A biological valve with synergistic anticoagulation and antioxidant functions and a preparation method thereof
By immobilizing bivalirudin and generating CeO2 nanoparticles in situ through EDC/NHS synergistic catalysis and enzymatic reaction, the problem of independent anticoagulant and antioxidant functions in bioprosthetic valves was solved, thereby improving the mechanical stability and long-term anticoagulant and antioxidant performance of bioprosthetic valves.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHENGDU MEDICAL COLLEGE
- Filing Date
- 2026-04-14
- Publication Date
- 2026-06-09
AI Technical Summary
Existing bioprosthetic valves have problems with their anticoagulant and antioxidant functions being independent and potentially weakening each other during long-term use. Furthermore, traditional cross-linking methods are complex and have poor repeatability, affecting the long-term durability and biocompatibility of the materials.
A one-step method using EDC/NHS synergistic catalytic reaction was employed to achieve matrix cross-linking and tyramine grafting. Subsequently, an enzymatic reaction was used to fix bivalirudin, which was then combined with in-situ generated CeO2 nanoparticles to form a synergistic anticoagulant and antioxidant functional layer.
This approach enhances the mechanical stability of bioprosthetic valves, strengthens their long-lasting anticoagulant and antioxidant properties, significantly improves their blood compatibility and durability, and avoids damage to the matrix structure caused by multi-step processing.
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Figure CN122163910A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a biological valve material, specifically a functionalized biomaterial for cardiovascular repair, particularly for artificial heart valves, and its preparation method, belonging to the field of biomedical materials technology. Background Technology
[0002] Heart valves are a crucial treatment for valvular heart disease, primarily classified into mechanical valves and bioprosthetic valves. Compared to mechanical valves, which require lifelong anticoagulation, bioprosthetic valves are increasingly widely used clinically due to their superior hemodynamic performance and lower postoperative anticoagulation requirements, especially suitable for elderly patients and young women wishing to conceive. However, the long-term durability of bioprosthetic valves remains a major bottleneck restricting their clinical application, with key failure mechanisms including structural degeneration, calcification, and thrombosis.
[0003] Traditional bioprosthetic valves are typically cross-linked with glutaraldehyde, which, while improving the material's mechanical strength and resistance to enzymatic degradation, has significant drawbacks. First, glutaraldehyde cross-linking can lead to cytotoxicity; residual free aldehyde groups may trigger chronic inflammation and immune responses, promoting calcification. Second, glutaraldehyde-crosslinked valves face complex physiological challenges after implantation: on one hand, the adhesion and activation of host immune cells (such as neutrophils and macrophages) on the material surface generates large amounts of reactive oxygen species (ROS), triggering oxidative stress, damaging collagen structure, and promoting calcium salt deposition; on the other hand, although bioprosthetic valves have a lower risk of thrombosis than mechanical valves, in areas with slow blood flow or in high-risk patients, platelet adhesion, activation, and the initiation of the coagulation cascade can still lead to valve thrombosis and functional failure.
[0004] To address these challenges, researchers have developed various modification strategies. In anticoagulation, heparinization is the most common method, but heparin's effectiveness depends on antithrombin III and carries a risk of bleeding, with limited long-term efficacy. Other anticoagulants, such as bivalirudin (a direct thrombin inhibitor), have attracted attention due to their direct action and controllability, but achieving stable and long-term fixation on material surfaces remains a significant challenge. In terms of antioxidation, introducing small-molecule antioxidants such as polyphenols is a common choice, but these molecules are prone to leaching and fail to provide long-term protection. In recent years, nanozymes with ROS scavenging capabilities, particularly cerium oxide (CeO2) nanoparticles, have gained attention due to their ability to scavenge ROS in redox cycles (Ce³⁺ / CeO2). 4 It has attracted much attention for its enzyme-like activity and stability during the ⁺ conversion.
[0005] However, most existing technologies employ a "stepwise modification" or "simple functional superposition" strategy. For example, glutaraldehyde is first used for cross-linking, followed by the introduction of amino groups or other active groups onto the surface through multiple chemical reactions, and finally, anticoagulant drugs or antioxidants are coupled. This multi-step process is not only complex and has poor reproducibility, but it may also damage the fine structure of the matrix, leading to a decline in mechanical properties. More importantly, there is often a lack of synergy between different functional components; anticoagulant and antioxidant functions are independent and may even weaken each other due to chemical incompatibility. For example, antioxidants may interfere with the activity of anticoagulant drugs, or the anticoagulant coating may affect the release and action of antioxidants. Furthermore, existing research usually treats CeO2 merely as an antioxidant additive, neglecting its potential impact on blood-material interface interactions.
