Porphyrin-Ru-based nano biocatalyst, vascular graft as well as preparation and application of porphyrin-Ru-based nano biocatalyst and vascular graft

Abstract

The invention belongs to the field of biocatalysis and vascular repair materials, and particularly relates to a porphyrin-Ru-based nano biocatalyst, a vascular graft as well as preparation and application of the porphyrin-Ru-based nano biocatalyst and the vascular graft. The porphyrin-Ru-based nano biocatalyst is a porous nano biocatalyst formed by loading a coordination polymer on nano carbon through pi-pi conjugation, wherein the coordination polymer is formed by connecting Ru with porphyrin through Ru-N coordination. According to the invention, a vascular graft doped with the porphyrin-Ru-based nano biocatalyst is then prepared by an electrospinning technology. The nano biocatalyst disclosed by the invention has a high-activity pi-conjugated Ru-N catalytic site, a pi-pi stacking effect and a porous structure, and the electron transfer capability and catalytic kinetics of the nano biocatalyst are synergistically enhanced, so that the vascular graft is endowed with ultrafast and broad-spectrum ROS removal capability; therefore, the composition has excellent anti-oxidative stress, anti-inflammatory and vascular repair and regeneration promoting capacities.

Description

Technical Field

[0001] The present invention belongs to the fields of biocatalysis and vascular repair materials, and particularly relates to a porphyrin-Ru-based nano-biocatalyst, a vascular graft, and their preparation and applications. Background Art

[0002] Cardiovascular diseases are the most common non-communicable diseases globally, mainly caused by vascular diseases such as atherosclerosis, thromboangiitis obliterans, traumatic arteriovenous fistula, and aortic dissection, accounting for nearly one-third of global mortality. Penetrating injuries by external sharp objects (including transplantation surgeries) can also cause massive bleeding and endanger life. Despite the help of medical treatments, in some cases, especially for large-area vascular injuries, vascular transplantation surgeries are still required. The ideal vascular graft material is autologous blood vessels, but the limitations of autologous graft materials and the occurrence of secondary injuries to patients are the main obstacles to their clinical applications. A feasible way to solve the shortage of autologous blood vessels is to construct artificial blood vessels in vitro and transplant them into the body (i.e., vascular tissue engineering). Current commercial vascular grafts mainly consist of synthetic materials: polyethylene terephthalate (Dacron) knitted fabrics and expanded polytetrafluoroethylene (e-PTFE) non-woven mesh. However, these grafts still face significant limitations, such as complications, poor tissue regeneration ability, and low patency rate. Therefore, exploring new biomaterials and related manufacturing strategies for vascular tissue engineering remains an urgent task.

[0003] The latest advances in tissue engineering have promoted in-depth research on vascular graft manufacturing technologies, including decellularized matrix methods, electrospinning techniques, and additive manufacturing through bioinks or 3D printing. Among them, electrospinning has become a versatile technique for fabricating membranes with high surface-to-volume ratios and hierarchical interconnected porous structures. By adjusting the polymer mixture during the electrospinning process, functionalized fiber materials can be obtained. Poly(ε-caprolactone) (PCL) is a biodegradable synthetic polymer that has been approved by the US Food and Drug Administration (FDA) and the Therapeutic Goods Administration (TGA). PCL has good biocompatibility and potential for processing and functionalization, meeting the strict requirements for vascular graft applications and being widely used in the manufacture of vascular grafts. However, oxidative stress and inflammatory responses at the vascular injury site can severely affect the effectiveness of vascular transplantation. Therefore, constructing an artificial vascular implant that not only has a vascular network structure but also can alleviate oxidative stress and inflammatory responses at the vascular injury site remains challenging.

[0004] Endogenous antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) play a key role in maintaining redox balance by catalytically neutralizing reactive oxygen species (ROS). These enzymes regulate oxidative stress pathways to reduce vascular inflammation and promote endothelial repair after injury. However, the application of natural antioxidant enzymes is still limited by their inherent limitations, including limited in vivo stability, antigenicity, lack of engineerability, and narrow ROS scavenging profiles, which together affect their application in inflammatory diseases. Therefore, chemists and materials scientists are actively involved in mimicking the mechanisms of natural antioxidant enzymes to create novel biocatalytic ROS-scavenging materials for antioxidant treatment. Summary of the Invention

[0005] To solve the above problems, the present invention first synthesized a novel nano-carbon-supported porous porphyrin-Ru-based nanobiocatalyst (abbreviated as SPPorRu in the present invention) by a coordination-driven self-assembly method, and then prepared a vascular graft incorporated with the nano-carbon-supported porous porphyrin-Ru-based nanobiocatalyst (abbreviated as SPPorRu@PCL in the present invention) by electrospinning technology. The SPPorRu of the present invention has highly active π-conjugated Ru-N catalytic sites, π-π stacking effects, and porous structures, which synergistically enhance its electron transfer ability and catalytic kinetics, thereby endowing SPPorRu@PCL with ultrafast and broad-spectrum ROS scavenging ability, making it have excellent antioxidant stress, anti-inflammatory, and pro-vascular repair and regeneration abilities.

[0006] Technical solution of the present invention:

[0007] The first technical problem to be solved by the present invention is to provide a nano-carbon-supported porous porphyrin-Ru-based nanobiocatalyst, which is a porous material formed by a coordination polymer in which Ru is connected to porphyrin through Ru-N coordination and is loaded on nano-carbon through π-π conjugation.

[0008] Further, the porphyrin is a porphyrin having a pyridyl group.

[0009] Further, the porphyrin is at least one of 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin, tetrakis(2-pyridyl)porphyrin, and 5,15-bis(4-pyridyl)-10,20-diphenylporphyrin.

[0010] Further, the nano-carbon is Ketjenblack, such as Ketjenblack ECP-600JD.

[0011] Further, the catalyst has SOD- and CAT-like activities and DPPH· scavenging performance.

[0012] The second technical problem to be solved by the present invention is to provide a method for preparing a nano-carbon-supported porous porphyrin-Ru-based nano-bio-catalyst as described herein, which comprises co-mixing and reacting a porphyrin compound, a Ru salt and nano-carbon in water to obtain the bio-catalyst.

[0013] Further, the molar ratio of the porphyrin compound to the Ru salt is 1:0.5 - 4, preferably 1:1.8 - 2.2.

[0014] Further, the porphyrin is a porphyrin having a pyridyl group.

[0015] Further, the porphyrin is at least one of 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin, tetrakis(2-pyridyl)porphyrin, 5,15-bis(4-pyridyl)-10,20-diphenylporphyrin.

