A heparin sodium-based nitric oxide donor, and a preparation method and application thereof

Abstract

The present application relates to a kind of heparin sodium-based nitric oxide donor and its preparation method and application, the method includes the following steps: (1) heparin sodium is dissolved in 2-(N-morpholino) ethanesulfonic acid buffer solution, adding activating agent, reaction is carried out, and reaction liquid A is obtained;(2) to reaction liquid A, continue to react to obtain reaction liquid B by adding β-mercaptoethylamine;(3) reaction liquid B is dialyzed, and solution C is obtained;(4) to solution C, nitrous acid tert-butyl ester is added, and the heparin sodium-based nitric oxide donor is obtained by reaction;Wherein, in step (1), the activating agent is 1-ethyl-3-(3-dimethylaminopropyl) carbonyl diimide or its hydrochloride salt.The heparin sodium-based nitric oxide donor prepared in the present application prolongs the release time of nitric oxide, and solves the problem of short half-life of nitric oxide.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a nitric oxide donor based on heparin sodium, its preparation method, and its application. Background Technology

[0002] Nitric oxide (NO) is an important endogenous messenger molecule and has long been a research hotspot in the scientific and medical communities. NO is widely involved in the regulation of physiological functions and intervention in pathological processes across multiple systems of the body, playing a central role in cell signaling, tissue protection, and the maintenance of homeostasis. In the cardiovascular system, NO can activate guanylate cyclase in vascular smooth muscle cells, promoting the production of cyclic guanosine monophosphate (cGMP), mediating vasodilation to regulate vascular tone, while simultaneously inhibiting platelet activation and aggregation and thrombus formation, protecting the integrity of vascular endothelial structure, and delaying the progression of diseases such as atherosclerosis. In the immune system, NO produced by immune cells such as macrophages can exert antibacterial and antiviral effects and participate in the regulation of immune responses by modulating the release of inflammatory factors. In the nervous system, NO, as a neurotransmitter, participates in nerve signal transmission and is of great significance for learning and memory, pain perception, and the protection of nerve cells. Furthermore, NO can promote local angiogenesis and tissue repair in wounds, inhibit excessive proliferation of smooth muscle cells, and play a crucial role in physiological metabolism and tissue regeneration. However, NO has an extremely short half-life (only 1-5 seconds) and is unstable, making it difficult to maintain an effective concentration at the site of action. This prevents it from continuously exerting physiological effects such as vasodilation and antithrombosis, which greatly limits its direct application.

[0003] Sodium heparin is a natural mucopolysaccharide extracted primarily from bovine lung or porcine small intestinal mucosa. It is a linear polymer formed by alternating 1-4 glycosidic bonds of D-glucosamine, L-iduronic acid, or D-glucuronic acid, with a large number of negative charges on its molecular chain. Due to its good anticoagulant activity, good biocompatibility, adjustable anticoagulant strength, and certain anti-inflammatory effects, sodium heparin has been extensively explored and researched for applications in cardiovascular anticoagulation therapy (such as thrombosis prevention), adjuvant anticoagulation in interventional procedures, extracorporeal circulation anticoagulation in hemodialysis, and drug carrier modification (such as enhancing targeting). However, there are currently no reports on the use of sodium heparin as a nitric oxide donor. Summary of the Invention

[0004] In view of the above-mentioned deficiencies of the prior art, the present invention provides a heparin sodium-based nitric oxide donor (HMA), its preparation method and application. The heparin sodium-based nitric oxide donor prepared by the present invention prolongs the nitric oxide release time and solves the problem of short nitric oxide half-life.

[0005] This invention provides a method for preparing a sodium heparin-based nitric oxide donor, the method comprising the following steps:

[0006] (1) Dissolve sodium heparin in 2-(N-morpholino)ethanesulfonic acid buffer solution, add activator, and react to obtain reaction solution A;

[0007] (2) Add β-mercaptoethylamine to reaction solution A and continue the reaction to obtain reaction solution B;

[0008] (3) Dialyze the reaction solution B to obtain solution C;

[0009] (4) Add tert-butyl nitrite to solution C, and after reaction, obtain the nitric oxide donor based on heparin sodium;

[0010] In step (1), the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its hydrochloride salt.

