Tissue engineering heart valve stent with sulfonated nano-enzyme coating as well as preparation method and application of tissue engineering heart valve stent

Abstract

The invention provides a tissue engineering heart valve stent with a sulfonated nano-enzyme coating as well as a preparation method and application of the tissue engineering heart valve stent, and belongs to the technical field of medical materials. The invention relates to a tissue engineering heart valve stent with a sulfonated nano-enzyme coating, which comprises an acellular matrix carrier, ruthenium / iridium-tannic acid nano-enzyme loaded on the acellular matrix carrier, and a sulfonated polymer coating fixed on the acellular matrix carrier and / or the surface of nano-particles through ultraviolet polymerization, the ruthenium / iridium-tannic acid nano-enzyme is formed by tannic acid TA, Ru < 3 + > and Ir < 3 + > through coordination. According to the invention, through double bionic activities of superoxide dismutase and nitric oxide synthase of ruthenium / iridium-tannic acid nano-enzyme and through the super-hydrophilicity and electrically neutral surface of the sulfonated polymer coating, collaborative regulation and control of O2-NO cascade catalysis and interface functions in a pathological microenvironment are realized; the structural and functional in-situ regeneration of the tissue engineering heart valve stent is promoted.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and specifically relates to a tissue-engineered cardiac valve stent with a sulfonated nanoenzyme coating, its preparation method, and its application. Background Technology

[0002] Valvular heart disease (VHD) is a serious cardiovascular disease that threatens human health worldwide. Currently, mechanical valve replacement and bioprosthetic valve replacement remain the main treatment methods in clinical practice. However, mechanical valves are prone to thrombosis due to insufficient biocompatibility of the materials, requiring patients to receive lifelong anticoagulation therapy, which significantly increases the risk of bleeding. While bioprosthetic valves have better blood compatibility, they have defects such as calcification and structural degradation, resulting in a short clinical lifespan.

[0003] In recent years, in situ tissue-engineered heart valves (TEHVs) have attracted widespread attention as a promising new treatment strategy. This technology guides the migration, proliferation, and differentiation of the patient's own cells through the implantation of a biodegradable cell-free scaffold, ultimately achieving in situ regeneration of the valve structure and restoration of function, and is expected to overcome the limitations of traditional artificial valves in terms of thrombosis risk and lifespan. Decellularized matrix (dECM) materials are considered an ideal TEHV scaffold material due to their similar mechanical properties to natural valves and good biodegradability.

[0004] However, clinical studies have shown significant individual differences in the effectiveness of heart valve regeneration, especially in elderly patients with a higher failure rate. This phenomenon is closely related to the patient's specific pathological microenvironment. Multiple pathological factors (such as diabetes, dyslipidemia, chronic kidney disease, and hypertension) and risk factors (such as aging and smoking) can significantly impair the cardiovascular system's repair capacity. The underlying mechanism is that such pathological states cause elevated levels of reactive oxygen species (ROS), inducing oxidative stress and thus creating a cardiovascular microenvironment unfavorable to tissue regeneration. In this type of microenvironment, superoxide anions (O2)... - Oxidative stress is a key toxic substance that damages endothelial cells (ECs). Excessive accumulation of oxytocin can lead to DNA oxidative damage, lipid peroxidation, and mitochondrial dysfunction in endothelial cells, thereby inhibiting endothelial cell proliferation and migration and promoting apoptosis. Simultaneously, oxidative stress also inhibits the activity of endothelial nitric oxide synthase (eNOS), reducing the production of nitric oxide (NO) and superoxide anion (O2). - Excessive accumulation of O2 - It can react rapidly with limited amounts of NO to produce highly toxic peroxynitrite (ONOO). -It can not only directly damage endothelial cells, further exacerbating endothelial dysfunction, but also cause the functional decoupling of eNOS, causing it to switch from producing NO to producing more O2. - This completely disrupts the biological homeostasis of NO. The loss of NO homeostasis weakens its key inhibitory role in platelet activation and aggregation, leading to a sharp increase in the risk of thrombosis in decellularized extracellular matrix (dECM) scaffolds in the early post-implantation period. Early thrombosis not only acutely obstructs the valve opening but also forms a physical barrier that severely hinders the migration and spread of endothelial cells to the scaffold surface, disrupts the endothelialization process, and severely impedes the in situ regeneration and functional recovery of the heart valves.

[0005] In existing technologies, researchers have attempted to use transition metal ions (such as Cu) 2+ Zn 2+ Fe 2+ This catalyzes the decomposition of endogenous S-nitrosothiols (RSNOs) to locally release NO, thereby promoting the endothelialization process of cardiovascular implants; however, in the presence of O2... - In excessively high levels of pathological microenvironment, NO is easily and rapidly quenched, leading to a significant decrease in its bioavailability, thus limiting the effectiveness of single NO release strategies; in addition, high concentrations of O2... - It is a powerful inflammatory signaling molecule that can drive macrophages to polarize towards a pro-inflammatory (M1) phenotype, continuously releasing inflammatory factors such as TNF-α and IL-1β, exacerbating the local inflammatory response, leading to excessive activation of fibroblasts and disordered extracellular matrix metabolism, causing abnormal collagen deposition and tissue fibrosis, and simultaneously accelerating the oxidative degradation of TEHVs scaffolds, ultimately resulting in the loss of elasticity and function of the regenerated valve. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the existing technology by providing a tissue-engineered cardiac valve scaffold with a sulfonated nanozyme coating, its preparation method, and its application. The scaffold is surface-loaded with ruthenium / iridium-tannic acid nanozymes and undergoes sulfonation modification, enabling the utilization of O2 in a pathological microenvironment. - - The synergistic regulation of NO cascade catalysis and interface function promotes the structural and functional in situ regeneration of tissue-engineered cardiac valve stents under complex pathological conditions, thereby improving the valve regeneration power under complex pathological conditions.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a tissue-engineered cardiac valve scaffold with a sulfonated nanoenzyme coating, comprising a decellularized matrix carrier, a ruthenium / iridium-tannic acid nanoenzyme loaded on the decellularized matrix carrier, and a sulfonated polymer coating fixed to the surface of the decellularized matrix carrier and / or the nanoparticles by ultraviolet light polymerization, wherein the ruthenium / iridium-tannic acid nanoenzyme is a mixture of tannic acid (TA) and Ru.3+ Ir 3+ Formed through coordination.

