLCs supernatant can polarize macrophages towards the M2 phenotype (anti-inflammatory phenotype). Next, this example further investigates whether HLCs supernatant can reverse macrophage polarization under inflammatory conditions. The results showed that LPS treatment upregulated the expression of IL 1β, TNFA, and TGFβ1 in THP-1, indicating that THP-1 (M0) was successfully induced to the M1 phenotype (pro-inflammatory phenotype). Notably, compared with the LPS-treated group, the expression of IL 1β, TNFA, and TGFβ1 in THP-1 treated with HLCs supernatant was significantly decreased, approaching the level of THP-1 (M0). In contrast, after HLCs supernatant treatment, the levels of IL 10 and MRC1 significantly increased to 23.6-fold and 21.1-fold, respectively. Figure 14 CD). Similar results were also obtained in RAW264.7 cells in this embodiment (CD). Figure 15 These results indicate that HLC supernatant can effectively promote the transformation of macrophage phenotype under inflammatory conditions.

[0115] Furthermore, this embodiment also investigated whether HLCs have a protective effect on hepatocytes under high oxidative stress. After H2O2 treatment, a large number of floating dead cells and debris were observed in AML12 cells cultured alone (control group), but no such cells were observed in cells co-cultured with HLCs or hADMSCs. Figure 16 Compared with AML12 cells cultured alone and AML12 cells co-cultured with hADMSCs, AML12 cells co-cultured with HLCs showed better cell viability. Figure 24 Through characterization of antioxidant gene expression in AML12 cells, this example found that when AML12 cells were co-cultured with HLCs, the mRNA expression of antioxidant genes SOD2, NQO1, and KEAP1 was significantly upregulated by 2.2-3.8 times. Figure 16 C). Therefore, in ALF treatment, HLCs may promote liver regeneration by enhancing the antioxidant capacity and cell viability of host hepatocytes.

[0116] Example 6

[0117] This embodiment provides the preparation of dECM hydrogels, their biocompatibility, and their effects on liver function, specifically including:

[0118] 1. Preparation of dECM hydrogel:

[0119] Approximately 200 grams of pork liver was gently chopped after removing attached tissue. The liver tissue underwent three cycles of freezing at -80°C and thawing at room temperature, rinsing with deionized water during this period. The liver tissue was decellularized for 3 days in deionized water containing 1% Triton X-100, 0.1% NH3·H2O, 1% penicillin / streptomycin, and 0.01 mM benzyl sulfonyl fluoride (PMSF), with the medium changed three times daily. Before freeze-drying, the decellularized liver tissue was washed in sterile water for 1 hour. All steps were performed with stirring at 150 rpm. To prepare the dECM stock solution, the decellularized liver tissue was further digested with 10% pepsin (Sigma-Aldrich, USA), added to 0.1 M hydrochloric acid at a dECM:pepsin (10:1) weight ratio, and stirred at room temperature for 48 hours. The morphology of the dECM hydrogel was characterized using scanning electron microscopy (SEM).

[0120] 2. To investigate the biocompatibility of dECM hydrogels and their effects on liver function, including:

[0121] Dilute the dECM stock solution with an alkaline medium to a final concentration of 5 mg / mL. -1 The pH was adjusted to 7.4 using 1M NaOH. HLCs or hADMSCs were then added at a concentration of 1×10⁻⁶. 6 cells mL -1 The HLCs were uniformly dispersed in dECM solution and added to 96-well plates, then incubated at 37°C for 1 hour to induce gelation. After a stable dECM hydrogel was formed, cell culture medium was added. Cell viability was measured using Calcein-AM / PI staining within 24 hours. On day 3, the culture supernatant was collected, and ALB and urea production by HLCs were detected. Simultaneously, frozen sections of the dECM hydrogel loaded with HLCs were prepared, and immunofluorescence staining for hepatocyte-related marker proteins, including ALB, HNF4α, ABCC2, and E-cadherin, was performed. hADMSCs cultured in dECM hydrogel served as a control.