[0011] Preferably, the molar ratio of heparin sodium to β-mercaptoethylamine is 1:(10~20).

[0012] Preferably, in step (1), the concentration of the 2-(N-morpholino)ethanesulfonic acid buffer solution is 0.05~0.2M, and the pH value is 5.0~6.0;

[0013] And / or, the reaction temperature is 20~30℃ and the reaction time is 0.5~1 hour.

[0014] Preferably, in step (2), the reaction temperature is 20~30℃ and the reaction time is 18~24 hours.

[0015] Preferably, in step (3), the dialysis is performed using a dialysis bag for 48-60 hours, with a molecular weight cutoff of 3500 Da.

[0016] Preferably, in step (4), before adding tert-butyl nitrite to solution C, the thiol content of solution C is determined, and the molar amount of tert-butyl nitrite added is 5 to 7 times the thiol content.

[0017] Preferably, in step (4), the pH value of the reaction is adjusted to 6-7;

[0018] And / or, the pH adjuster used for pH adjustment is selected from sodium hydroxide solution and potassium hydroxide solution;

[0019] And / or, the reaction is carried out under inert gas protection and / or light protection conditions, and the reaction time is 8 to 12 hours.

[0020] Preferably, in step (4), the reaction further includes a post-processing step;

[0021] And / or, the post-processing steps include: dialysis and / or lyophilization;

[0022] And / or, the dialysis is performed using a dialysis bag for 48-60 hours, with a molecular weight cutoff of 3500 Da;

[0023] And / or, the freeze-drying temperature is -20 to -50°C, and the freeze-drying time is 48 to 72 hours.

[0024] The present invention also provides a sodium heparin nitric oxide donor prepared by the method described in any of the above-mentioned embodiments.

[0025] The present invention also provides the application of the method described in any one of the above claims or the above-described heparin sodium-based nitric oxide donor in the preparation of biomedical materials, wherein the biomedical materials are selected from wound dressings, vascular stents or artificial blood vessels.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The heparin sodium-based nitric oxide donor (HMA) prepared in this invention prolongs the nitric oxide release time, solving the problem of short nitric oxide half-life. The preparation method employed in this invention is simple to operate, with mild and easily controllable reaction conditions. It is not only easy to implement but also effectively avoids damage to the heparin sodium molecular structure during preparation. The heparin sodium-based nitric oxide donor prepared in this invention possesses an SNO active group, which can release NO under certain conditions. Simultaneously, heparin sodium can bind to coagulation factors in the blood environment and inhibit the coagulation process, exhibiting both anti-inflammatory and anticoagulant effects. It holds promise for the preparation of multifunctional antithrombotic medical materials, such as those used in hemodialysis tubing, cardiac interventional catheters, or as an anticoagulant adjuvant in surgical procedures.

[0028] The heparin sodium-based nitric oxide donor (HMA) provided by this invention combines the antithrombotic activity of heparin sodium with the controlled-release NO function of a nitric oxide donor. Heparin sodium exerts potent anticoagulant and antithrombotic effects by enhancing antithrombin III activity and inhibiting coagulation factor Xa / IIa and platelet adhesion and aggregation. Simultaneously, HMA can stably release nitric oxide (NO) under physiological conditions, inhibiting platelet activation and aggregation by increasing intracellular cGMP levels, and also dilating blood vessels and improving endothelial function. This forms a synergistic anticoagulant and antithrombotic effect with heparin sodium, significantly improving overall anticoagulant performance and achieving a dual-mechanism synergistic prevention and treatment of thrombosis. It can be widely used in biomedical materials selected from blood-contact materials, such as artificial blood vessels, vascular stents, and wound dressings, which possess excellent anticoagulant effects. Attached Figure Description

[0029] Figure 1 The infrared spectrum of the heparin sodium-based nitric oxide donor (HMA) and heparin sodium raw material (Hep) prepared in Example 1 of this invention;

[0030] Figure 2 The effect of the molar ratio of heparin sodium to β-mercaptoethylamine on the mass fraction of thiol in Examples 1-6 of this invention;

[0031] Figure 3 The effect of the molar ratio of tert-butyl nitrite to mercapto groups on the total NO content in Examples 1 and 7-10 of this invention;

[0032] Figure 4 The graph shows the NO release performance of the heparin sodium-based nitric oxide donor (HMA) prepared in Example 1 of this invention in PBS (pH=7.4) buffer and ascorbic acid (Asc) solution.