[0006] Therefore, developing a novel modification technology that can simultaneously achieve bioprosthetic valve matrix stabilization, long-lasting anticoagulation, and highly effective antioxidant effects, with synergistic enhancement effects among the functional components, is of great significance for improving the long-term durability and biocompatibility of bioprosthetic valves. This invention is based on this need and proposes an innovative integrated functionalization strategy. Summary of the Invention
[0007] The purpose of this invention is to provide a method for preparing a biological valve that has both synergistic anticoagulant and antioxidant functions.
[0008] This invention is implemented as follows:
[0009] A method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions, comprising:
[0010] Step 1: Immerse the decellularized bioprosthetic valve matrix in a buffer solution containing carbodiimide (EDC), N-hydroxysuccinimide (NHS), and tyramine.
[0011] In this step, two key processes are achieved simultaneously through the synergistic catalytic effect of EDC / NHS: a) activating the carboxyl groups on the matrix collagen to form amide bonds with the amino groups of adjacent molecular chains, thereby achieving internal cross-linking of the matrix and enhancing mechanical stability; b) covalently grafting the amino groups of the added tyramine molecules onto the matrix through amide bonds, thereby uniformly introducing a large number of tyramine groups rich in phenolic hydroxyl groups into the entire cross-linking network as "active anchors" for subsequent enzymatic fixation.
[0012] Step 2: Immerse the tyramine-grafted bioprosthetic valve matrix obtained in Step 1 in a solution containing the direct thrombin inhibitor bivalirudin for 1 hour at room temperature. Then add tyrosine oxidase to catalyze the reaction and react at room temperature for 8-24 hours.
[0013] This enzymatic reaction oxidizes the phenolic hydroxyl group of the tyramine group to a quinone structure, which then undergoes Michael addition or Schiff base reaction with amino groups on the bivalirudin molecule, thereby efficiently, stably, and covalently fixing bivalirudin on the surface and inside of the matrix.
[0014] Step 3: In-situ construction of the synergistic functional layer: The bioprosthetic valve with immobilized bivalirudin obtained in Step 2 was immersed in a cerium nitrate solution and reacted at room temperature for 1 hour, allowing Ce³⁺ ions to be loaded onto the matrix network through physical adsorption and coordination. Subsequently, by adjusting the pH to an alkaline environment, the loaded Ce³⁺ was situ formed into cerium oxide (CeO₂) nanoparticles.
[0015] A further step is:
[0016] The bioprosthetic valve matrix includes porcine pericardium, porcine aortic valve, bovine pericardium, and bovine aortic valve.
[0017] A further step is:
[0018] In step two, the concentration of bivalirudin solution is 0.5-5 mg / mL.
[0019] A further step is:
[0020] The amount of tyrosine oxidase added is 10-100 U / mL.
[0021] A further step is:
[0022] In step three, the concentration of the cerium nitrate solution is 1-100 mM.
[0023] A further step is:
[0024] In step three, the pH is adjusted by adding ammonia.
[0025] A further step is:
[0026] In step three, the alkaline environment is pH 8.0-10.0.
[0027] A key finding of this invention is that CeO2 nanoparticles generated in situ not only act as highly efficient ROS scavengers (through their Ce³⁺ / Ce⁻ ratio) 4 The redox pair imparts antioxidant properties to the material and, unexpectedly, produces a significant synergistic anticoagulant effect with the covalently fixed bivalirudin, jointly enhancing the overall antithrombotic performance of the material.
[0028] The present invention also provides a bioprosthetic valve with synergistic anticoagulation and antioxidant functions, which is prepared by the preparation method of the bioprosthetic valve with synergistic anticoagulation and antioxidant functions provided by the present invention.
[0029] Compared with the prior art, the outstanding advantages and innovations of this invention are as follows:
[0030] The "one-step" integrated modification method innovatively utilizes the reaction characteristics of EDC / NHS to simultaneously enhance the mechanical properties of the matrix (crosslinking) and construct a bioactive interface (introducing tyramine) in a single reaction step. This method is simple and efficient, avoids potential damage to the delicate biological matrix structure caused by multi-step processing, and lays a solid foundation for subsequent precise enzymatic fixation.