[0008] Preferably, the ruthenium / iridium-tannic acid nanozyme has dual bioactivity as both superoxide dismutase and nitric oxide synthase; in the ruthenium / iridium-tannic acid nanozyme, the molar ratio of ruthenium to iridium is 1:4 to 4:1, and the ratio of the total molar amount of ruthenium and iridium to the molar amount of tannic acid is 1:1.

[0009] Preferably, the sulfonated polymer coating is a 3-sulfonopropyl methacrylate potassium salt polymer coating.

[0010] Preferably, the decellularized matrix carrier is one of a decellularized pericardium, a decellularized aortic valve, a decellularized pulmonary valve, a decellularized mitral valve, or a tricuspid valve.

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned tissue-engineered cardiac valve stent with a sulfonated nanozyme coating, comprising the following steps: 1) The decellularized matrix carrier was immersed in a ruthenium / iridium-tannic acid nanozyme solution, and the adsorption reaction was carried out by shaking. After washing with deionized water, the nanozyme-loaded scaffold was obtained. 2) The nanozyme-loaded scaffold obtained in step 1) was immersed in N-(3-aminopropyl)methacrylamide hydrochloride solution for a period of time. After being taken out and washed with deionized water, the scaffold was immersed in a mixed solution containing sulfonated monomer and photoinitiator I2959 for 2 hours. After being taken out, it was sandwiched between two pieces of quartz glass for ultraviolet light irradiation crosslinking reaction. After washing with deionized water, the tissue-engineered heart valve scaffold with sulfonated nanozyme coating was obtained.

[0012] Preferably, the preparation method of the ruthenium / iridium-tannic acid nanozyme is as follows: a quantitative amount of tannic acid, RuCl3 and IrCl3 are dissolved in ultrapure water, then the solutions are mixed evenly, the pH of the mixed solution is adjusted to 8-10 with an alkaline solution, and the reaction is carried out at 80-90℃ for 24-48 hours. Finally, the reaction product is purified by dialyzing for 3-5 days to obtain ruthenium / iridium-tannic acid nanoparticles.

[0013] Preferably, the concentration of the ruthenium / iridium-tannic acid nanozyme solution in step 1) is 5-20 cm⁻¹. 2 / mL, with a shaking adsorption time of 1-4h.

[0014] Preferably, in step 2), the concentration of photoinitiator I2959 in the mixed solution is 5-10 mg / mL, the concentration of sulfonic acid monomer is 100-200 mg / mL, the sulfonic acid monomer is potassium 3-sulfopropyl methacrylate with a mass concentration of 5-10 mg / mL, and the soaking time is 4-8 h; the ultraviolet light wavelength is 365 nm, and the irradiation intensity is 5 mW / cm².2 The irradiation time is 15 minutes.

[0015] A third aspect of the present invention provides the application of the tissue-engineered cardiac valve stent with the above-mentioned sulfonated nanoenzyme coating, the stent being used to prepare cardiovascular repair medical devices or tissue-engineered valve transplantation medical devices.

[0016] Furthermore, the stent is used to prepare cardiovascular repair medical devices or tissue-engineered valve transplantation medical devices suitable for pathological microenvironments.