[0016] Further, the Ru salt is RuCl3·xH2O.

[0017] Further, the nano-carbon is Ketjenblack.

[0018] Generally, the addition amount of the nano-carbon is not limited as long as it can form a porous nano-bio-catalyst as described herein through π-π conjugation with the Ru-N coordination polymer. In some specific embodiments, the addition amount of the nano-carbon in the co-mixing system can be 25 - 75% (w / v), preferably 50% (w / v).

[0019] Further, the reaction conditions are to react at room temperature for 6 - 24 h.

[0020] Further, the preparation method further comprises adding an acid and a polymer surfactant to the co-mixing solution.

[0021] Further, the acid is hydrochloric acid, and the concentration of the hydrochloric acid is 0.01 M - 0.1 M.

[0022] Further, the polymer surfactant is polyvinylpyrrolidone.

[0023] Further, the preparation method comprises dissolving the porphyrin compound and the polymer surfactant in an aqueous solution of the acid to form a homogeneous solution A; dispersing the nano-carbon in solution A; dissolving the Ru salt in water to obtain solution B; then dropwise adding solution B to solution A under stirring, and obtaining the bio-catalyst after reaction.

[0024] Further, the dispersion includes ultrasonic dispersion.

[0025] Further, the stirring speed is 200 - 800 rpm.

[0026] Further, the conditions for the reaction are to react at room temperature for 6 - 24 h.

[0027] Further, the preparation method further includes centrifuging to collect the reaction product after the reaction, and washing and drying it.

[0028] The third technical problem to be solved by the present invention is to provide a vascular graft, which is a polycaprolactone fiber membrane incorporated with a nano-carbon loaded porous porphyrin-Ru-based nano-bio-catalyst as described herein.

[0029] The fourth technical problem to be solved by the present invention is to provide a preparation method of the vascular graft as described herein, which includes dispersing the nano-carbon loaded porous porphyrin-Ru-based nano-bio-catalyst as described herein in an organic solvent, adding polycaprolactone and stirring well, and obtaining the vascular graft by electrospinning.

[0030] Further, the mass ratio of polycaprolactone to the bio-catalyst is 5:1 - 2.

[0031] Further, the mass fraction of polycaprolactone in the organic solvent is 10 - 20%.

[0032] Further, the organic solvent is hexafluoroisopropanol.

[0033] Further, the time for stirring well is 12 - 24 h.

[0034] Further, the stirring speed is 100 - 200 rpm.

[0035] Further, the parameters of the electrospinning are: reciprocating movement distance 20 cm, roller rotation speed 200 rpm, opening the injection pump 2 mL / h, and setting the voltage 15 mV.

[0036] The fifth technical problem to be solved by the present invention is to provide the use of the nano-carbon loaded porous porphyrin-Ru-based nano-bio-catalyst or the vascular graft as described herein in the preparation of a biomaterial with antioxidant activity.

[0037] Further, the antioxidant activity includes SOD and CAT-like activities.

[0038] Further, the antioxidant activity includes ROS scavenging activity.

[0039] The sixth technical problem to be solved by the present invention is to provide the use of the vascular graft as described herein in the preparation of a biomaterial with antioxidant stress, anti-inflammatory, and functions of promoting vascular injury repair and regeneration.

[0040] Advantages of the present invention

[0041] The SPPorRu biocatalyst prepared by the present invention has highly active π-conjugated Ru-N catalytic sites, π-π stacking effect and porous structure, which synergistically enhance its electron transfer ability and catalytic kinetics, thus endowing SPPorRu@PCL with ultrafast and broad-spectrum ROS scavenging ability. The biomimetic extracellular matrix structure and ROS scavenging activity of SPPorRu@PCL have excellent functions of protecting cells, inhibiting inflammation, promoting blood vessel repair and regeneration during the treatment of vascular injury. The present invention verifies that the porous nanocarbon and π-conjugated porphyrin structure promote the effective utilization of Ru catalytic sites, thus effectively improving the enzymatic catalytic activity of SPPorRu. Therefore, SPPorRu@PCL also exhibits high-efficiency and broad-spectrum SOD and CAT mimetic activities in scavenging ROS. The resulting SPPorRu@PCL has high biosafety, can effectively rescue the function of human umbilical vein endothelial cells (HUVEC), can effectively relieve oxidative stress in a high-ROS environment, reduce DNA and mitochondrial damage and cell apoptosis, and promote cell adhesion of human umbilical vein endothelial cells. This makes SPPorRu@PCL show excellent biocompatibility, anti-inflammatory and blood vessel repair-promoting abilities in animal models of treating vascular injury, providing a promising approach for the development of transplantation materials for treating vascular injury and related diseases. Brief Description of the Drawings

[0042] Figure 1 Shows the preparation and characterization of SPPorRu and SPPorRu@PCL. a) Schematic diagram of the synthesis of SPPorRu; b) Average sizes of SPPorRu and KB; c) HAADF-TEM and EDS elemental mapping of Ru, N and C elements on SPPorRu; d) N2 adsorption / desorption isotherm; e) FTIR spectrum; f) High-resolution XPS pattern of N 1s and g) Ru 3p; h) Raman spectrum; i) EDS elemental mapping of Ru, N and C elements and their merged images on SPPorRu@PCL; j) Mercury intrusion porosimetry.

[0043] Figure 2 Shows the SEM images of KB, PPorRu and SPPorRu.

[0044] Figure 3 Shows the pore size distributions of SPPorRu and PPorRu.

[0045] Figure 4Shows the antioxidant-like activities of SPPorRu and SPPorRu@PCL. a) CAT-like properties and scavenging activities normalized by Ru content; b) Catalytic center poisoning test of SPPorRu biocatalyst with KSCN; c) Typical Michaelis-Menten curves and double-reciprocal plots for determining the kinetic constants of SPPorRu with H2O2 as the substrate; d) O2 generation of SPPorRu and SPPorRu@PCL; e) Comparison of CAT-like and SOD-like activities and DPPH scavenging characteristics of kSPPorRu and SPPorRu@PCL; f) Recycling activities of SPPorRu-5:1, SPPorRu-5:1.5, and SPPorRu-5:2; g) Comparison of SPPorRu with state-of-the-art ROS-scavenging nanocatalysts. Data are represented as mean ± SD from three independent experiments (n = 3).

[0046] Figure 5 Shows the live / dead staining of HUVECs incubated with SPPorRu@PCL loaded with different nanobiocatalyst contents for 24 h. Scale bar = 100 μm.