[0031] The synergistic anticoagulant effect of "1+1>2": This is the core discovery of this invention. Experiments have confirmed (e.g., by measuring activated partial thromboplastin time (APTT) and thrombin time (TT)) that, compared to samples with only immobilized bivalirudin or only loaded CeO2, bioprosthetic valves containing both bivalirudin and CeO2 nanoparticles exhibit a significantly enhanced anticoagulant index. This goes beyond the traditional understanding of CeO2 as merely an antioxidant. It is speculated that Ce³⁺ / CeO2 may achieve a synergistic effect on anticoagulant function by influencing the intrinsic pathway of the coagulation cascade reaction, forming a complementary and enhancing mechanism with bivalirudin, which directly inhibits thrombin activity.
[0032] Multifunctional integration and long-term stability: The final bioprosthetic valve integrates three major advantages: a) a stable three-dimensional network structure, derived from EDC / NHS cross-linking, providing long-term mechanical support; b) long-lasting and highly effective low thrombotic activity, derived from the synergistic effect of bivalirudin and CeO2; c) excellent antioxidant stress resistance, derived from the ROS scavenging function of CeO2 nanoparticles. This organic combination of multiple functions, especially the introduction of a synergistic anticoagulant effect, is expected to significantly improve the bioprosthetic valve's blood compatibility, anti-calcification ability, and long-term durability. Attached Figure Description
[0033] Figure 1 This is a schematic diagram illustrating the expression of ROS in endothelial cells grown on the material in an embodiment of the present invention. Detailed Implementation
[0034] The present invention will be further described in detail below with reference to specific embodiments.
[0035] Example 1
[0036] Preparation of EDC cross-linked biovalve
[0037] 1) Wash fresh porcine pericardium with deionized water and blot dry with filter paper; prepare a mixed aqueous solution containing 0.5% sodium deoxycholate, 0.02% EDTA·2Na and 0.5% Triton X-100, immerse the tissue material washed in step 1) in the mixed aqueous solution and place it in a constant temperature incubator at 37 ℃ with continuous shaking for 24 h, then wash thoroughly with deionized water, and repeat the above steps once; 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 and pH = 7.6, and continue to immerse the tissue material obtained in step 2) in this mixed solution with shaking for 48 h, changing the mixed solution every 24 h; finally, wash thoroughly with deionized water to obtain decellularized porcine pericardium.
[0038] 2) Decellularized porcine pericardium was immersed in MES buffer containing EDC (200 mM) and NHS (200 mM) (pH = 5.5) and reacted with shaking at room temperature for 12 hours to obtain EDC cross-linked bioprosthetic valve (CP).
[0039] Example 2
[0040] Preparation of EDC cross-linked and bivalirudin-modified anticoagulant bioprosthetic valves
[0041] 1) The decellularization method shall be carried out in step 1) of Example 1.
[0042] 2) Decellularized porcine pericardium was immersed in MES buffer containing EDC (200 mM), NHS (200 mM), and tyrosine hydrochloride (100 mM) (pH = 5.5) and reacted at room temperature with shaking for 12 hours to obtain a cross-linked bioprosthetic valve with tyrosine groups introduced simultaneously.
[0043] 3) The valve material obtained in step 2 was immersed in a PBS solution containing 1 mg / mL bivalirudin and reacted at room temperature for 1 h. Then, tyrosine oxidase at a concentration of 50 U / mL was added and reacted at room temperature for 12 h to obtain an EDC cross-linked and bivalirudin-modified anticoagulant bioprosthetic valve (BD-CP).
[0044] Example 3
[0045] Preparation of EDC cross-linked modified cerium oxide nanoenzymes for antioxidant biovalve
[0046] 1) Proceed according to steps 1-2 of Example 2.
[0047] 2) The valve material obtained in step 1 was immersed in a 10 mM cerium nitrate hexahydrate aqueous solution and shaken at room temperature for 1 h. Then, ammonia was added dropwise to adjust the pH of the solution to between 8.0 and 10.0 and the reaction was shaken for 24 h to obtain an antioxidant biovalve (Ce-CP) with EDC crosslinking and cerium oxide modification.
[0048] Example 4
[0049] Preparation of EDC crosslinked and combined with bivalirudin / cerium oxide dual modification for anticoagulation and antioxidant bioprosthetic valves
[0050] 1) Proceed according to steps 1-3 of Example 2.
[0051] 2) The valve material obtained in step 1 was immersed in a 10 mM cerium nitrate hexahydrate aqueous solution and reacted with shaking at room temperature for 1 h. Then, ammonia was added dropwise to adjust the pH of the solution to between 8.0 and 10.0, and the reaction was continued with shaking for 24 h to obtain an EDC cross-linked and bivalirudin / cerium oxide dual-modified anticoagulant and antioxidant bioprosthetic valve (BD / Ce). n -CP, where the subscript n represents the concentration of the cerium nitrate solution.