[0017] The beneficial effects of this invention are as follows: 1. The tissue-engineered cardiac valve scaffold with a sulfonated nanozyme coating provided by this invention achieves O2 under pathological microenvironment by loading ruthenium / iridium-tannic acid nanozymes onto the surface of a decellularized matrix carrier and performing sulfonation modification. - Synergistic regulation of NO cascade catalysis and interfacial function; Ruthenium / iridium-tannic acid nanozymes can efficiently scavenge superoxide anions and continuously generate nitric oxide in situ, fundamentally reversing oxidative stress and restoring O2. - -NO promotes biological homeostasis, thereby significantly improving the proliferation and migration of endothelial cells and inhibiting their apoptosis, accelerating the re-endothelialization process at the cellular functional level. Simultaneously, the sulfonated polymer coating, through its hydrophilic properties, significantly inhibits non-specific protein adsorption and platelet adhesion, providing immediate antithrombotic protection and promoting selective adhesion and migration of endothelial cells. The synergistic effect of these two factors overcomes early thrombosis under pathological conditions and significantly improves endothelialization efficiency in the pathological microenvironment. Furthermore, this valve stent effectively regulates macrophage polarization to reduce inflammatory responses, inhibits pathological fibrosis and abnormal calcification, and significantly promotes the structural and functional in-situ regeneration of tissue-engineered cardiac valve stents under complex pathological conditions, improving valve regeneration efficiency under complex pathological conditions. 2. This invention utilizes a dual-activity ruthenium / iridium-tannic acid nanozyme loaded with superoxide dismutase (SOD) and nitric oxide synthase (eNOS) to efficiently and continuously catalyze the dismutation of excess superoxide anions in the pathological microenvironment into hydrogen peroxide and oxygen. Its eNOS-mimicking activity mimics endothelial cell function, utilizing endogenous arginine to catalyze the in-situ generation of nitric oxide, directly replenishing the NO deficiency caused by inhibited eNOS activity. Together, they constitute intracellular O2. - -NO cascade catalytic reaction, effectively stopping O2 - The vicious cycle of NO reacting with NO to form peroxynitrite restores O2. - -NO biocompatibility significantly improves the bioavailability of NO, significantly reduces the DNA damage, mitochondrial dysfunction and apoptosis effects of oxidative stress on endothelial cells, and fundamentally improves the regenerative microenvironment; 3. In this invention, the sulfonated polymer coating, through its superhydrophilic and electrically neutral surface, can physically inhibit and block the non-specific adsorption and activation of plasma proteins, platelets and inflammatory cells on the material surface in the early stage of stent implantation, providing immediate antithrombotic protection, effectively preventing the formation of early thrombi, avoiding acute thrombosis of valve openings by thrombi and the physical barrier formed by them that hinders cell migration. At the same time, the sulfonated polymer coating can promote endothelial cell adhesion, and under the synergistic effect of NO homeostasis, it ensures that endothelial cells quickly and completely cover the stent surface, significantly accelerating the formation of a complete functional endothelial layer. 4. The ruthenium / iridium-tannic acid nanoenzyme in this invention removes O2. - At the same time, it eliminates the key signal that drives macrophages to polarize to a pro-inflammatory phenotype, prompts macrophages to transform to an anti-inflammatory phenotype that promotes tissue repair, reduces the release of inflammatory factors, thereby significantly alleviating the chronic inflammatory response around the implant, avoiding tissue damage and scaffold degradation caused by continuous inflammatory stimulation, while avoiding the continuous abnormal activation of fibroblasts, effectively inhibiting abnormal collagen deposition and tissue fibrosis, ensuring the orderly regeneration of functional valve tissue and the long-term structural and functional stability of the regenerated valve; 5. This invention uses a decellularized matrix derived from natural tissues as a carrier, providing an ideal three-dimensional structure and biological basis for cell migration and ingrowth; it utilizes the abundant catechol groups in tannic acid to achieve strong and universal adhesion to various matrices, ensuring the firm and uniform loading of ruthenium / iridium-tannic acid nanozymes on the carrier; and it uses ultraviolet light-initiated polymerization technology to construct a sulfonated polymer coating on the surface, which forms a protective network on the surface of the decellularized matrix fibers that enhances mechanical stability and prevents premature expansion. Attached Figure Description

[0018] Figure 1 The image shows a transmission electron microscope (TEM) image of the ruthenium / iridium-tannic acid nanozyme prepared in Example 2. Figure 2 This is an FDA staining image of endothelial cells cultured under high glucose conditions using the decellularized matrix carrier prepared in Example 1. Figure 3 This is an FDA staining image of endothelial cells cultured under high glucose conditions using the Ru / Ir@TA scaffold prepared in Example 2. Figure 4 FDA staining image of Ru / Ir@TA-SPM scaffold prepared in Example 2 after culturing endothelial cells under high glucose conditions; Figure 5 This is a DHE staining image of endothelial cells cultured under high glucose conditions using the decellularized matrix carrier prepared in Example 1. Figure 6This is a DHE staining image of endothelial cells cultured under high glucose conditions using the Ru / Ir@TA-SPM scaffold prepared in Example 2. Figure 7 The image shows the DAF-FMDA staining of endothelial cells cultured on the decellularized matrix carrier prepared in Example 1 under high glucose conditions. Figure 8 This is a DAF-FMDA staining image of endothelial cells cultured under high glucose conditions using the Ru / Ir@TA-SPM scaffold prepared in Example 2. Figure 9 The image shows the F4 / 80 immunofluorescence staining of diabetic mice one month after the decellularized matrix carrier prepared in Example 1 was implanted subcutaneously. Figure 10 The image shows the F4 / 80 immunofluorescence staining of a Ru / Ir@TA-SPM scaffold implanted subcutaneously in diabetic mice one month after Example 2. Figure 11 The image shows the CD31 immunohistochemical staining of the decellularized matrix carrier prepared in Example 1 implanted into the abdominal aorta of a diabetic rabbit two months later. Figure 12 The image shows the CD31 immunohistochemical staining of the Ru / Ir@TA-SPM stent prepared in Example 2, two months after its insertion into the abdominal aorta of a diabetic rabbit. Detailed Implementation

[0019] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description. In the following embodiments, reagents and instruments not specifically mentioned are commercially available, and experimental operations not specifically mentioned are performed according to the manufacturer's instructions or conventional techniques in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention; the endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of various ranges, the endpoint values ​​of various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein; the technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0020] This invention provides a tissue-engineered cardiac valve scaffold with a sulfonated nanozyme coating, comprising a decellularized matrix carrier, a ruthenium / iridium-tannic acid nanozyme loaded on the decellularized matrix carrier, and a sulfonated polymer coating fixed to the surface of the decellularized matrix carrier and / or the nanoparticles by ultraviolet light polymerization, wherein the ruthenium / iridium-tannic acid nanozyme is a mixture of tannic acid (TA) and Ru. 3+ Ir 3+ Formed through coordination.

[0021] In some preferred embodiments of the present invention, the ruthenium / iridium-tannic acid nanozyme has dual bioactivity of superoxide dismutase and nitric oxide synthase; in the ruthenium / iridium-tannic acid nanozyme, the molar ratio of ruthenium to iridium is 1:4 to 4:1, and the ratio of the total molar amount of ruthenium and iridium to the molar amount of tannic acid is 1:1.