[0047] Figure 6 Shows in vitro ROS scavenging and cell protection by SPPorRu@PCL. a) Flow cytometry of ROS levels after DCFH-DA staining and b) Statistical analysis (n = 3); c) Flow cytometry cell apoptosis analysis of HUVECs under different treatment conditions and d) Quantitative analysis (n = 5); e) Levels of TNF-α and IL-1β in cell culture medium after different treatments (n = 8). P values were calculated using one-way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0048] Figure 7 Shows the HUVEC adhesion behavior. a) SEM false-color images of HUVECs adhered to PCL and SPPorRu@PCL and b) Statistics of cytoskeleton length and area (n = 6, n = 7); c) Live / dead staining of HUVECs on the membrane on the first day and growth on tubular scaffolds at 1, 3, and 5 days, green: calcein AM.

[0049] Figure 8It shows the expression of adhesion-related proteins on the membrane material under ROS stimulation. a) Representative immunofluorescence images of F-actin, CD31, and DAPI co-staining of HUVECs adhered to the membrane, and b) statistical analysis of cell spreading area (n = 7), c) cytoskeleton length (n > 3), and d) mean fluorescence of CD31 (n = 3); e) representative immunofluorescence images of vinculin of HUVECs adhered to the membrane and f) statistical analysis of fluorescence intensity of proteins expressed by single cells (n = 4). P values were calculated using one-way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0050] Figure 9 It shows the in vivo biocompatibility and anti-inflammatory effect of SPPorRu@PCL. a) In vivo demonstration of SPPorRu@PCL implantation in a rat model; b) representative implantation images and color Doppler ultrasound images showing patency of sham operation, PCL, and SPPorRu@PCL; c) representative H&E staining of blood vessel sections after implantation; d) IHC staining of TNF-α on blood vessel tissues (black arrows point to the puncture holes, scale bar = 50 μm) and e) quantitative statistics (n = 6); f) SEM images of PCL and SPPorRu@PCL after implantation (scale bar = 200 μm). P values were calculated using one-way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0051] Figure 10 It shows the effect of SPPorRu@PCL on promoting the repair of blood vessel tissues. a) Representative immunofluorescence staining of CD31 and b) quantitative statistics of the CD31 positive ratio (n = 3); c) representative immunofluorescence staining of α-SMA and d) quantitative statistics of the α-SMA positive ratio (n = 3). White arrows point to the puncture holes, scale bar = 50 μm. P values were calculated using one-way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0052] Figure 11 It shows the effect of SPPorRu@PCL on in vivo macrophage polarization in blood vessel tissues. a) Representative immunofluorescence staining of CD68 (M0 macrophages) and b) quantitative statistics of the CD68 positive ratio (n = 4); c) representative immunofluorescence co-staining of CD86 (M1 macrophages) and CD206 (M2 macrophages) and d) quantitative statistics of the CD86+ / CD206+ ratio (n = 4). P values were calculated using one-way ANOVA, *P < 0.05, **P < 0.01, ****P < 0.0001.

[0053] Figure 12Schematically shows the synthesis process of SPPorRu@PCL and the working mechanism of alleviating vascular inflammation. Detailed implementation manners

[0054] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0055] The raw materials and reagents used in the examples, comparative examples and performance tests of the present invention: RuCl3·xH2O and hexafluoroisopropanol were purchased from Aladdin Chemical Co., Ltd. (Shanghai, China). Ketjen Black (KB, ECP-600JD) was purchased from Kande New Energy Technology Co., Ltd. (Guangdong, China). 5,10,15,20-Tetrakis(4-pyridyl)-21H,23H-porphyrin (Por) was purchased from Yanshen Technology Co., Ltd. (Jilin, China). Polyvinylpyrrolidone (PVP, Mw = 58000) and hydrochloric acid (37%) were provided by Alfa Aesar (MA, USA). Polycaprolactone (PCL, Mw = 80000) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). All aqueous solutions were prepared with deionized water (DI). Unless otherwise specified, the remaining reagents were provided by Aladdin Chemical Co., Ltd. (Shanghai, China). All reagents were of analytical purity and did not require further treatment.

[0056] Example 1: Preparation of SPPorRu nanobiocatalyst

[0057] The SPPorRu nanobiocatalyst was synthesized by a coordination-driven self-assembly method. The 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin tetradentate organic ligand with a planar strong conjugate structure was coordinated and self-assembled with ruthenium on the highly conductive and highly specific carbon support Ketjen Black through π-π stacking driving to form a homogeneous porphyrin-based Ru-N coordination polymer nanobiocatalyst SPPorRu. The synthesis process is as Figure 1As shown in a. Briefly, 5,10,15,20 - tetra(4 - pyridyl) - 21H,23H - porphyrin (Por, 20 mg) and polyvinylpyrrolidone (PVP, 10 mg) were dissolved in 10 mL of 0.1 M hydrochloric acid (HCl) to form a homogeneous solution A. Subsequently, 10 mg of conductive Ketjenblack (KB) was dispersed into solution A by sonication for 30 minutes to ensure uniform distribution of the nanoparticles. Meanwhile, RuCl3·xH2O (13 mg) was dissolved in 10 mL of deionized water (DI) to prepare solution B, which was then added dropwise to solution A under constant stirring (500 rpm, 25 °C). The mixture was allowed to react for 12 hours to promote the Ru 3+ directed coordination with the porphyrin ligand and PVP / KB - mediated hierarchical assembly. The resulting SPPorRu nanocomposite was purified by performing three cycles of centrifugation (1100 rpm, 10 min) and washing with deionized water (30 mL × 3), and then vacuum - dried at 60 °C for 12 h to obtain a stable dark - purple powder, which is the SPPorRu nanobiocatalyst. Table 1 below shows the elemental atomic content of the nanobiocatalyst measured by XPS.

[0058] Table 1: Elemental atomic content of the nanobiocatalyst measured by XPS

[0059]

[0060] Example 2: Preparation of SPPorRu@PCL vascular graft

[0061] Poly(ε - caprolactone) (PCL) electrospun fiber membranes have been proven to be excellent materials for vascular grafts. SPPorRu nanoparticles were incorporated into PCL to evaluate their redox - regulating effects on HUVEC proliferation and inflammatory responses under high ROS conditions. SPPorRu / PCL precursor solutions were prepared in hexafluoroisopropanol (HFIP) using a 15% PCL concentration (w / v) and different SPPorRu nanoparticle loadings (0%, approximately 20%, approximately 30%, approximately 40% w / v). After homogenization by magnetic stirring, the solutions were loaded into 10 - mL syringes equipped with needles, and SPPorRu@PCL was obtained after electrospinning. Briefly, according to the addition amounts shown in Table 1, SPPorRu was added to 5 mL of hexafluoroisopropanol (HFIP) solvent, ultrasonically dispersed for 10 min, and 450 mg of polycaprolactone (PCL) was added. After stirring overnight at 200 rpm, the speed was adjusted to 100 rpm and stirred for 4 h for electrospinning (HZ - 03, QDHZDF); the electrospinning parameters were adjusted as follows: reciprocating distance 20 cm, roller speed 200 rpm, injection pump opened at 2 mL / h, and voltage set at 15 mV; the nanofiber membrane was collected after electrospinning.