[0122] 3. Determination of the ROS scavenging ability of Cu NZs@PLGA nanofiber-reinforced dECM hydrogel:

[0123] This experiment evaluated the protective effect of CuNZs@PLGA nanofibers on HLCs cultured in dECM hydrogels under oxidative stress. (The experiment used a 1×10⁻⁶ nanofiber ratio.) 6 mL -1HLCs were encapsulated in dECM at a cell density of 150 ng / mL and added to 96-well plates to form a gel at 37 °C. This gel was then coated with Cu NZs@PLGA nanofibers (equivalent Cu NZs concentration of 150 ng / mL). -1 Cells were pre-incubated overnight. Cells were stimulated with 500 μM H2O2 for 24 h, and cell viability was detected by Calcein-AM / PI staining. Intracellular ROS were detected by DCFH-DA staining, and the average fluorescence intensity was measured by a microplate reader for ROS quantification.

[0124] 4. Characterization results of Cu NZs@PLGA nanofiber reinforced dECM hydrogel are as follows: Figure 17-18 .

[0125] This embodiment uses liver dECM hydrogel ( Figure 17 A) In situ delivery of HLCs: Liver dECM hydrogels preserved the bioactive components of the liver, and the 3D culture environment promoted the maturation of HLCs. After incubation at 37°C for 1 hour, the resulting dECM hydrogel exhibited a relatively large storage modulus (G') greater than the loss modulus (G"). Figure 17 B, Figure 18 A). SEM images show that the dECM hydrogel has a porous structure, and no residual cells were found after decellularization. Figure 17 C). However, gels with an elastic modulus less than 100.0 Pa may not be suitable for transplantation surgery. Therefore, we used Cu NZs@PLGA nanofibers as mechanical support for the dECM hydrogel ( Figure 18 B). After cell embedding, HLCs are uniformly distributed in the Cu NZs@PLGA nanofiber-bound dECM hydrogel. Figure 17 D).

[0126] 5. Liver dECM hydrogel and biocompatibility results are as follows: Figure 17-18 .

[0127] After 3 days of culture in dECM hydrogel (3D culture), HLCs showed good cell viability. Figure 18 C), hepatocyte-related markers remained highly expressed, including ALB, HNF4α, E-cadherin, and ABCC2 (C). Figure 17 EH). HLCs cultured in dECM hydrogels showed more than 8.0-fold higher mRNA expression levels of ALB and CK18 genes compared to those cultured in 2D plates. Simultaneously, the drug metabolism-related gene CYP3A4 was also significantly upregulated. Figure 17 I). Furthermore, the secretion of ALB and urea in the supernatant of HLC culture medium also increased further ( Figure 17(J) In particular, the urea content of 3D-cultured HLCs increased by 2.3 times compared to 2D-cultured HLCs. Therefore, dECM hydrogels have good biocompatibility and are beneficial for maintaining the phenotype of HLCs and enhancing hepatocyte-related functions.

[0128] 6. The cell-protective effect of Cu NZs@PLGA nanofibers on HLCs in dECM hydrogel was determined, and the results are as follows: Figure 19 .

[0129] This experiment used Cu NZs@PLGA nanofibers (equivalent Cu NZs concentration of 150 ng / mL) -1 After pretreatment, compared with the control group, Cu NZs@PLGA nanofibers significantly reduced H2O2-induced HLC death. Figure 19 A), this is because Cu NZs significantly alleviated the high intracellular ROS level ( Figure 19 B).

[0130] Example 7

[0131] This embodiment provides an experiment on the treatment of ALF mice with orthotopic cell transplantation drugs (HLCs / Cu NZs@fiber / dECM), specifically including:

[0132] 1. All animal experiments were conducted according to the protocol approved by the Animal Protection and Use Committee of Sun Yat-sen University (SYSU-IACUC-2022-001799). Intraperitoneal injection of 30% (v / v) CCl4 corn oil solution (5 μL g) was administered. -1 Mice were treated for 24 hours to establish an ALF mouse model. Male C57BL / 6 mice (6-8 weeks old, 18-22g) were randomly divided into 6 groups, including (1) blank (n=5): healthy mice; (2) control group (n=5): ALF mice with sham surgery; (3) fiber / dECM (n=5): ALF mice implanted with PLGA nanofiber combined with dECM hydrogel; (4) Cu NZs@fiber / dECM (n=5): ALF mice implanted with Cu NZs@PLGA nanofiber combined with dECM hydrogel; (5) hADMSCs / Cu NZs@fiber / dECM (n=5): ALF mice implanted with hADMSCs / Cu NZs@fiber / dECM; (6) HLCs / Cu NZs@fiber / dECM (n=5): ALF mice implanted with HLCs / CuNZs@fiber / dECM. One or three days after transplantation, administer an intraperitoneal injection of 0.6% sodium pentobarbital solution (Aladdin, China) at a dose of 10 μL / g. -1Blood and organs were collected. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and albumin (ALB) levels were measured according to the manufacturers' protocols using an AST assay kit (CH0101202, Maccura, China), an ALT assay kit (CH0101201, Maccura, China), and an ALB assay kit (CH0101002, Maccura, China). Collected heart, liver, spleen, lung, and kidney tissues were fixed in 4% PFA, paraffin-embedded, and stained with hematoxylin and eosin (G1005, Servicebio, China). Hepatocyte apoptosis was detected using a TUNEL apoptosis detection kit (G1501, Servicebio, China). Hepatocyte proliferation was detected by Ki67 staining (GB11141, Servicebio, China). Furthermore, TNF-α (GB11188, Servicebio, China) and IL-6 (GB11141, Servicebio, China) immunofluorescence staining was performed on liver sections to assess the degree of inflammation. ROS levels in frozen liver sections were detected using dihydroethylenediamine (DHE, GDP1018, Servicebio, China). Mean fluorescence intensity was quantified using Image J. Immunofluorescence staining (anti-CD31 antibody, GB11063-1, Servicebio, China) was performed on liver tissue surrounding implanted HLCs / Cu NZs@fiber / dECM to illustrate angiogenesis and vascularization of the implant.

[0133] 2. Results of ALF treatment using Cu NZs@PLGA nanofiber-reinforced dECM hydrogel to deliver HLCs: Figure 20-26 .

[0134] The above examples demonstrate that Cu NZs@PLGA nanofibers possess strong ROS scavenging efficiency and angiogenesis-promoting effects, and that dECM hydrogel can enhance liver function in HLCs. Therefore, this example assesses the therapeutic potential of HLCs / Cu NZs@fiber / dECM synergistic therapy for CCl4-induced ALF. Figure 20 A). After CCl4 treatment, the liver became lighter in color and harder in texture, with extensive cell necrosis near the vascular area. Figure 21 According to HE staining images, the implantation of CuNZs@PLGA nanofibers significantly reduced the necrotic area by 17.9%–29.9% on day 1. Figure 20B, E). Similarly, TUNEL and Ki67 staining also showed that, compared with the "control group" and the "fiber / dECM" group, the ALF mice in the "Cu NZs@fiber / dECM" group had fewer apoptotic cells and more proliferating cells in their livers. This suggests a cytoprotective role of Cu NZs in the initial stage of ALF oxidative stress. Notably, compared with hADMSCs / CuNZs@fiber / dECM, ALF mice implanted with HLCs / Cu NZs@fiber / dECM exhibited less tissue necrosis and more significant cell proliferation, indicating that the implanted HLCs have a compensatory effect on liver function and additional protection against host hepatocytes. Figure 20 CD, FG). These results are consistent with in vitro experimental results. Mice treated with HLCs / Cu NZs@fiber / dECM showed decreased serum AST and ALT levels and restored serum ALB levels, further demonstrating the superior therapeutic effect of HLCs. Figure 20 HJ). Regarding inflammation and oxidative stress, compared to the untreated control group, the implantation of Cu NZs@PLGA nanofibers significantly reduced the accumulation of inflammatory factors such as TNF-α, IL-6, and intracellular ROS on day 1. Figure 21 Furthermore, the mRNA expression levels of TNFA, IL-6, and COX2 genes in the hepatocytes of untreated control mice were more than 10.0 times higher than those in normal mice. After treatment with Cu NZs@PLGA nanofibers, the mRNA expression levels of these genes decreased significantly. Figure 22 GI). In particular, the mRNA expression of IL1 and INOS genes returned to the normal range. Figure 22 JK), while the level of the anti-inflammatory gene IL10 in the "Cu NZs@fiber" group increased by more than 5.0 times compared with the untreated control group (JK). Figure 22 Furthermore, HLC implantation further reduced the mRNA expression of pro-inflammatory genes TNFA and IL1 in the liver tissue of ALF mice, and upregulated the expression of the anti-inflammatory gene IL10. Compared with the control group, HLCs / Cu NZs@fiber / dECM treatment significantly reduced the expression levels of TNFA and IL1 genes by 2.6-fold and 2.0-fold, respectively, while significantly upregulating the expression level of IL10 gene by 9.3-fold, indicating that HLCs / Cu NZs@fiber / dECM has an advantage in regulating the harsh local microenvironment.