[0022] In some preferred embodiments of the present invention, the sulfonated polymer coating is a 3-sulfonopropyl methacrylate potassium salt polymer coating.

[0023] In some preferred embodiments of the present invention, the decellularized matrix carrier is one of decellularized pericardium, decellularized aortic valve, decellularized pulmonary valve, decellularized mitral valve, or tricuspid valve.

[0024] The decellularized matrix can be prepared using conventional decellularization methods. For example, fresh porcine pericardium, aortic valve, and pulmonary valve are washed with deionized water and the surface moisture is blotted dry with filter paper. A mixed aqueous solution containing 0.5% sodium deoxycholate, 0.02% EDTA·2Na, and 0.5% Triton X-100 is prepared. The washed tissue material is immersed in the solution and placed in a 37°C incubator with continuous shaking for 24 hours. Then, it is thoroughly washed with deionized water, and the above steps are repeated once. A 200 U / mL DNase + 20 µg / mL RNase solution is prepared using a buffer solution (10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2, pH=7.6). The tissue obtained in the previous step is further immersed in this solution with shaking for 48 hours, and the enzyme solution is changed every 24 hours. Finally, it is thoroughly washed with deionized water to obtain the decellularized matrix.

[0025] The present invention also provides a method for preparing the above-mentioned tissue-engineered cardiac valve stent with sulfonated nanoenzyme coating, comprising the following steps: 1) The decellularized matrix carrier was immersed in a ruthenium / iridium-tannic acid nanozyme solution, and the adsorption reaction was carried out by shaking. After washing with deionized water, the nanozyme-loaded scaffold was obtained. 2) The nanozyme-loaded scaffold obtained in step 1) was immersed in N-(3-aminopropyl)methacrylamide hydrochloride solution for a period of time. After being taken out and washed with deionized water, the scaffold was immersed in a mixed solution containing sulfonated monomer and photoinitiator I2959 for 2 hours. After being taken out, it was sandwiched between two pieces of quartz glass for ultraviolet light irradiation crosslinking reaction. After washing with deionized water, the tissue-engineered heart valve scaffold with sulfonated nanozyme coating was obtained.

[0026] Among them, N-(3-aminopropyl)methacrylamide hydrochloride can combine with phenolic hydroxyl and quinone groups on the surface of decellularized matrix carriers or ruthenium / iridium-tannic acid nanoenzymes, thereby firmly anchoring it on the scaffold surface and introducing C=C double bonds. The double bonds can undergo copolymerization with sulfonated monomers under the action of ultraviolet photoinitiator I2959, thereby firmly grafting the sulfonated polymer onto the scaffold surface through covalent bonds, which significantly improves the durability and stability of the sulfonated polymer coating.

[0027] In some preferred embodiments of the present invention, the preparation method of the ruthenium / iridium-tannic acid nanozyme is as follows: a quantitative amount of tannic acid, RuCl3 and IrCl3 are dissolved in ultrapure water, then the solutions are mixed uniformly, the pH of the mixed solution is adjusted to 8-10 with an alkaline solution, and the reaction is carried out at 80-90℃ for 24-48 hours. Finally, the reaction product is purified by dialyzing for 3-5 days to obtain ruthenium / iridium-tannic acid nanoparticles.

[0028] In some preferred embodiments of the present invention, the concentration of the ruthenium / iridium-tannic acid nanozyme solution in step 1) is 5-20 cm⁻¹. 2 / mL, with a shaking adsorption time of 1-4h.

[0029] In some preferred embodiments of the present invention, the concentration of photoinitiator I2959 in the mixed solution of step 2) is 5-10 mg / mL, the concentration of sulfonic acid monomer is 100-200 mg / mL, the sulfonic acid monomer is potassium 3-sulfopropyl methacrylate with a mass concentration of 5-10 mg / mL, and the soaking time is 4-8 h; the ultraviolet light wavelength is 365 nm, and the irradiation intensity is 5 mW / cm². 2 The irradiation time is 15 minutes.

[0030] The present invention also provides the application of the tissue-engineered cardiac valve stent with the above-mentioned sulfonated nanoenzyme coating; specifically, the stent is used to prepare cardiovascular repair medical devices or tissue-engineered valve transplantation medical devices; further, the stent is used to prepare cardiovascular repair medical devices or tissue-engineered valve transplantation medical devices suitable for pathological microenvironments.

[0031] The following specific embodiments further illustrate the tissue-engineered cardiac valve stent with sulfonated nanozyme coating, its preparation method, and its application.

[0032] Example 1 Preparation of decellularized matrix carriers 1) Wash the fresh pig pericardium with deionized water and blot dry the surface of the membrane with filter paper; 2) Prepare a mixed aqueous solution containing 0.5% sodium deoxycholate, 0.02% EDTA·2Na and 0.5% Triton X-100. Immerse the tissue material cleaned in step 1) in the mixed aqueous solution and place it in a constant temperature oven at 37°C with continuous shaking for 24 hours. Then rinse thoroughly with deionized water and repeat the above steps once. 3) Prepare a mixed solution containing 200 U / mL DNase and 20 µg / mL RNase using a buffer solution containing 10 mM Tris-HCl, 2.5 mM MgCl2 and 0.5 mM CaCl2 at pH=7.6. Continue to soak the tissue material obtained in step 2) in this mixed solution and shake for 48 h, changing the mixed solution every 24 h. Finally, wash thoroughly with deionized water to obtain the decellularized matrix carrier.