[0062] Table 2: Added weight percentages of PCL and SPPorRu in the nanofiber membrane.

[0063] HFIP (mL) PCL (mg) SPPorRu (mg) PCL:SPPorRu PCL 5 450 SPPcRu@PCL - 5:1 5 450 90 5∶1 SPPcRu@PCL - 5:1.5 5 450 140 5∶1.5 SPPcRu@PCL - 5:2 5 450 170 5∶2

[0064] Comparative Example 1:

[0065] Except for not adding Ketjenblack (KB) to Solution A, the PPorRu material was prepared by the same method as in Example 1 as a control for the SPPorRu nanobiocatalyst.

[0066] Performance test:

[0067] Test method:

[0068] 1. Characterization of SPPorRu and SPPorRu@PCL

[0069] Fourier transform infrared (FTIR) spectroscopy (4000 - 500 cm -1 , Nicoleti S50, KBr pellets), powder X-ray diffraction (PXRD; Bruker D8 Advance, Cu Kα, 5° - 80°), X-ray photoelectron spectroscopy (XPS; Thermo K-Alpha+, Al Kα, 1486.6 eV), scanning / transmission electron microscopy with EDS elemental mapping (SEM: ApreoS HiVoc; TEM: Talos F200S, 200 kV) and hydrodynamic diameter analysis (Malvern Zetasizer NanoZS) were used to characterize the chemical bonds, crystallinity, elemental state, morphology / composition and colloidal stability of Por, PPorRu and SPPorRu, respectively. Raman spectra were recorded on a Horiba XploRA PLUS system (Thermo Fisher, USA) with a 532 nm laser. The fiber pore size distribution was determined by mercury intrusion porosimetry (PoreMaster 33, Quantachrome, USA).

[0070] 2. Enzyme-like catalytic performance of SPPorRu

[0071] The catalase-mimicking activity of the nanobiocatalyst was evaluated by quantitative H2O2 decomposition (measured at 405 nm by Ti(SO4)2-based spectrophotometry) and O2 generation (real-time dissolved oxygen monitoring). A reaction mixture of PBS (pH 7.4, 37 °C) containing 50 μg / mL catalyst and 10 mM H2O2 was incubated for 30 minutes, and then the residual H2O2 was detected by forming a titanium(IV) oxysulfate complex (50 μL sample + 100 μL 13.9 mM Ti(SO4)2 solution) at reaction times of 0.5, 1, 2, 3, 4, and 5 minutes. The absorbance of the solution at 405 nm was measured to evaluate the remaining H2O2 concentration. The evaluation of O2 production from H2O2 catalysis was as follows: 100 mM H2O2 and 2.5 μg mL -1 of the nanobiocatalyst were mixed in 20 mL of PBS, and then the dissolved oxygen (DO) concentration was continuously monitored for 5 minutes using a calibrated oxygen meter (model JPSJ-605F, Yidian Scientific Instrument Co., Ltd., Shanghai, China), and data were recorded at intervals of 5 / 10 seconds throughout the measurement period. The cycling test was the CAT activity measured after every 30 minutes of reaction.

[0072] In-situ Fourier transform infrared (FTIR) reflection spectra were obtained using a Nicolet iS50 spectrometer (Thermo Fisher Scientific, USA) equipped with a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector. The CAT-like activity tested by in-situ FTIR was used as the CAT-like catalytic activity test.

[0073] The reaction rate values and substrate concentrations were fitted to the Michaelis-Menten equation to calculate the kinetic constants Vmax and TON (the maximum number of substrate exchanges per unit active catalytic center) of the enzymatic reaction, and the CAT-like activity of the nanobiocatalyst was evaluated as follows, where [E0] represents the molar concentration of the metal in the nanomaterial.

[0074]

[0075] The SOD-like activity was tested by evaluating ·O2 - scavenging. ·O2 - was generated from 1 mg of KO2 (Sigma-Aldrich, MO, USA) in 1 mL of 18-crown-6 (Aladdin, Shanghai, China) dimethyl sulfoxide (DMSO) solution (3 mg mL -1 ). Then 50 μgmL -1 of the nanobiocatalyst was dispersed into the KO2 / DMSO solution. After 5 minutes of reaction, 10 μL of nitroblue tetrazolium (NBT)-DMSO solution (10 mg mL -1NBT (Aladdin, Shanghai, China) was used to measure the remaining ·O2 - The absorbance of the test solution at 680 nm was then measured, and the ·O2 - scavenging ability was quantitatively evaluated.

[0076] The antioxidant activity of the nanobiocatalyst was evaluated by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Briefly, 1 mL of DPPH ethanol solution (50 μg / mL; Aladdin Biochemical Technology Co., Ltd.) was mixed with 1 mL of nanobiocatalyst suspension (PBS solution at 12.5 μg / mL, pH 7.4) to form a 2 mL reaction system. After incubation in the dark at 25 °C for 30 minutes, the absorbance at 519 nm was measured using a UV-vis spectrophotometer (Shimadzu UV-2600).

[0077] 3. Determination of biocompatibility and blood compatibility.

[0078] To evaluate the biocompatibility of the fiber membrane, human umbilical vein endothelial cells (HUVECs) were cultured in complete medium containing 90% DMEM and 10% fetal bovine serum (FBS). HUVECs seeded at 2.0×10 4 cells / well were incubated with sterilized PCL, SPPorRu@PCL-5:1, SPPorRu@PCL-5:1.5, and SPPorRu@PCL-5:2 for 1 day. HUVECs were also cultured in untreated wells as a control (normal group). The Calcein-AM / PI kit (Yeasen, China) was used for live / dead staining assays.