[0052] Experimental Example 1
[0053] The stability of the components of the uncrosslinked decellularized valve, Example 1, Example 2, Example 3, and Example 4 was characterized;
[0054] The experimental method was as follows: Uncrosslinked decellularized valve membranes (Examples 1, 2, 3, and 4) were cut to 1 cm × 1 cm size, thoroughly rinsed with deionized water, and then frozen and lyophilized at −80 ℃ for 24 h. The initial dry weight of the sample was recorded as W0. Collagenase was prepared in 50 mM Tris-HCl buffer (pH 7.4), and 10 mM CaCl2 was added to a final concentration of 200 U / mL. Each sample was placed in a 2 mL centrifuge tube, and 1.0 mL of collagenase solution was added to completely submerge the sample; a sample with only buffer added and no collagenase was used as a blank control. The samples were placed in a 37 ℃ constant temperature shaker and incubated at 100 rpm for 24 h. After incubation, the samples were removed, rinsed three times with deionized water, and then frozen and lyophilized again at −80 ℃ for 24 h. The remaining dry weight was recorded as W1. The weight loss rate of the sample was calculated using the following formula to evaluate the anti-collagenase degradation ability of the valve material:
[0055] Collagenase degradation rate (%) = [(W0 − W1) / W0] × 100%
[0056] Each group should have at least 6 samples tested in parallel, and the results should be expressed as mean ± standard deviation.
[0057] Table 1. Weight loss rate after collagenase degradation in blank examples and different embodiments.
[0058] Weight loss rate (%) after collagenase degradation Uncrosslinked decellularized valve 98.51 ± 3.82 CP 6.84 ± 1.25 BD-CP 6.11 ± 1.46 Ce-CP 4.72 ± 1.64 BD / Ce-CP 2.90 ± 0.82
[0059] As shown in Table 1, the uncrosslinked decellularized valve was almost completely degraded after 24 h of collagenase treatment, with a weight loss rate as high as 98.51±3.82%, indicating that the untreated valve matrix is difficult to resist enzymatic degradation. After crosslinking treatment, the degradation rate of the CP group was significantly reduced to 6.84±1.25%, indicating that crosslinking can effectively improve the valve's resistance to enzymatic degradation. Based on this, the degradation rate of the BD-CP group obtained in Example 2 decreased to 6.11±1.46%; the degradation rate of the Ce-CP group obtained in Example 3 decreased to 4.72±1.64%; and the degradation rate of the combined modified BD / Ce-CP group in Example 4 decreased to 2.90±0.82%. This shows that the functional coating constructed in this invention can further enhance the anti-collagenase degradation performance of crosslinked valves and improve the structural stability and long-term performance of biological valves.
[0060] Experiment Example 2
[0061] The anticoagulant properties of the uncrosslinked decellularized valves, Example 1, Example 2, Example 3, and Example 4 were characterized.
[0062] The experimental method was as follows: Uncrosslinked decellularized valves, and samples from Examples 1 (CP), 2 (BD-CP), 3 (Ce-CP), and 4 (BD / Ce-CP) were cut into 10 mm diameter discs and placed in 24-well plates. Fresh rabbit whole blood was collected from healthy adult New Zealand white rabbits via the ear vein into centrifuge tubes containing 3.8 wt% sodium citrate anticoagulant (anticoagulant to whole blood volume ratio 1:9). The anticoagulant was centrifuged at 3000 rpm for 15 min, and the supernatant plasma was collected. 0.5 mL of plasma was added to the surface of each sample, and the samples were incubated at 37 ℃ for 30 min to ensure sufficient contact between the sample and plasma. After incubation, the plasma was collected for coagulation parameter determination. Activated partial thromboplastin time (APTT) and thrombin time (TT) were measured using an automated coagulation analyzer. Each group was tested in parallel at least four times. Results are expressed as mean ± standard deviation, as shown in Table 2 below.
[0063] Table 2. Anticoagulant properties of blank examples and different embodiments.