[0135] The efficacy results of HLCs / Cu NZs@fiber / dECM in treating CCl4-induced ALF after 3 days are as follows: Figure 23 Liver necrosis was significantly reduced, with inflammatory cell infiltration being the dominant feature. Figure 23 AB). Tunel-positive cells were fewer, while Ki67-positive cells were widely distributed in liver tissues of all groups. Figure 23 CD). Serum AST and ALT levels in the HLCs / Cu NZs@fiber / dECM group were similar to normal values ​​but significantly lower than in other treatment groups. Figure 23 EF). Meanwhile, serum ALB levels in mice treated with HLCs / Cu NZs@fiber / dECM were completely restored ( Figure 23 G). HLCs / Cu NZs@fiber / dECM also slowed weight loss in ALF mice in the initial phase and accelerated weight recovery starting from day 3. Figure 23 H). Furthermore, the mitigation of inflammatory storm and excessive intracellular ROS by Cu NZs@PLGA nanofibers was confirmed by TNF-α, IL-6, and DHE staining on day 3. Figure 24 AC). Compared with the hADMSCs / Cu NZs@fiber / dECM group, the expression of TNFA, COX2, and NLRP3 gene mRNA in mouse hepatocytes of the HLCs / Cu NZs@fiber / dECM group was significantly reduced. Figure 24 Therefore, HLCs / Cu NZs@fiber / dECM can sustainably and effectively alleviate inflammatory storms, inhibit oxidative stress, and establish an ideal microenvironment for host hepatocytes, thereby promoting liver regeneration. Furthermore, the angiogenic Cu NZs also promote the migration of host CD31-positive endothelial cells to the HLCs-loaded dECM hydrogel (HNF4α-positive cells) and the formation of blood vessels throughout the implant, suggesting that the therapeutic effect of HLCs / Cu NZs@fiber / dECM may be partly attributable to the adequate vascularization of the implant. Figure 25 HE staining of major organs was used to evaluate the systemic toxicity of HLCs / Cu NZs@fiber / dECM. After 3 days of treatment, there were no significant changes in the heart, spleen, lungs, and kidneys of mice, indicating that HLCs / Cu NZs@fiber / dECM has good biocompatibility in vivo. Figure 26 In summary, these analyses of anti-inflammatory, liver function, proliferation, and apoptosis results based on blood and liver tissue confirm that HLCs / Cu NZs@fiber / dECM has a good synergistic therapeutic effect on ALF by combining the antioxidant and angiogenesis activities of Cu NZs with the anti-inflammatory and hepatocyte function of HLCs.

[0136] Example 8

[0137] This application provides transcriptome analysis of liver tissue from the attachment regions of the treatment groups (HLCs / Cu NZs@fiber / dECM: HLC-1 and HLC-2) and the control group (control: Ctrl-1 and Ctrl-2) one day after treatment in the above embodiments, to explore the therapeutic mechanism of the synergistic therapeutic effect of HLCs / Cu NZs@fiber / dECM on ALF mice.

[0138] 1. RNA sequencing:

[0139] Liver tissues (n=2) from the control group and the HLCs / Cu NZs@fiber / dECM group were isolated and milled in TRIzon reagent to extract mRNA. Sequencing libraries were constructed using high-quality RNA samples. Illumina Novaseq was used for sequencing. TM 6000 (Illumina, USA) was used for 2×150bp end sequencing (PE150) according to the vendor's recommended protocol. All data were processed using the R-package provided by Lc-Bio Technology (Hangzhou, China).

[0140] 2. Statistical Analysis:

[0141] All statistical analyses were performed using GraphPad Prism 9 (GraphPad software, USA). Data are expressed as mean ± standard deviation and comparisons were made using Student's t-test or one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, or # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001 indicates a statistically significant difference. ns indicates no statistically significant difference.