[0033] Figure 5 This is a SEM image of the HMA / PCL nanofiber sample prepared in Example 11 of this invention.

[0034] Figure 6 The effect of NO release in the HMA / PCL nanofiber sample prepared in Example 11 of this invention on the proliferation of human umbilical vein endothelial (HUVEC) cells;

[0035] Figure 7 The effect of NO release from the HMA / PCL nanofiber sample prepared in Example 11 of this invention on the proliferation of human umbilical artery smooth muscle (HUASMCs) cells. Detailed Implementation

[0036] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0037] The first aspect of this invention provides a method for preparing a sodium heparin-based nitric oxide donor, the method comprising the following steps:

[0038] (1) Dissolve sodium heparin in 2-(N-morpholino)ethanesulfonic acid buffer solution, add activator, and react to obtain reaction solution A;

[0039] (2) Add β-mercaptoethylamine to reaction solution A and continue the reaction to obtain reaction solution B;

[0040] (3) Dialyze the reaction solution B to obtain solution C;

[0041] (4) Add tert-butyl nitrite to solution C, and after reaction, obtain the nitric oxide donor based on heparin sodium;

[0042] In step (1), the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its hydrochloride salt.

[0043] In some embodiments, the molar ratio of sodium heparin to β-mercaptoethylamine is 1:(10~20), for example, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc.

[0044] In some embodiments, the molar ratio of heparin sodium to β-mercaptoethylamine is 1:(12~20).

[0045] In some embodiments, in step (1), the concentration of the 2-(N-morpholino)ethanesulfonic acid buffer solution is 0.05~0.2M, for example, 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.10M, 0.12M, 0.14M, 0.16M, 0.18M, ​​0.2M, etc.; and the pH value is 5.0~6.0, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, etc.

[0046] In some embodiments, in step (1), the reaction temperature is 20~30°C and the reaction time is 0.5~1 hour, for example, 0.5 hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1 hour, etc.

[0047] In some embodiments, in step (2), the reaction temperature is 20~30°C and the reaction time is 18~24 hours, for example, 18 hours, 21 hours, 24 hours, etc.

[0048] In some embodiments, in step (3), the dialysis is performed using a dialysis bag for 48 to 60 hours, for example, 48 hours, 54 hours, 60 hours, etc., with a molecular weight cutoff of 3500 Da.

[0049] In some embodiments, in step (4), before adding tert-butyl nitrite to solution C, the thiol content of solution C is first determined, and the molar amount of tert-butyl nitrite added is 5 to 7 times the thiol content, for example, 5 times, 5.1 times, 5.2 times, 5.3 times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6 times, 6.1 times, 6.2 times, 6.3 times, 6.4 times, 6.5 times, 6.6 times, 6.7 times, 6.8 times, 6.9 times, 7 times, etc.

[0050] In some embodiments, in step (4), before adding tert-butyl nitrite to solution C, the thiol content of solution C is first determined, and the molar amount of tert-butyl nitrite added is 5 times the thiol content.

[0051] In some embodiments, in step (4), the pH value of the reaction is adjusted to 6-7, for example, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc.

[0052] In some embodiments, the pH adjuster used for pH adjustment is selected from sodium hydroxide solution and potassium hydroxide solution.

[0053] In some embodiments, in step (4), the reaction is carried out under inert gas protection and / or light protection conditions, and the reaction time is 8 to 12 hours, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.

[0054] In some embodiments, in step (4), the reaction is carried out under inert gas protection and light-protected conditions.

[0055] In some embodiments, in step (4), the reaction is carried out under inert gas protection conditions.

[0056] In some embodiments, the reaction in step (4) is carried out under light-protected conditions.

[0057] In some implementations, step (4) further includes a post-processing step after the reaction.

[0058] In some embodiments, the post-processing steps include dialysis and / or lyophilization.

[0059] In some embodiments, the post-processing steps include dialysis and lyophilization.

[0060] In some embodiments, the dialysis is performed using a dialysis bag for 48 to 60 hours, for example, 48 hours, 54 hours, 60 hours, etc., with a molecular weight cutoff of 3500 Da.