[0033] Example 2 This embodiment uses the method of Example 1 to prepare a decellularized matrix carrier; 1) Synthesis of Ruthenium / Iridium-tannic acid nanozymes (Ru / Ir@TANPs) 0.1 mmol of tannic acid was dissolved in 8 mL of ultrapure water. 0.05 mmol of RuCl3 and 0.05 mmol of IrCl3 were dissolved in 1 mL of ultrapure water respectively. The solutions were mixed evenly, and the pH of the mixture was adjusted to 8 with 10 M NaOH solution. The mixture was heated to 80 °C in a water bath and stirred for 24 h. Finally, the reaction product was dialyzed for 3 days to obtain ruthenium / iridium-tannic acid nanozymes (Ru / Ir@TANPs).

[0034] 2) Fabrication of nanozyme-supported scaffolds The Ru / Ir@TANPs prepared in step 1) were resuspended in deionized water to prepare a Ru / Ir@TANPs solution with a concentration of 1 mg / mL; a 18 cm² area was used as the substrate. 2 The decellularized matrix carrier was immersed in 3 ml of Ru / Ir@TANPs solution, and after shaking and adsorption reaction for 1 hour, the product was washed with deionized water to obtain the nanozyme-loaded scaffold (Ru / Ir@TA scaffold). 3) Sulfonation modification The Ru / Ir@TA scaffold prepared in step 2) above was immersed in a 5 mg / mL solution of N-(3-aminopropyl)methacrylamide hydrochloride for 8 hours to introduce double bonds. After removal, it was washed with deionized water, and then immersed in a mixed solution containing 100 mg / mL of 3-sulfopropyl methacrylate potassium salt and 5 mg / mL of initiator I2959 for 1 hour. After removal, it was sandwiched between two pieces of quartz glass and reacted at a wavelength of 365 nm and 5 mW / cm². 2 After irradiation with ultraviolet light for 15 minutes at the specified intensity, followed by washing with deionized water, tissue-engineered cardiac valve stents with sulfonated nanozyme coatings (Ru / Ir@TA-SPM stents) were obtained.

[0035] Example 3 This embodiment uses the method of Example 1 to prepare a decellularized matrix carrier; 1) Synthesis of Ruthenium / Iridium-tannic acid nanozymes (Ru / Ir@TANPs) 0.1 mmol of tannic acid was dissolved in 8 mL of ultrapure water. 0.08 mmol of RuCl3 and 0.02 mmol of IrCl3 were dissolved in 1 mL of ultrapure water respectively. The solutions were mixed evenly, and the pH of the mixture was adjusted to 9 with 10 M NaOH solution. The mixture was heated to 90 °C in a water bath and stirred for 24 h. Finally, the reaction product was dialyzed for 3 days to obtain ruthenium / iridium-tannic acid nanozymes (Ru / Ir@TANPs).

[0036] 2) Fabrication of nanozyme-supported scaffolds The Ru / Ir@TANPs prepared in step 1) were resuspended in deionized water to prepare a Ru / Ir@TANPs solution with a concentration of 1 mg / mL; a 18 cm² area was used as the substrate. 2 The decellularized matrix carrier was immersed in 3 ml of Ru / Ir@TANPs solution, and after shaking and adsorption reaction for 2 h, the product was washed with deionized water to obtain the nanozyme-loaded scaffold (Ru / Ir@TA scaffold). 3) Sulfonation modification The Ru / Ir@TA scaffold prepared in step 2) above was immersed in a 10 mg / mL N-(3-aminopropyl)methacrylamide hydrochloride solution for 4 hours to introduce double bonds. After rinsing with deionized water, the scaffold was then immersed in a mixed solution containing 150 mg / mL potassium 3-sulfopropyl methacrylate and 8 mg / mL initiator I2959 for 1 hour. After removal, it was sandwiched between two pieces of quartz glass and reacted at a wavelength of 365 nm and 5 mW / cm². 2 After irradiation with ultraviolet light for 15 minutes at the specified intensity, followed by washing with deionized water, tissue-engineered cardiac valve stents with sulfonated nanozyme coatings (Ru / Ir@TA-SPM stents) were obtained.

[0037] Example 4 This embodiment uses the method of Example 1 to prepare a decellularized matrix carrier; 1) Synthesis of Ruthenium / Iridium-tannic acid nanozymes (Ru / Ir@TANPs) 0.1 mmol of tannic acid was dissolved in 8 mL of ultrapure water. 0.02 mmol of RuCl3 and 0.08 mmol of IrCl3 were dissolved in 1 mL of ultrapure water respectively. The solutions were mixed evenly, and the pH of the mixture was adjusted to 10 with 10 M NaOH solution. The mixture was heated to 80 °C in a water bath and stirred for 48 h. Finally, the reaction product was dialyzed for 3 days to obtain ruthenium / iridium-tannic acid nanozymes (Ru / Ir@TANPs).

[0038] 2) Fabrication of nanozyme-supported scaffolds The Ru / Ir@TANPs prepared in step 1) were resuspended in deionized water to prepare a Ru / Ir@TANPs solution with a concentration of 1 mg / mL; a 18 cm² area was used as the substrate. 2 The decellularized matrix carrier was immersed in 3 ml of Ru / Ir@TANPs solution, and after shaking and adsorption reaction for 4 hours, the product was washed with deionized water to obtain the nanozyme-loaded scaffold (Ru / Ir@TA scaffold). 3) Sulfonation modification The Ru / Ir@TA scaffold prepared in step 2) above was immersed in a 5 mg / mL solution of N-(3-aminopropyl)methacrylamide hydrochloride for 8 hours to introduce double bonds. After rinsing with deionized water, the scaffold was then immersed in a mixed solution containing 200 mg / mL of potassium 3-sulfopropyl methacrylate and 10 mg / mL of initiator I2959 for 1 hour. After removal, it was sandwiched between two pieces of quartz glass and reacted at a wavelength of 365 nm and 5 mW / cm². 2 After irradiation with ultraviolet light for 15 minutes at the specified intensity, followed by washing with deionized water, tissue-engineered cardiac valve stents with sulfonated nanozyme coatings (Ru / Ir@TA-SPM stents) were obtained.