[0064] APTT(s) TT(s) Uncrosslinked decellularized valve 10.8 ± 0.64 17.65 ± 0.91 CP 10.36 ± 0.57 17.87 ± 1.15 BD-CP 20.63 ± 3.5 54.97 ± 4.84 Ce-CP 12.93 ± 1.12 21.37 ± 1.53 BD / Ce-CP 25.47 ± 1.19 146.9 ± 3.97
[0065] The data in the table show that the APTT and TT of the Ce-CP group were slightly prolonged compared to the unmodified and simple cross-linked groups, at 12.93±1.12 s and 21.37±1.53 s, respectively, indicating that the introduction of antioxidant components alone has limited improvement on anticoagulation performance. The APTT and TT of the BD-CP group were significantly prolonged to 20.63±3.5 s and 54.97±4.84 s, respectively, indicating that the introduction of anticoagulation components can effectively inhibit intrinsic coagulation pathways and fibrin formation. Furthermore, the BD / Ce-CP group, which is jointly modified with Ce, exhibited the best anticoagulation performance, with an APTT of 25.47±1.19 s and a TT of 146.9±3.97 s, both significantly higher than other groups. These results demonstrate that the functional coating constructed in this invention can significantly delay the coagulation process. The bivalirudin anticoagulation component is the main factor improving anticoagulation performance, while the CeO2 antioxidant component, in synergy with it, can further enhance the anticoagulation effect. The BD / Ce-CP group combines optimal anticoagulant properties with functional synergy, and has the potential to be more suitable for blood-contact bioprosthetic valve materials.
[0066] Experimental Example 3
[0067] The antioxidant properties of the uncrosslinked decellularized valve, Example 1, Example 2, Example 3, and Example 4 were characterized.
[0068] The experimental method was as follows: Uncrosslinked decellularized valves (Examples 1, 2, 3, and 4) were sterilized by soaking in 75% ethanol for 6 hours. After washing with sterile PBS, the materials were 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, and seeded at a concentration of 20,000 cells per well. After culturing at 37°C and 5% CO2 for 24 h, 1 mM H2O2 was added to the culture medium. The expression of ROS in the endothelial cells grown on the material was qualitatively observed by DCFH-DA staining.
[0069] The results are attached. Figure 1 As shown, the unmodified CeO group exhibited strong green fluorescence after stimulation with 1 mM H2O2, indicating a high level of intracellular ROS. In contrast, the fluorescence of the Example 3 (Ce-PP) and Example 4 (BD / Ce-CP) groups was significantly weakened, demonstrating that the introduction of CeO2 can effectively scavenge intracellular ROS and significantly improve antioxidant performance. This indicates that the valve material of the present invention can effectively reduce ROS, alleviate cellular oxidative damage, and restore endothelial cell repair activity.
[0070] Although the present invention has been described herein with reference to illustrative embodiments, the above embodiments are merely preferred embodiments of the present invention, and the implementation of the present invention is not limited to the above embodiments. It should be understood that those skilled in the art can devise many other modifications and implementations, which will fall within the scope and spirit of the principles disclosed in this application.
Claims
1. A method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions, characterized in that... include: Step 1: Immerse the decellularized bioprosthetic valve matrix in a buffer solution containing carbodiimide, N-hydroxysuccinimide, and tyramine; Step 2: Immerse the tyramine-grafted bioprosthetic valve matrix obtained in Step 1 in a solution containing the direct thrombin inhibitor bivalirudin for 1 hour at room temperature; then add tyrosine oxidase to catalyze the reaction and react at room temperature for 8-24 hours. Step 3: In-situ construction of synergistic functional layer: The bioprosthetic valve with immobilized bivalirudin obtained in Step 2 is immersed in cerium nitrate solution and reacted at room temperature for 1 hour, so that Ce³⁺ ions are loaded onto the matrix network through physical adsorption and coordination; then, by adjusting the pH to an alkaline environment, the loaded Ce³⁺ is formed in-situ into cerium oxide nanoparticles.
2. The method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions according to claim 1, characterized in that: The bioprosthetic valve matrix includes porcine pericardium, porcine aortic valve, bovine pericardium, and bovine aortic valve.
3. The method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions according to claim 1, characterized in that: In step two, the concentration of bivalirudin solution is 0.5-5 mg / mL.
4. The method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions according to claim 1, characterized in that: The amount of tyrosine oxidase added is 10-100 U / mL.
5. The method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions according to claim 1, characterized in that: In step three, the concentration of the cerium nitrate solution is 1-100 mM.
6. The method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions according to claim 1, characterized in that: In step three, the pH is adjusted by adding ammonia.
7. The method for preparing a bioprosthetic valve with synergistic anticoagulant and antioxidant functions according to claim 1, characterized in that: In step three, the alkaline environment is pH 8.0-10.
0.
8. A bioprosthetic valve with synergistic anticoagulant and antioxidant functions, characterized in that: It is prepared by the method for preparing a biological valve with synergistic anticoagulant and antioxidant functions as described in any one of claims 1 to 7.