[0079] Hemolysis assays and complete blood counts were used to evaluate the blood compatibility of the membranes. Blood cells were collected from human volunteers at West China Hospital and approved by the Institutional Review Board of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences (IRB, Ethical Approval No. 202024)

[0080] 4. In vitro ROS scavenging activity

[0081] The DCFH-DA kit (Beyotime, China) was used to detect intracellular ROS and evaluate the ROS scavenging ability of SPPorRu@PCL. Cells seeded at 2.0×10 4HUVECs seeded at a density of [number of cells] per well were incubated overnight and then incubated with PCL and SPPorRu@PCL membranes (area 1 cm × 1 cm) in the presence of 200 μM H2O2 for 3 hours. Subsequently, the DCFH-DA fluorescent indicator was added to the culture chamber and co-cultured with the cells at 37 °C for 30 minutes. Then, intracellular fluorescence was detected by an automated inverted fluorescence microscope (Olympus IX83, Japan). DCFH-DA quantification under 300 μM H2O2 was detected by flow cytometry.

[0082] 5. Flow cytometry for apoptosis analysis

[0083] HUVECs seeded at a density of 1×10 4 cells / mL were allowed to adhere overnight and then incubated with the fibrous membrane in the presence of 400 μM H2O2 for 1 day. Then, HUVECs were stained with an Annexin V-FITC / PI apoptosis detection kit (Yeasen, China) for flow cytometry analysis.

[0084] 6. ELISA kit detection

[0085] HUVECs seeded at a density of 1×10 4 cells / mL were allowed to adhere overnight and then incubated with the fibrous membrane in the presence of 400 μM H2O2 for 1 day. After that, the supernatant was taken for analysis. The concentrations of TNF-α and IL-1β in the culture supernatant were measured using an ELISA kit (Ruixinbio, China) according to the standardized procedure provided by the supplier. Serum obtained by centrifuging rat blood was collected for detection using rat IL-1β and TGF-β ELISA kits.

[0086] 7. Adhesion behavior of HUVECs on SPPorRu

[0087] SEM images of HUVECs adhered to the film were obtained by the following procedure. HUVECs at a density of 1.0×10 4 cells per well were seeded on sterilized PCL and SPPorRu@PCL membranes placed in 24-well plates and incubated together overnight. They were washed 3 times with PBS, fixed with 4% Paraformal Dehyde (Biosharp, China) at room temperature for 15 minutes, the fixative was aspirated, rinsed 3 times with PBS, dehydrated stepwise with ethanol (ethanol concentrations were 30%-50%-70%-80%-90%-100% in sequence), and left at each concentration for 15 minutes. Finally, the ethanol was aspirated, air-dried naturally, and observed by SEM (Thermo Scientific, USA).

[0088] CLSM images of cell growth on the fibrous membranes and tubular materials were obtained through the following procedures. HUVECs were seeded onto sterile PCL and SPPorRu@PCL membranes (including tubular constructs) pre-positioned in 24-well plates at a density of 1.0×104 cells / well and then incubated overnight under standard culture conditions. The live / dead staining assay of cells on the membranes was obtained using the Calcein-AM / PI Kit (Beyotime, China), and the live cell staining assay of cells on the tubular materials was obtained by Calcein-AM staining, and then detected by confocal laser scanning microscopy (CLSM, Leica, Germany).

[0089] 8. In vivo implantation safety

[0090] Seven-week-old male Sprague-Dawley rats (280 - 320 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd., and all animal experiments were conducted according to the protocol approved by the Institutional Animal Care and Use Committee of Sichuan University (No. 20230818003). Color Doppler ultrasound (Mindray, China) was used to monitor the patency and diameter of blood vessels at the material implantation site. The rats were first anesthetized with 2% isoflurane, and then the blood vessels at the material embedding site were detected in color Doppler mode. Histopathological evaluation of major organs (heart, liver, spleen, lung, and kidney) was performed by hematoxylin and eosin (H&E) staining to evaluate the systemic toxicity after material implantation. And routine blood tests were used to evaluate the blood compatibility of the materials.

[0091] 9. Histological analysis

[0092] Seven days after implantation, the grafts were fixed in 4% paraformaldehyde (PFA), and the vascular tissues around the grafts were processed for histopathological and immunofluorescence evaluations. Scanning electron microscopy (SEM; Hitachi SU8010) was used to examine the surface of the fibrous grafts.

[0093] Histological evaluation included H&E staining, as well as immunohistochemistry (IHC) and immunofluorescence (IF) to analyze the local inflammatory response and vascular repair. Vascular inflammation was quantified by immunostaining for TNF-α and inducible nitric oxide synthase (iNOS). Multiplex immunofluorescence staining was performed to characterize cell components: anti-CD31 for endothelial cells (ECs), anti-α-SMA for smooth muscle cells (SMCs), anti-CD68 for pan-macrophages, anti-CD86 for M1 pro-inflammatory macrophages, and anti-CD206 for M2 anti-inflammatory macrophages.

[0094] The antibodies and reagents used in the in vivo experiments of this study were anti-CD68 antibody [ED1] (Abcam, UK), anti-CD86 antibody [OX48] (Abcam, UK), CD206 / MRC1 (E6T5J) Rabbit monoclonal antibody (CST, USA), recombinant anti-CD31 antibody [EPR17259] (Abcam, UK), α-smooth muscle actin (D4K9N) Rabbit monoclonal antibody (CST, USA), IL-6 rabbit monoclonal antibody (Abclonal, China), TNF-α rabbit pAb (Abclonal, China). Immunofluorescence co-staining used secondary antibody dyes, including opal 520 reagent (Akoya Biosciences, USA), opal 570 reagent (Akoya Biosciences, USA) and opal 650 reagent (Akoya Biosciences, USA)

[0095] 10. Statistical analysis

[0096] Statistical analysis was performed in GraphPad Prism 9.0. Data represent mean ± SD of three independent biological replicates. Inter-group differences were evaluated by one-way ANOVA with Tukey's post hoc test (for multiple groups) or unpaired two-tailed t-test (for two groups), and significance was set at p < 0.05.

[0097] Test results:

[0098] 1. Structural characterization of SPPorRu nano-bio-catalyst and SPPorRu@PCL membrane

[0099] Structural characterization of SPPorRu. The nano-bio-catalyst SPPorRu with an average size of 47 nm was revealed by scanning electron microscopy (SEM) ( Figure 1 b, Figure 2 ), indicating that its diameter is larger than that of KB (25 nm), and compared with PPorRu, the particle morphology is smaller and more uniform. Images of high-angle annular dark-field TEM (HAADF-TEM) and the corresponding energy-dispersive spectroscopy (EDS) ( Figure 1 c) revealed the uniform distribution of C, N and Ru elements on KB. In addition, the nitrogen adsorption-desorption isotherms of SPPorRu and PPorRu at 77 K ( Figure 1 d, Figure 3 ) revealed their microporous structures. The surface area of PPorRu was only about 23 m 2 g -1 , while the addition of nano-carbon increased the surface area of SPPorRu (130 m 2 g -1)。The large specific surface area and porous structure of SPPorRu are beneficial for the more exposure of active sites and the complete contact of reactants. These results indicate that the successful encapsulation of PPorRu on KB results in SPPorRu showing a smaller and more uniform nanoparticle morphology, as well as a larger specific surface area and pore size, thus contributing to the more exposure of active sites.