[0061] In some embodiments, the freeze-drying temperature is -20 to -50°C, for example, -20°C, -30°C, -40°C, -50°C, etc.; the freeze-drying time is 48 to 72 hours, for example, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, etc.

[0062] A second aspect of the present invention provides a sodium heparin nitric oxide donor prepared according to the method described in any of the preceding claims.

[0063] A third aspect of the present invention provides the use of the heparin sodium-based nitric oxide donor as described above in the preparation of biomedical materials.

[0064] In some embodiments, the biomedical material is selected from blood-contact materials.

[0065] In some embodiments, the biomedical material is selected from one or more of artificial blood vessels, vascular stents, and wound dressings.

[0066] In some embodiments, a heparin sodium-based nitric oxide donor is blended with a polymer and prepared into fibrous, membrane, or porous materials using electrospinning, freeze-drying, or casting methods, for use in the preparation of artificial blood vessels, vascular stents, or wound dressings.

[0067] In some embodiments, heparin sodium-based nitric oxide donor / polymer nanofibers are prepared by electrospinning for use in the fabrication of artificial blood vessels, vascular stents, or wound dressings.

[0068] The method of the present invention will be described below through specific embodiments. It should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0069] In the following examples, unless otherwise specified, all temperatures are in Celsius; unless otherwise specified, all starting materials and reagents are commercially available or synthesized according to known methods; commercially available materials and reagents are used directly without further purification; unless otherwise specified, commercially available manufacturers include, but are not limited to, Sinopharm Group, Bailingwei Technology Co., Ltd., TCI (Shanghai) Chemical Industry Development Co., Ltd., Shanghai Bid Pharmaceutical Technology Co., Ltd., and Shanghai Mairui Chemical Technology Co., Ltd.

[0070] Example 1

[0071] (1) Dissolve 800 mg of heparin sodium (0.066 mmol) in 40 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (0.1 M, pH 5.5), add 383.2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 2.47 mmol) activator, and react for 0.5 h to obtain reaction solution A;

[0072] (2) 61.6 mg of β-mercaptoethylamine (MEA, 0.8 mmol) was added to reaction solution A and reacted at room temperature for 24 h to obtain reaction solution B;

[0073] (3) Place the reaction solution B from step (2) into a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da, change the ultrapure water every 12 hours, dialyze at room temperature for 48 hours, and obtain solution C after dialysis.

[0074] (4) The grafting rate (mass fraction of thiol) of heparin sodium in solution C was determined by the Ellman method. Then, tert-butyl nitrite with a thiol content of 5 times was added, and the pH of the solution was adjusted to 6.5 with 1M sodium hydroxide solution. The reaction was carried out at room temperature for 12 hours under nitrogen protection and in the dark. The solution was dialyzed with ultrapure water at room temperature for 48 hours using a dialysis bag with a molecular weight cutoff of 3500 Da. The solution was then freeze-dried at -20℃ for 48 hours to obtain a nitric oxide donor based on heparin sodium.

[0075] The heparin sodium-based nitric oxide donor (HMA) and heparin sodium raw material (Hep, as a control group) prepared in Example 1 were characterized by infrared spectroscopy using a tablet compression method. Figure 1 As shown, HMA at 1500cm -1 The presence of a distinct NO stretching vibration characteristic peak at the heparin sodium site, while the absence of this characteristic peak at the Hep site, confirms that the -SNO active group is fixed on the heparin sodium, indicating that the synthesis of the heparin sodium nitric oxide donor (HMA) was successful.

[0076] Examples 2-10

[0077] The specific differences between Examples 2-10 and Example 1 are shown in Table 1. All contents not mentioned in Table 1 are the same as those in Example 1.

[0078] Table 1

[0079]

[0080] like Figure 2 As shown, the analysis of the thiol grafting rate (thiol mass fraction) of sodium heparin in solution C measured by the Ellman method in step (4) of Examples 1-6 of the present invention shows that: when the molar ratio of sodium heparin to β-mercaptoethylamine is 1:(10~20), the thiol grafting rate (thiol mass fraction) is relatively high; when the molar ratio of sodium heparin to β-mercaptoethylamine is 1:12, the thiol grafting rate (thiol mass fraction) is the highest.