[0039] Experimental Example 1 The morphology of the ruthenium / iridium-tannic acid nanozyme prepared in Example 2 was characterized by transmission electron microscopy (TEM). The results are as follows Figure 1 As shown: The ruthenium / iridium-tannic acid nanozyme prepared by this invention is spherical with a particle size of about 300 nm and uniform particle size.

[0040] Experiment Example 2 Endothelial cell proliferation performance was characterized for the decellularized matrix carrier prepared in Example 1, the Ru / Ir@TA scaffold prepared in Example 2, and the Ru / Ir@TA-SPM scaffold. The experimental method was as follows: Scaffold material or decellularized matrix was cut into 1 cm diameter discs and sterilized by immersing in 75% ethanol for 3 hours. The material was then thoroughly washed with sterile PBS and transferred to 48-well plates for later use. Human umbilical vein endothelial cells (purchased from Huatuo Biotechnology Co., Ltd., catalog number HTX3606) were digested and resuspended in normal complete culture medium. The cells were seeded onto the material surface at a density of 20,000 cells per well and cultured at 37°C and 5% CO2 for 24 hours. The old culture medium was replaced with 50 mM glucose complete culture medium, and the cells were cultured for another 48 hours. Endothelial cell proliferation on the material was qualitatively observed using FDA (Fluoride Chemicals) assays. result Figure 2-4 As shown, under high glucose-induced microenvironment, the proliferation of endothelial cells on the surface of the unmodified decellularized matrix carrier is limited, and the cell number is sparse. The number of endothelial cells on the surface of the Ru / Ir@TA scaffold modified with ruthenium / iridium-tannic acid nanozyme increases, indicating that the nanozyme, through its dual biomimetic catalytic activity of superoxide dismutase and nitric oxide synthase, can effectively remove excess superoxide anions and promote NO generation, thus improving the survival and proliferation of endothelial cells under high glucose conditions. Moreover, the Ru / Ir@TA-SPM scaffold prepared in this invention exhibits the best proliferation-promoting effect, with a significant increase in the number of endothelial cells on its surface. This indicates that the synergistic effect of the sulfonated polymer coating and the nanozyme can significantly improve cell survival and proliferation under high glucose conditions, providing favorable conditions for the rapid formation of a complete functional endothelial layer.

[0041] Experimental Example 3 The decellularized matrix carrier prepared in Example 1, the Ru / Ir@TA scaffold prepared in Example 2, and the Ru / Ir@TA-SPM scaffold were characterized for endothelial cell migration performance. The experimental method was as follows: Scaffold material or decellularized matrix was cut into 1 cm diameter discs and sterilized by soaking in 75% ethanol for 3 hours. The material was then thoroughly washed with sterile PBS and transferred to 48-well plates for later use. Human umbilical vein endothelial cells (purchased from Huatuo Biotechnology Co., Ltd., catalog number HTX3606) were digested and resuspended in normal complete culture medium. The cells were seeded at a density of 20,000 cells per well on half of the material surface, while the other half was shielded. The cells were cultured at 37°C and 5% CO2 for 24 hours. After removing the shielding material, the old culture medium was replaced with serum-free medium with a glucose concentration of 50 mM, and the endothelial cells were cultured for another 48 hours. FDA staining was used for qualitative observation, and the migration distance of the endothelial cells on the material was measured. The results are shown in Table 1 below. Table 1. Migration distance of endothelial cells on sample materials sample Migration distance (μm) Ru / Ir@TA-SPM bracket 117.1±10.2 Ru / Ir@TA bracket 90.6±5.1 Decellularized matrix carrier 37.8±5.6 As shown in Table 1, the endothelial cell migration distance on the surface of the Ru / Ir@TA-SPM scaffold (117.1±10.2µm) was significantly higher than that of the unmodified group (37.8±5.6µm) and the group loaded with nanozymes only (90.6±5.1µm). This indicates that the tissue-engineered cardiac valve scaffold with sulfonated nanozyme coating prepared in this invention can significantly enhance the migration ability of endothelial cells through the synergistic effect of the sulfonated polymer coating and ruthenium / iridium-tannic acid nanozyme, which is beneficial to accelerating the endothelial coverage process on the surface of the valve scaffold.