[0100] In addition, we continued to study the specific chemical structure of SPPorRu. First, the N-H absorption peak at 3300 cm -1 of PPorRu and SPPorRu measured by Fourier transform infrared spectroscopy (FTIR) disappeared ( Figure 1 e), proving the formation of pyrrole Ru-N sites in the porphyrin. In addition, X-ray photoelectron spectroscopy (XPS) was used to study the Ru coordination environment. According to the N1s spectrum of XPS ( Figure 1 f), compared with porphyrin (Por), PPorRu showed peak shifts of ─N═ from 396.72 eV to 397.09 eV, pyrrole nitrogen from 397.62 eV to 398.59 eV, and ─NH from 398.77 to 399.1 eV, respectively, confirming the successful assembly of the Ru-N coordination polymer network. In addition, due to the π-electron interaction between PPorRu and KB, compared with PPorRu, SPPorRu has peak shifts of ─N═ (398.25 eV), pyrrole N (399.6 eV), and ─NH (400.13 eV). In addition, XPS analysis of the Ru3p spectrum shows that Ru is in the cationic state ( Figure 1 g). In addition, Raman spectroscopy reveals the π-π stacking interaction between the sp 2 -hybridized porphyrin-Ru complex (PPorRu) and the highly conductive Ketjen black (KB) support. As shown in the Raman spectrum ( Figure 1 h), compared with KB, SPPorRu shows an 8 cm -1 blue shift in the in-plane vibration (G band at 1582 cm -1 ), indicating that PPorRu attached to KB shows electron-donating properties. In addition, some small peaks also appear in the spectrum of SPPorRu at the Raman band positions corresponding to PPorRu: 1001.3 cm -1 ; 1144.6 cm -1 ; 1247.4 cm -1 ; 1450.4 cm -1 ; 1498.3 cm -1 ; 1553.8 cm -1These results indicate that PPorRu is connected to KB through π-π stacking interactions to form SPPorRu. There is no significant difference in the ID / IG intensity ratio between KB (1.20) and SPPorRu (1.07), indicating that SPPorRu retains the average sp2 domain size and thus can provide high electrical conductivity and fast electron transfer ability. These characterizations demonstrate the successful synthesis of the nanoporous porphyrin-Ru nanobiocatalyst supported on nanocarbon.

[0101] Structural characterization of SPPorRu@PCL. First, structural characterization was carried out to verify the successful encapsulation of SPPorRu nanoparticles into PCL fibers. The EDS mapping results ( Figure 1 i) show that Ru and N accumulate at the nanoparticles, while C exhibits a selective distribution pattern for the fibers, indicating the successful incorporation of SPPorRu into PCL fibers. The pore size distribution of the membrane was characterized by mercury intrusion porosimetry ( Figure 1 j), and the incorporation of SPPorRu led to a larger pore size of the membrane (average pore size 29404 nm) compared to the PCL membrane (average pore size 12741 nm).

[0102] 2. Evaluation of the biocatalytic activities of SPPorRu and SPPorRu@PCL

[0103] After verifying the chemical structures of the nanobiocatalysts, we further tested how their structures affect the enzyme-catalytic properties for scavenging ROS and the broad-spectrum ROS-scavenging catalytic activities of SPPorRu and SPPorRu@PCL. The ROS-scavenging ability, including CAT-like and SOD-like abilities, was scientifically studied and compared. First, the H2O2 scavenging rate was used to determine the CAT-like activity. Both PPorRu and SPPorRu exhibited high CAT-like H2O2 scavenging activities, but after normalization according to the Ru content measured by XPS (Table 1), the catalytic efficiency of SPPorRu was about twice higher than that of PPorRu ( Figure 4 a). This result confirms that the addition of KB enhances the exposure of active sites, thus improving the catalytic efficiency of Ru atoms. In addition, the importance of Ru in the biocatalyst was demonstrated by using potassium thiocyanate (KSCN, which can irreversibly adsorb to the metal active center through strong coordination competition, resulting in a decrease in catalytic activity) to poison the catalytic active center. After adding KSCN, the SPPorRu biocatalyst showed a sharp decrease in CAT-like activity ( Figure 4 b), thus indicating that the Ru-N site is the main catalytic active center for scavenging ROS.

[0104] In addition, catalytic kinetics was evaluated by determining the maximum reaction rate (Vmax) and turnover number (TON, the number of molecules converted per active site per second). SPPorRu with a large Vmax (36.80 μM·s -1 ) and a high TON (12.65 s -1 ) exhibited excellent CAT-like activity ( Figure 4 c), indicating that the highly active Ru-N catalytic sites with π-conjugation and the π-π stacking effect synergistically promoted the electron transfer efficiency and the exposure of active sites, thus enhancing the catalytic kinetics of the SPPorRu nanobiocatalyst.

[0105] Then, after verifying the excellent catalytic ability of SPPorRu, we next explored the ROS scavenging performance of SPPorRu@PCL functionalized with the SPPorRu biocatalyst. The SPPorRu@PCL fiber membrane showed good O2 generation ( Figure 4 d). The SPPorRu nanobiocatalyst and the SPPorRu@PCL fiber membrane showed CAT-like and SOD-like activities and DPPH· scavenging ( Figure 4 e). To reveal the long-term ROS scavenging activity, the cycling activity was evaluated, and the results showed that there was no significant change in the activity between the fiber membranes during 5 cycles, indicating a stable catalytic ability ( Figure 4 f). In addition, the TON values of SPPorRu and other recently reported antioxidant enzyme-like nanobiocatalysts (such as Ru@CoSe nanolayers, Mn3O4 nanoflowers, and Ru-CN nanoparticles) were studied ( Figure 4 g), which clearly showed that SPPorRu had the most prominent CAT-like activity among the reported biocatalysts. Overall, these results indicated that the SPPorRu nanoparticles and SPPorRu@PCL exhibited efficient and broad-spectrum ROS scavenging capabilities.