[0142] The results are as follows Figure 27-30 As shown in the volcano plot, 833 upregulated genes and 1184 downregulated genes were differentially expressed in the liver of the HLCs / Cu NZs@fiber / decm treatment group. Figure 27 A). Furthermore, KEGG (the Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that the top 20 differentially enriched pathways involved in cell cycle, liver biosynthesis, drug and substance metabolism, apoptosis, and liver regeneration. Figure 27B). Furthermore, the high expression of drug metabolism-related genes in the HLCs / Cu NZs@fiber / decm treatment group and their gene set enrichment analysis (GSEA) indicate the important role of cytochrome P450-mediated drug metabolism in ALF treatment. Figure 27 C, Figure 28 Besides the Hippo signaling pathway, which is crucial for liver regeneration, the p53 signaling pathway, associated with apoptosis, also plays a significant role in treatment. GSEA results showed that after implantation of HLCs / Cu NZs@fiber / dECM, 88% of key genes in the p53 signaling pathway were differentially downregulated. Figure 27 C, Figure 29 Notably, some genes in the Wnt signaling pathway, a key pathway mediating liver regeneration, were significantly upregulated. Figure 30 The AXIN2 gene, a marker of hepatocyte proliferation / self-renewal, was highly expressed in the HLCs / Cu NZs@fiber / dECM group. These results are consistent with TUNEL and Ki67 staining findings. Figure 20 C and 20D). Furthermore, transcriptomic analysis showed that in the livers of mice implanted with HLCs / Cu NZs@fiber / dECM, genes associated with inflammation or oxidative stress, such as IL33 and HIF1A, were significantly downregulated, while antioxidant genes such as SOD1, SOD2, GPX1, and CAT, as well as angiogenesis-related genes VEGFA and ANG, were highly expressed. Figure 8 D). More importantly, in the liver tissue of mice in the HLCs / Cu NZs@fiber / dECM group, the expression of liver regeneration and hepatocyte-related genes such as ALB, HNF4A, DPP4, ABCC2, TTR, UGT1A1, GSTM1, and SULT1A1 was significantly increased. Figure 27 D).

[0143] In summary, excessive ROS, severe cytokine storm, and extensive liver necrosis are the main pathological features of ALF. Therefore, clearing ROS to reduce hepatocellular necrosis, replenishing hepatocellular cells to achieve liver function compensation, and thus promoting liver regeneration are key to ALF treatment. Therefore, this application presents a synergistic therapeutic platform composed of nanofibers loaded with antioxidant metal nanozymes and a gel loaded with functional cells (HLCs / Cu NZs@fiber / dECM) for treating CCl4-induced ALF, and demonstrates the good therapeutic effect of HLCs / Cu NZs@fiber / dECM.

[0144] This embodiment successfully synthesized and characterized Cu NZs with good biocompatibility and high ROS scavenging efficiency. Even when loaded onto PLGA nanofibers, the antioxidant effect of Cu NZs remained unaffected. Cu NZs@PLGA nanofibers achieved an equivalent Cu NZs concentration as low as 150 ng mg. -1 It can effectively remove ROS, improving the survival rate of hepatocytes both in vitro and in vivo. In addition, Cu NZs@PLGA nanofibers can significantly promote the migration and proliferation of HUVECs, which is beneficial for the rapid vascularization of implants in vivo.

[0145] Since most cells can be cleared by macrophages or phagocytes after transplantation into the spleen, and the inflammatory environment of the liver can also impair the viability of transplanted cells, this embodiment loads HLCs onto a drug-loaded gel (such as dECM hydrogel) and combines it with CuNZs@PLGA nanofibers for in situ delivery of HLCs. Results show that dECM, as a carrier of HLCs, provides a good 3D culture environment due to its porous structure and natural components, maintaining / enhancing the liver function of HLCs and facilitating liver function compensation in damaged livers. Furthermore, this embodiment demonstrates that CuNZs@PLGA nanofibers in dECM hydrogel have a scavenging effect on H2O2 and a cytoprotective effect on the loaded HLCs. In addition, dECM hydrogel has low mechanical strength, making it difficult to maintain its structure during in situ transplantation; CuNZs@PLGA nanofiber-reinforced dECM hydrogel improves the operability of in vivo transplantation. CuNZs@PLGA nanofibers can also reduce implant adhesion to adjacent organs or tissues, promoting the integration of HLCs with the host liver.