[0042] Experiment Example 4 The decellularized matrix carrier prepared in Example 1 and the Ru / Ir@TA-SPM scaffold prepared in Example 2 were used to characterize the endothelial cell apoptosis performance. The experimental method was as follows: Orthotopic tissue-engineered valve scaffold material or decellularized matrix was cut into 1 cm diameter discs, sterilized by soaking in 75% ethanol for 3 hours, and then washed with sterile PBS before being placed in 48-well plates. Human umbilical vein endothelial cells (purchased from Huatuo Biotechnology Co., Ltd., catalog number HTX3606) were digested and resuspended in normal complete culture medium, seeded at a concentration of 20,000 cells per well, and cultured at 37°C and 5% CO2 for 24 hours. The culture medium was then replaced with 50 mM glucose complete culture medium, and the endothelial cells were cultured for another 48 hours. Flow cytometry was performed using Annexin V-FITC / 7-AAD double staining to determine the apoptosis rate of the cells. The results are shown below: Table 2 Apoptosis rate of endothelial cells on sample materials sample Apoptosis rate (%) Ru / Ir@TA-SPM bracket 32.8±1.4 Decellularized matrix carrier 8.1±0.6 As shown in Table 2, under high glucose conditions, the apoptosis rate of endothelial cells on the surface of the unmodified decellularized matrix carrier was as high as 32.8±1.4, indicating that the pathological microenvironment induced significant cell apoptosis. In contrast, the apoptosis rate of endothelial cells on the surface of the Ru / Ir@TA-SPM scaffold prepared in this invention was only 8.1±0.6. This result shows that under high glucose conditions, the tissue-engineered cardiac valve scaffold with sulfonated nanozyme coating prepared in this invention can significantly reduce the apoptosis rate of endothelial cells under pathological conditions and has good anti-apoptotic ability.

[0043] Experimental Example 5 The decellularized matrix carrier prepared in Example 1 and the Ru / Ir@TA-SPM scaffold prepared in Example 2 were subjected to intracellular superoxide anion (O2) treatment. - Sweeping performance characterization; The experimental method was as follows: In situ tissue-engineered valve scaffold material or decellularized matrix was cut into 1 cm diameter discs, sterilized by soaking in 75% ethanol for 3 hours, and then washed with sterile PBS before being placed in 48-well plates. Human umbilical vein endothelial cells (purchased from Huatuo Biotechnology Co., Ltd., catalog number HTX3606) were digested and resuspended in normal complete culture medium, seeded at a concentration of 20,000 cells per well, and cultured at 37°C and 5% CO2 for 24 hours. The culture medium was then replaced with 50 mM glucose complete culture medium, and the endothelial cells were cultured for another 48 hours. DHE staining was used to qualitatively observe the presence of superoxide anions (O2) in the endothelial cells grown on the material. - The expression of ); result Figure 5-6 As shown: Under a high-glucose environment, intracellular O2 in endothelial cells on the surface of unmodified decellularized matrix carriers... - The level is high, and the fluorescence signal is strong; while the O2 in the endothelial cells on the surface of the Ru / Ir@TA-SPM scaffold is high. - The fluorescence signal was significantly weakened, indicating that the ruthenium / iridium-tannic acid nanozyme in the scaffold of this invention has excellent SOD-mimicking properties, and can efficiently and continuously remove excess O2 expressed in the pathological microenvironment. - It blocks the oxidative stress chain reaction at its source, thus maintaining intracellular redox homeostasis.

[0044] Experimental Example 6 The intracellular NO production performance of the decellularized matrix carrier prepared in Example 1 and the Ru / Ir@TA-SPM scaffold prepared in Example 2 was characterized. The experimental method was as follows: Orthotopic tissue-engineered valve scaffold material or decellularized matrix was cut into 1 cm diameter discs, sterilized by soaking in 75% ethanol for 3 hours, and then washed with sterile PBS before being placed in 48-well plates. Human umbilical vein endothelial cells (purchased from Huatuo Biotechnology Co., Ltd., catalog number HTX3606) were digested and resuspended in normal complete culture medium, seeded at a concentration of 20,000 cells per well, and cultured at 37℃ and 5% CO2 for 24 hours. The old culture medium was then replaced with 50 mM glucose complete culture medium, and the endothelial cells were cultured for another 48 hours. The expression of NO in the endothelial cells grown on the material was qualitatively observed by DAF-FMDA staining. result Figure 7-8 As shown, the NO expression level of endothelial cells on the surface of the Ru / Ir@TA-SPM scaffold prepared in this invention was significantly higher than that of endothelial cells grown on the surface of unmodified decellularized matrix. This indicates that under high glucose conditions, the Ru / Ir@TA-SPM scaffold prepared in this invention can effectively promote the production of NO in endothelial cells and restore cellular O2. - -NO maintains biological homeostasis and preserves normal cell function.

[0045] Experimental Example 7 The anti-inflammatory properties of the decellularized matrix carrier prepared in Example 1 and the Ru / Ir@TA-SPM scaffold prepared in Example 2 were characterized. The experimental method was as follows: a diabetic mouse model was induced by intraperitoneal injection of streptozotocin; the materials were cut into 1*1cm pieces. 2 Square-shaped materials were sterilized by soaking in 75% ethanol for 3 hours, washed with sterile saline, and implanted subcutaneously into diabetic mice for 1 month. The inflammation status after implantation was identified by F480 / DAPI double staining. result Figure 9-10 As shown in the figure, the proportion and density of F4 / 80 positive cells represent the degree of inflammation; the more positive cells there are, the more severe the inflammation. As shown in 9-10, in the subcutaneous implantation model of diabetic mice, a large number of F4 / 80 positive cells accumulated around the unmodified decellularized matrix carrier, indicating a strong inflammatory response on the material. However, the density of F4 / 80 positive cells around the Ru / Ir@TA-SPM scaffold prepared in this invention was significantly reduced one month after implantation. This indicates that under high glucose conditions, the Ru / Ir@TA-SPM scaffold prepared in this invention can effectively regulate macrophage polarization, reduce the inflammatory response of the implanted material, and create a favorable immune microenvironment for tissue regeneration.