[0081] Detection Example 1

[0082] Weigh 5 mg of the heparin sodium-based nitric oxide donor (HMA) prepared in Examples 1 and 7-10 of this invention, dissolve it in 5 mL of deionized water, place it in a reaction vessel, and heat at 120 °C for 12 h. After cooling, transfer 1 mL of the reaction solution to a 15 mL centrifuge tube and dilute with 9 mL of deionized water. Take 50 μL of the diluted solution, add 100 μL of Griess reagent, react in the dark for 15 min, and measure the absorbance (OD) value at 540 nm using an ELISA reader. Calculate the total NO content according to the standard curve. Each sample was measured in triplicate. The total NO content of the sample in Example 1 was found to be 27.8 μmol / L.

[0083] like Figure 3 As shown, the analysis of the total NO content test results of samples 1 and 7-10 in Examples shows that when the molar ratio of tert-butyl nitrite to mercapto is (5-7):1, the total NO content is relatively high; among them, when the molar ratio of tert-butyl nitrite to mercapto is 5:1, the total NO content reaches the highest level.

[0084] Detection Example 2

[0085] The NO release behavior of the heparin sodium-based nitric oxide donor (HMA) prepared in Example 1 was detected by dialysis combined with Griess reagent. 5 mg of the heparin sodium-based nitric oxide donor (HMA) prepared in Example 1 was weighed and dissolved in 2 mL of PBS buffer (pH=7.4) and 2 mL of 250 μg / mL ascorbic acid (Asc) solution, respectively. The solutions were then transferred to dialysis bags with a molecular weight cutoff (MWCO) of 3500 Da and placed in centrifuge tubes. The corresponding volume of PBS buffer or Assc solution was added outside the dialysis bag. At set time points, 50 μL samples were taken, and 100 μL of Griess reagent was added. The reaction was carried out in the dark for 15 min, and the OD value was measured using a microplate reader at 540 nm. The corresponding NO concentration was calculated based on the standard curve. Each sample was measured in triplicate, with an equal volume of the corresponding solution added after each sampling.

[0086] like Figure 4 As shown, the heparin sodium-based nitric oxide donor is relatively stable in PBS buffer and can stably release nitric oxide for up to 8 hours under ascorbic acid (Asc) catalysis.

[0087] Example 11

[0088] Heparin sodium nitric oxide donor (HMA) / polymer nanofibers were prepared by electrospinning, and the preparation method is as follows:

[0089] Polycaprolactone (PCL) and heparin sodium-based nitric oxide donor (HMA) prepared in Example 1 of this invention were weighed and blended at a mass ratio of 4:1. The blend was dissolved in hexafluoroisopropanol and stirred thoroughly to prepare a homogeneous spinning solution with a solid content of 8 wt%. Subsequently, electrospinning was performed under the process conditions of applying a voltage of 20 kV and receiving distance of 20 cm to finally obtain HMA / PCL nanofiber samples.

[0090] The morphology of HMA / PCL nanofiber samples was characterized using scanning electron microscopy (SEM). Figure 5 As shown, the prepared HMA / PCL nanofibers have continuous morphology, uniform fiber diameter distribution, smooth surface, and good morphology, and can be used for artificial blood vessels, vascular stents, or wound dressings.

[0091] Detection Example 3

[0092] This test example evaluates the effect of the HMA / PCL nanofiber sample prepared in Example 11 on cell growth behavior. The steps include:

[0093] Circular PCL and HMA / PCL nanofiber samples with a diameter of 1.5 cm were placed in 24-well plates and washed three times with PBS, with three replicate wells for each group. Subsequently, human umbilical vein endothelial cells (HUVECs) in the logarithmic growth phase were selected, digested with 0.25% trypsin, and then diluted to 1×10⁶ cells with RPMI medium. 4 Cells / mL. 1 mL of cell suspension was added to each well, and the cells were incubated for 72 h. Ascorbic acid (+Asc) was added to the culture system to continuously release NO from the nitric oxide donor loaded in the catalytic material. A negative control group (-Asc) was used without ascorbic acid. After incubation, 1 mL of fresh cell culture medium was replaced in each well, and 100 μL of CCK-8 solution was added. Cells were incubated for another 2 h, and the OD value of each well was measured at 450 nm using a microplate reader. Cell viability was calculated based on the measured OD values ​​to evaluate the proliferative activity of each group of cells.