[0146] Furthermore, this embodiment evaluated the synergistic therapeutic effect of HLCs / Cu NZs@fiber / dECM in in vivo experiments. On one hand, the antioxidant nanozyme Cu NZs can efficiently remove excess ROS and prevent the worsening of liver tissue necrosis. For example... Figure 6 and Figure 23 As shown, reduced apoptosis leads to a decrease in the accumulation of inflammatory factors, thereby preventing severe inflammatory storms in the liver. Figure 22 , 24 An improved microenvironment also benefits the survival of HLCs. For example... Figure 25 As shown, Cu NZs can also promote angiogenesis and vascularization of the implant. On the other hand, HLCs have hepatocyte-related functions, which are further maintained or enhanced in dECM hydrogels. Figure 20 and Figure 23The results showed that HLCs significantly increased ALB levels in ALF mice. Furthermore, HLCs possess inherent anti-inflammatory activity, thereby synergistically protecting host hepatocytes from oxidative stress and accelerating liver regeneration. As expected, HLCs / Cu NZs@fiber / dECM exhibited excellent therapeutic effects against CCl4-induced ALF. In conclusion, the synergistic effect of HLCs / Cu NZs@fiber / dECM contributes to effective liver regeneration in ALF mice and promotes long-term liver function integration of transplanted cells in vivo.

[0147] This embodiment further performs transcriptome analysis to explore potential therapeutic mechanisms. From the differential gene expression heatmap, such as... Figure 27 As shown in Figure D, differentially regulated genes related to oxidative stress and inflammation correspond to the ROS elimination and anti-inflammatory capabilities of HLCs / Cu NZs@fiber / dECM. High expression of angiogenesis-related genes indicates the pro-angiogenic effect of Cu NZs. KEGG and GSEA analyses were used to further explore the pathways by which HLCs / Cu NZs@fiber / dECM affects apoptosis. Studies have shown that significant hepatocyte senescence in acute liver injury may disrupt the balance between hepatocyte regeneration and exhaustion. Our results show that after treatment with HLCs / Cu NZs@fiber / dECM, differentially regulated genes enriched in the p53 signaling pathway and significantly upregulated AXIN2 were observed. Upregulation of AXIN2 in hepatocytes is considered a surface marker of hepatocyte self-renewal, cell cycle reentry, and regeneration. These results suggest that HLCs / Cu NZs@fiber / dECM may inhibit hepatocyte senescence and promote liver regeneration by influencing the Wnt signaling pathway. Furthermore, abundant differentially expressed genes were also observed in pathways such as liver biosynthesis and drug metabolism. Notably, genes related to the cytochrome p450 drug metabolism pathway were differentially upregulated in the livers of the HLCs / Cu NZs@fiber / dECM group. Furthermore, hepatocyte-related genes were also highly expressed, suggesting that the potential mechanism of liver function compensation in HLCs may be related to substance biosynthesis and drug metabolism. Overall, transcriptomic analysis also confirmed the synergistic effects of HLCs / Cu NZs@fiber / dECM in ALF treatment, including antioxidant, angiogenesis-promoting, anti-inflammatory, and hepatocyte-related functional responses.

Claims

1. A drug for orthotopic cell transplantation, characterized in that, include: Biomimetic nanoenzyme fibers and drug-loaded gels loaded with functional cells; The preparation method of the biomimetic nanoenzyme fiber includes: compounding and fiberizing an antioxidant metal nanoenzyme and a biodegradable polymer under solvent conditions to obtain a biomimetic nanoenzyme fiber; the antioxidant metal nanoenzyme is loaded on the biomimetic nanoenzyme fiber; the antioxidant metal nanoenzyme is selected from copper-based nanoenzymes; the biodegradable polymer is selected from polylactic acid-polyglycolic acid copolymer (PLGA). The functional cells are selected from functional hepatocyte-like cells; The drug-loaded gel is selected from decellularized matrix dECM hydrogel.

2. The orthotopic cell transplantation drug according to claim 1, characterized in that, The solvent is selected from one or more of hexafluoroisopropanol (HFIP), dichloromethane, and tetrahydrofuran.

3. The orthotopic cell transplantation drug according to claim 1, characterized in that, The fiberization is selected from electrospinning, 3D printing, or spin coating.

4. The use of the orthotopic cell transplantation drug according to claim 1 in the preparation of drugs for treating solid organ diseases, characterized in that, The diseases of solid organs include liver diseases.

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