[0106] 3. In vitro ROS Scavenging, Anti-inflammatory, and Cytoprotective Effects of SPPorRu@PCL

[0107] After demonstrating that SPPorRu@PCL showed significant and multifunctional antioxidant enzyme mimicking activities, we systematically evaluated its application potential in scavenging ROS and regulating human umbilical vein endothelial cells (HUVECs) under oxidative stress conditions. First, the cell biocompatibility of the nanobiocatalyst on HUVECs was analyzed. After incubating HUVECs with SPPorRu@PCL with different nanobiocatalyst contents for 24 hours, almost no dead cells were observed by live / dead assay ( Figure 5), which confirmed the non-toxicity of the SPPorRu@PCL fiber membrane (especially the SPPorRu@PCL-5:1.5 fiber membrane) as a favorable cell proliferation substrate. Therefore, SPPorRu@PCL-5:1.5 loaded with 30% SPPorRu was used as the experimental group in subsequent experiments.

[0108] Thereafter, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was applied as a fluorescent probe to explore the catalytic ROS scavenging ability of SPPorRu@PCL on the intracellular ROS level of HUVEC cells under H2O2 treatment. After treatment with H2O2, quantitative analysis by flow cytometry could observe obvious green fluorescence in HUVEC Figure 6 a), indicating a very high intracellular ROS level. Notably, the green fluorescence signal could be significantly weakened after treatment with SPPorRu@PCL, while the PCL group had no obvious effect on reducing the ROS level Figure 6 b). These results indicate that SPPorRu@PCL exhibits very effective ROS scavenging ability and can be used to regulate the fate of HUVEC under conditions of high ROS levels. Long-term and high ROS stimulation exacerbated oxidative stress and further induced apoptosis of HUVEC, while cells co-incubated with SPPorRu@PCL significantly reduced apoptosis caused by oxidative stress Figure 6 c, 6d). Overall, our results show that SPPorRu@PCL can reduce DNA and mitochondrial damage, and even apoptosis caused by oxidative stress, through ROS scavenging.

[0109] In addition, the ROS microenvironment may also lead to severe inflammatory responses in cells, and our results show that SPPorRu@PCL treatment reduced the expression of TNF-α and IL-1β Figure 6 e). In summary, the above results indicate that SPPorRu@PCL can act as an antioxidant to reduce ROS, thereby reducing DNA and mitochondrial damage, and even apoptosis induced by high ROS conditions, thus showing its anti-inflammatory effect.

[0110] 4. In vitro growth regulation of HUVEC by SPPorRu@PCL

[0111] Electrospun fiber membranes not only serve as a supporting matrix for cell proliferation and migration, but the interfaces between the fibers also provide sufficient roughness and physical sites for cell adhesion and growth. Therefore, we investigated the growth status of HUVEC on the fiber membranes. After culturing on the fiber membranes for 1 day, SEM showed that most of the HUVEC in the exposed PCL membranes were spherical and not spread, while the cells in the SPPorRu@PCL had more cell pseudopodia, with a diffuse and elongated morphology, and a larger spreading area and aspect ratioFigure 7 a, 7b). Live / dead staining on the cell membrane also provided further evidence of good cell viability. Then, HUVECs were seeded onto the membrane and cultured continuously for 5 days for fluorescence staining and confocal imaging. Figure 7 Panel c shows that HUVECs grew well on the fibrous membrane, gradually growing to cover the fibrous membrane wall, with a good cell density on SPPorRu@PCL, forming an integrated vascular endothelial morphology, which provided the feasibility of artificial blood vessels.

[0112] The extracellular matrix environment stimulates a series of cell signal transduction reactions, and the expression of PECAM-1 (CD31) and cytoskeletal proteins in HUVECs play key roles in cell movement, spreading, and differentiation. Therefore, the growth and adhesion of HUVECs were further evaluated under elevated ROS conditions to elucidate the protective effect of SPPorRu@PCL. As Figure 8 shown in panel a, under high ROS conditions, HUVECs on PCL showed reduced spreading, cytoskeletal retraction (red), and decreased cell area, as well as a significant decrease in CD31 expression (green). In contrast, HUVECs grown on SPPorRu@PCL maintained their characteristic polygonal shape morphology and showed significantly elongated cytoskeletal organization, as well as a larger spreading area and higher CD31 expression ([[]] Figure 8 b-d).

[0113] Focal adhesion protein is crucial for focal adhesion complex assembly and signal transduction. Under high ROS conditions, H2O2-treated HUVECs showed lower focal adhesion protein expression on the PCL membrane ( Figure 8 e, 8f), but showed higher fluorescence intensity on the SPPorRu@PCL fibrous membrane, indicating enhanced adhesion ability of HUVECs under oxidative stress conditions. These results suggest that compared with PCL alone, SPPorRu@PCL can provide effective protection against ROS damage and defend against the oxidative stress stretching state of vascular endothelial cells.

[0114] 5. In vivo vascular injury model for SPPorRu@PCL treatment

[0115] Given the good protective effect of SPPorRu@PCL on HUVEC cells in an oxidative stress environment, we further explored its in vivo anti-inflammatory and vascular repair effects induced by stressors such as vascular injury and inflammation in a Sprague-Dawley rat animal model. Due to the difficulty of transplantation surgery, we referred to a previously reported vascular injury model in which holes were punched in the abdominal aortic blood vessels, and then SPPorRu@PCL or PCL was wrapped around the injured blood vessels to prevent leakage and failure. The schematic diagram details the step-by-step surgical procedure performed in the rat model ( Figure 9a). Color Doppler ultrasound examination 7 days later showed that at 7 days after surgery, both types of membranes remained patent around the perforation, did not cause blood vessel swelling, and there was no difference in blood flow rate ( Figure 9 b).

[0116] Vascular tissue at the puncture site was obtained and stained with H&E. Compared with the sham operation group, there was obvious infiltration of inflammatory cells at the vascular puncture site in the PCL group, while the inflammation in the SPPorRu@PCL was significantly alleviated. Figure 9 c). Further immunohistochemical (IHC) staining and statistical analysis of the pro-inflammatory cytokine TNF-α showed that the expression in the SPPorRu@PCL group was significantly lower than that in the PCL group (9d, 9e). These results indicate that SPPorRu@PCL can be considered an antioxidant that can protect vascular tissue from inflammation at the puncture site. In addition, adhesion of blood-related substances (including blood cells, platelets, and proteins) was examined by SEM imaging, and no obvious cell adhesion was observed. Figure 9 f).