[0046] Experimental Example 8 The decellularized matrix carrier prepared in Example 1 and the Ru / Ir@TA-SPM scaffold prepared in Example 2 were characterized for in vivo endothelialization. The experimental method was as follows: a diabetic rabbit model was induced by intravenous injection of alloxan; the in situ tissue-engineered valve scaffold material or decellularized matrix material was cut into rectangles of 1cm*1.5cm and wrapped around the outer surface of a 2.5cm diameter metal coronary stent. The covered stent was then implanted into the abdominal aorta of the diabetic rabbit. The sample was removed 2 months after implantation, and the endothelial cell coverage of the material was observed by CD31 staining. result Figure 11-12 As shown, the unmodified decellularized matrix carrier surface has fewer CD31-positive areas and incomplete endothelial layer coverage, indicating that the endothelialization process on this material surface is hindered. In contrast, the Ru / Ir@TA-SPM scaffold prepared in this invention exhibits a continuous, complete, and regularly shaped endothelial cell layer two months after implantation, with cells arranged along the blood flow direction and significantly enhanced CD31 expression. These results fully demonstrate that the scaffold material of this invention can still effectively promote endothelial cell migration, proliferation, and functional arrangement in the pathological microenvironment, significantly accelerating the endothelialization process of the scaffold material and possessing good clinical translational potential.

[0047] In summary, it should be noted that the above description is only a preferred embodiment of the present invention and should not be used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still make some simple deductions, substitutions, or equivalent substitutions of some technical features for the technical solutions described in the foregoing embodiments without departing from the concept of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tissue-engineered cardiac valve scaffold with a sulfonated nanozyme coating, comprising a decellularized matrix carrier, characterized in that: It also includes a ruthenium / iridium-tannic acid nanozyme loaded on the decellularized matrix carrier, and a sulfonated polymer coating fixed to the surface of the decellularized matrix carrier and / or the nanoparticles by ultraviolet light polymerization, wherein the ruthenium / iridium-tannic acid nanozyme is composed of tannic acid (TA) and Ru. 3+ Ir 3+ Formed through coordination.

2. The tissue-engineered cardiac valve stent with a sulfonated nanoenzyme coating according to claim 1, characterized in that: The ruthenium / iridium-tannic acid nanozyme exhibits dual bioactivity as both superoxide dismutase and nitric oxide synthase. In the ruthenium / iridium-tannic acid nanozyme, the molar ratio of ruthenium to iridium is 1:4 to 4:1, and the ratio of the total molar amount of ruthenium and iridium to the molar amount of tannic acid is 1:

1.

3. The tissue-engineered cardiac valve stent with a sulfonated nanoenzyme coating according to claim 1, characterized in that: The sulfonated polymer coating is a 3-sulfonic acid propyl methacrylate potassium salt polymer coating.

4. The tissue-engineered cardiac valve stent with a sulfonated nanoenzyme coating according to claim 1, characterized in that: The decellularized matrix carrier is one of the following: decellularized pericardium, decellularized aortic valve, decellularized pulmonary valve, decellularized mitral valve, or tricuspid valve.

5. A method for preparing a tissue-engineered cardiac valve stent with a sulfonated nanozyme coating as described in any one of claims 1-4, characterized in that: Includes the following steps: 1) The decellularized matrix carrier was immersed in a ruthenium / iridium-tannic acid nanozyme solution, and the adsorption reaction was carried out by shaking. After washing with deionized water, the nanozyme-loaded scaffold was obtained. 2) The nanozyme-loaded scaffold obtained in step 1) is immersed in N-(3-aminopropyl)methacrylamide hydrochloride solution for a period of time. After rinsing with deionized water, the scaffold is immersed in a mixed solution containing sulfonated monomer and photoinitiator I2959 for 1-2 hours. After rinsing with deionized water, the tissue-engineered heart valve scaffold with sulfonated nanozyme coating is obtained.

6. The method for preparing a tissue-engineered cardiac valve stent with a sulfonated nanozyme coating according to claim 5, characterized in that: The preparation method of the ruthenium / iridium-tannic acid nanozyme is as follows: a quantitative amount of tannic acid, RuCl3 and IrCl3 are dissolved in ultrapure water, and then the solutions are mixed evenly. The pH of the mixed solution is adjusted to 8-10 with an alkaline solution, and the reaction is carried out under water bath heating at 80-90℃ with stirring for 24-48 hours. Finally, the reaction product is purified by dialyzing for 3-5 days to obtain the ruthenium / iridium-tannic acid nanozyme.

7. The method for preparing a tissue-engineered cardiac valve stent with a sulfonated nanozyme coating according to claim 5, characterized in that: In step 1), the concentration of the ruthenium / iridium-tannic acid nanozyme solution is 1-10 mg / mL, and the shaking adsorption time is 1-4 h.

8. The method for preparing a tissue-engineered cardiac valve stent with a sulfonated nanozyme coating according to claim 5, characterized in that: In step 2), the concentration of photoinitiator I2959 in the mixed solution is 5-10 mg / mL, the concentration of sulfonic acid monomer is 100-200 mg / mL, and the sulfonic acid monomer is potassium 3-sulfopropyl methacrylate with a mass concentration of 5-10 mg / mL. The soaking time is 4-8 hours. The ultraviolet light wavelength is 365 nm, and the irradiation intensity is 5 mW / cm². 2 The irradiation time is 15 minutes.

9. The application of the tissue-engineered cardiac valve stent with a sulfonated nanozyme coating as described in any one of claims 1-4, characterized in that: The stent is used to prepare cardiovascular repair medical devices or tissue-engineered valve transplantation medical devices.

10. The application of the tissue-engineered cardiac valve stent with sulfonated nanoenzyme coating according to claim 9, characterized in that: The stent is used to prepare cardiovascular repair medical devices or tissue-engineered valve transplantation medical devices suitable for pathological microenvironments.

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