[0094] The procedure for detecting the proliferative activity of human umbilical artery smooth muscle cells (HUASMCs) is the same as above.

[0095] like Figure 6-7As shown, the cell viability of HUVECs on HMA / PCL nanofiber samples increased to 164.33%, significantly higher than that of the PCL control group; the cell viability of HUASMCs on HMA / PCL nanofiber samples decreased to 75.91%; compared with the PCL group, the HMA / PCL group, under the condition of catalytic release of NO, can significantly promote the proliferation of human umbilical vein endothelial cells and inhibit the excessive proliferation of human umbilical artery smooth muscle cells.

[0096] Detection Example 4

[0097] According to ISO 10993-4, the hemolysis rate of non-hemolytic biomaterials must be less than 5%. This test example evaluates the in vitro hemolysis of the HMA / PCL nanofiber sample prepared in Example 11. The steps include:

[0098] A 2% (v / v) red blood cell suspension was prepared using physiological saline. PCL and HMA / PCL nanofiber sample membranes were then immersed in a mixture of 2.5 mL of red blood cell suspension and 2.5 mL of physiological saline, respectively. After incubation at 37°C for 1 h, the membranes were centrifuged at 1000 rpm, and 100 μL of the supernatant was transferred to a 96-well plate. The OD value was measured at 540 nm using a microplate reader. Physiological saline and distilled water were used as negative and positive controls, respectively, and the hemolysis rate was calculated.

[0099] The results showed that the hemolysis rates of PCL and HMA / PCL nanofiber samples were 2% and 1.3%, respectively, both far below 5%. This indicates that both materials have good blood compatibility, and the HMA / PCL nanofiber sample has a lower hemolysis rate.

[0100] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for preparing a sodium heparin-based nitric oxide donor, characterized in that, The method includes the following steps: (1) Dissolve sodium heparin in 2-(N-morpholino)ethanesulfonic acid buffer solution, add activator, and react to obtain reaction solution A; (2) Add β-mercaptoethylamine to reaction solution A and continue the reaction to obtain reaction solution B; (3) Dialyze the reaction solution B to obtain solution C; (4) Add tert-butyl nitrite to solution C, and after reaction, obtain the nitric oxide donor based on heparin sodium; In step (1), the activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its hydrochloride salt; The molar ratio of heparin sodium to β-mercaptoethylamine is 1:(10~20). In step (4), before adding tert-butyl nitrite to solution C, the thiol content of solution C is first determined, and the molar amount of tert-butyl nitrite added is 5 to 7 times the thiol content.

2. The method according to claim 1, characterized in that, In step (1), the concentration of the 2-(N-morpholino)ethanesulfonic acid buffer solution is 0.05~0.2M and the pH value is 5.0~6.

0.

3. The method according to claim 1, characterized in that, In step (1), the reaction temperature is 20~30℃ and the reaction time is 0.5~1 hour.

4. The method according to claim 1, characterized in that, In step (2), the reaction temperature is 20~30℃ and the reaction time is 18~24 hours.

5. The method according to claim 1, characterized in that, In step (3), the dialysis is performed using a dialysis bag for 48-60 hours, with a molecular weight cutoff of 3500 Da.

6. The method according to claim 1, characterized in that, In step (4), the pH value of the reaction is adjusted to 6-7; The pH adjustment agent used is a sodium hydroxide solution or a potassium hydroxide solution.

7. The method according to claim 1, characterized in that, In step (4), the reaction is carried out under inert gas protection and / or light protection conditions, and the reaction time is 8 to 12 hours.

8. The method according to claim 1, characterized in that, In step (4), the reaction also includes a post-processing step; The post-processing steps include: dialysis and / or lyophilization; The dialysis was performed using a dialysis bag for 48-60 hours, with a molecular weight cutoff of 3500 Da. The freeze-drying temperature is -20 to -50°C, and the freeze-drying time is 48 to 72 hours.

9. A sodium heparin-based nitric oxide donor prepared by the method of any one of claims 1 to 8.

10. The application of the method according to any one of claims 1 to 8 or the heparin sodium-based nitric oxide donor according to claim 9 in the preparation of biomedical materials, characterized in that, The biomedical material is selected from wound dressings, vascular stents, or artificial blood vessels.

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