[0117] Next, we measured the vascular repair effect. Immunofluorescence staining images showed that there were more CD31-positive cells lining the intima in the SPPorRu@PCL group than in the PCL group. Figure 10 a, 10b). In the images stained with α-SMA and quantitative statistics, more positive cells were observed, mainly smooth muscle cells and myofibroblast phenotypes infiltrated into the proliferative SPPorRu@PCL layer to increase the stability and contractile function of the new intima. Figure 10 c, 10d).

[0118] Macrophages play a key role in vascular inflammation and remodeling. Therefore, we further evaluated macrophage infiltration and polarization in different vascular grafts. Immunofluorescence staining was used to identify the numbers of pan-macrophages (M0), pro-inflammatory, and anti-inflammatory macrophages of CD68, CD86, and CD206, respectively. The number of CD68+ cells in the SPPorRu@PCL graft was lower than that in the PCL graft, indicating a reduction in inflammatory cell infiltration. Figure 11 a, 11b). Importantly, more CD86-positive cells (M1 macrophages) and CD206-positive cells (M2 macrophages) infiltrated into the same SPPorRu@PCL area compared with PCL. Figure 11 c). However, the ratio of M2 / M1 phenotypes of SPPorRu@PCL was higher than that of PCL, confirming that the polarization switch provides an anti-inflammatory microenvironment for the remodeling response. Figure 11d). Therefore, in vivo experiments showed that the SPPorRu@PCL membrane could reduce macrophage recruitment, promote the polarization of macrophages from M1 to M2, reduce inflammation, thereby promoting the proliferation of endothelial cells and smooth muscle cells, facilitating vascular repair, and could be used as a vascular graft.

[0119] 7. Conclusions

[0120] The research of the present invention shows that the synthesized novel antioxidant enzyme-like SPPorRu@PCL is an ideal and effective vascular graft, which can mimic anti-ROS enzymes to combat oxidative stress and inflammatory vascular damage in the HUVEC microenvironment. Structural and functional analyses show that SPPorRu@PCL exhibits excellent H2O2 and ·O2 - scavenging activities, which are attributed to the loaded SPPorRu integrating π-conjugated Ru-N catalytic sites, π-π stacking effects, and porous structures. These characteristics synergistically enhance the electron transfer ability and catalytic kinetics (Vm ax = 36.80 μM·s -1 and TON = 12.65 s -1 ), which are superior to all reported enzyme-mimicking antioxidants in scavenging ROS. In vitro and in vivo experiments also showed that SPPorRu@PCL could significantly reduce oxidative stress and inflammation, rescue the survival state of HUVECs, relieve inflammation, and promote the regeneration and repair of vascular tissues, Figure 12 summarizing the synthesis process of SPPorRu@PCL and the working mechanism of alleviating vascular inflammation. This vascular graft modified with an efficient artificial antioxidant of the present invention will have promising clinical significance for vascular injury.

[0121] It should be noted that the description and drawings of the present invention give preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments do not serve as additional limitations to the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. Moreover, the above technical features continue to be combined with each other to form various embodiments not listed above, all of which are regarded as the scope described in the description of the present invention; further, for those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. A nano-carbon supported porous porphyrin-Ru-based nano-bio-catalyst, characterized in that, The catalyst is a porous material formed by a coordination polymer in which Ru is connected to porphyrin through Ru-N coordination and is supported on nano-carbon through π-π conjugation.

2. The nano-biological catalyst according to claim 1, wherein The porphyrin is a porphyrin having a pyridyl group; Further, the porphyrin is at least one of 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin, tetrakis(2-pyridyl)porphyrin, and 5,15-bis(4-pyridyl)-10,20-diphenylporphyrin.

3. The nano-biological catalyst according to claim 1, characterized in that, The nano-carbon is Ketjen black, such as Ketjen black ECP-600JD.

4. The nano-biological catalyst according to claim 1, characterized in that, The catalyst has SOD and CAT-like activities and DPPH· scavenging performance.

5. The preparation method of the nano-carbon supported porous porphyrin-Ru-based nano-bio-catalyst according to any one of claims 1-4, characterized in that, It includes co-blending and reacting a porphyrin compound, a Ru salt, and nano-carbon in water to obtain the biocatalyst.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the porphyrin compound to the Ru salt is 1:0.5 to 4; Preferably, the molar ratio of the porphyrin compound to the Ru salt is 1:1.8 to 2.2; Further, the porphyrin is a porphyrin having a pyridyl group; Further, the porphyrin is at least one of 5,10,15,20-tetrakis(4-pyridyl)-21H,23H-porphyrin, tetrakis(2-pyridyl)porphyrin, and 5,15-bis(4-pyridyl)-10,20-diphenylporphyrin; Further, the Ru salt is RuCl3·xH2O; Further, the nano-carbon is Ketjen black, such as Ketjen black ECP-600JD; Further, the reaction conditions are to react at room temperature for 6 - 24 h; Further, the preparation method further includes adding an acid and a polymer surfactant to the co-blended solution; Further, the acid is hydrochloric acid, and the concentration of the hydrochloric acid is 0.01 M - 0.1 M; Further, the polymer surfactant is polyvinylpyrrolidone; Further, the preparation method includes dissolving a porphyrin compound and a polymer surfactant in an aqueous solution of an acid to form a homogeneous solution A; Dispersing the nano-carbon in solution A; Dissolving the Ru salt in water to obtain solution B; Then, dropwise adding solution B to solution A under stirring, and obtaining the biocatalyst after reaction.

7. A vascular graft, characterized in that, The vascular graft is a polycaprolactone fiber membrane incorporated with the nano-carbon-supported porous porphyrin-Ru-based nano-biocatalyst as described in any one of claims 1 - 4.

8. The method for preparing a vascular graft according to claim 7, wherein It includes dispersing the nano-carbon-supported porous porphyrin-Ru-based nano-biocatalyst as described in any one of claims 1 - 4 in an organic solvent, adding polycaprolactone, fully stirring and mixing, and obtaining the vascular graft by electrospinning; Further, the mass ratio of polycaprolactone to the biocatalyst is 5:1 to 2; Further, the organic solvent is hexafluoroisopropanol.

9. Use of the nano-carbon-supported porous porphyrin-Ru-based nano-biocatalyst as described in any one of claims 1 - 4 or the vascular graft as described in claim 7 in the preparation of a biomaterial having antioxidant activity; Further, the antioxidant activity includes SOD and CAT-like activities; Further, the antioxidant activity includes ROS scavenging activity.

10. Use of the vascular graft according to claim 7 in the preparation of a biomaterial having antioxidant stress, anti-inflammatory, and functions of promoting vascular injury repair and regeneration.

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