Keratin-based nitric oxide donor as well as preparation method and application thereof

Abstract

The invention relates to a keratin-based nitric oxide donor as well as a preparation method and application thereof. The preparation method of the keratin-based nitric oxide donor comprises the following steps: S1, reacting activated thiomalic acid with keratin to obtain a conjugate; s2, the conjugate obtained in the step S1 reacts with tert-butyl nitrite, and the keratin-based nitric oxide donor is obtained. The method provided by the invention is simple to operate and mild in condition, and the obtained NO donor is higher in NO loading capacity, more in release amount and long-acting.

Description

Technical Field

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

[0002] Nitric oxide (NO), as a gaseous signaling molecule in the human body, plays a crucial role in physiological and pathological processes. Its functions include vasodilation, inhibition of platelet aggregation and adhesion, regulation of immune inflammatory responses, promotion of angiogenesis, and antibacterial activity. Based on these broad biological functions, the development of exogenous NO donors that can be stably stored and controllably released at target sites has become a research hotspot in fields such as cardiovascular implantable devices (e.g., artificial blood vessels, stents) and wound dressings.

[0003] Currently, exogenous NO donors mainly include small molecule compounds and NO donors bound to macromolecular carriers. While small molecule NO donors have some therapeutic effects, they generally suffer from short half-lives, poor stability, uncontrollable release, and potential systemic toxicity, limiting their long-term, localized clinical application. To overcome these shortcomings, researchers have focused on covalently grafting NO donor groups (such as nitrosyl groups) onto biological macromolecular carriers to construct macromolecular NO donors. Among these, nitrosothiols have attracted considerable attention due to their relative stability under physiological conditions and their ability to trigger NO release through light, heat, metal ions, or ascorbic acid.

[0004] Keratin, an abundant, biocompatible, and biodegradable natural protein, contains a large number of cysteine ​​residues in its molecular chain. After reduction treatment, it exposes abundant thiol groups, providing an ideal reaction site for constructing S-nitrosolated keratin via nitrosation. Existing technologies have reported the preparation of S-nitrosolated keratin by reacting reduced keratin with nitrosating agents (such as tert-butyl nitrite or sodium nitrite / acid systems), and its application in anticoagulant and wound-healing materials (e.g., Chinese invention patent CN107141345A).

[0005] However, existing NO donor technologies based on reduced keratin still have significant shortcomings, such as low NO loading capacity and poor NO release performance. Therefore, there is an urgent need in the field to develop a NO donor with high NO loading capacity, good NO release performance and controllable release. Summary of the Invention

[0006] In view of the deficiencies of the prior art mentioned above, the present invention provides a keratin-based nitric oxide (NO) donor (KM) with high loading capacity, good NO release performance, and biocompatibility, as well as its preparation method and application.

[0007] Solution for solving the problem: This invention provides a method for preparing keratin-based nitric oxide donors, the method comprising the following steps: Step S1: React activated thiomalic acid with keratin to obtain a conjugate; Step S2: The conjugate obtained in step S1 is reacted with tert-butyl nitrite to obtain the keratin-based nitric oxide donor.

[0008] Preferably, in step S1, the reaction is controlled to be carried out at room temperature.

[0009] Preferably, in step S1, the reaction time is 10 to 30 hours.

[0010] Preferably, step S1 is carried out under a nitrogen atmosphere.

[0011] Preferably, the molar ratio of the thiomalic acid to the keratin is 1 to 10:1.

[0012] Preferably, the reaction in step S1 further includes a post-processing step, which includes purifying the reaction product and freeze-drying it to obtain the conjugate.

[0013] Preferably, the reaction in step S2 is carried out in the presence of a solvent selected from diethyl ether and / or methanol.

[0014] Preferably, the reaction in step S2 is carried out under dark conditions.

[0015] Preferably, the reaction in step S2 is carried out under a nitrogen atmosphere.

[0016] Preferably, in step S2, the stoichiometric molar ratio of the thiol group to tert-butyl nitrite in the coupling compound is 1:1~3.

[0017] Preferably, the reaction in step S2 further includes a post-processing step, which includes concentration and drying steps; the concentration includes evaporation concentration and / or vacuum low-temperature concentration; the drying includes vacuum drying.

[0018] The present invention provides a keratin-based nitric oxide donor prepared according to any one of the above methods.

[0019] This invention provides the application of the keratin-based nitric oxide donor described above in the preparation of biomedical materials, wherein the biomedical materials are selected from one or more of artificial blood vessels, vascular stents, and wound dressings.

[0020] The effects of the invention: This invention involves an amide reaction between keratin and activated thiomalic acid to form a keratin-thiomalic acid conjugate (pre-KM), which is then reacted with tert-butyl nitrite to generate a keratin-based NO donor. The preparation method is simple, mild, and easy to implement. Compared to existing small-molecule NO donors, the keratin-based NO donor provided by this invention exhibits high stability and low biotoxicity. Compared to NO donors directly prepared from reduced keratin and nitrosating agents, the NO donor provided by this invention has a higher NO loading capacity, greater release, and longer-lasting effect, effectively promoting cell proliferation.

[0021] The keratin-based NO donor provided by this invention can be widely used in other biomedical materials, such as vascular contact materials and wound dressings, and has excellent anticoagulant effects. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the synthetic route for the keratin-based NO donor of the present invention; Figure 2 A schematic diagram of the ultraviolet spectra of keratin and the keratin-based NO donor prepared in this invention; Figure 3 The graph shows the NO release performance of KM and KSNO in PBS (pH=7.4) buffer solution; Figure 4 SEM images of KM / PCL blend electrospinning; Figure 5 The graph shows the NO release performance of KM / PCL blend electrospinning in PBS (pH=7.4) buffer solution; Figure 6 The hemolysis rate of KM / PCL blend electrospinning (a) and platelet adhesion electron micrograph (b) are shown, where the yellow arrow in b indicates platelets that are clearly adhered to the fibrous membrane; Figure 7 This is a schematic diagram illustrating the effect of KM / PCL blend electrospinning on cell proliferation. Detailed Implementation

[0023] 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.

[0024] Through extensive and in-depth research and numerous experimental screenings, the inventors of this invention have, for the first time, proposed a novel keratin-based biomacromolecule nitric oxide donor (KM), its preparation method, and its applications. The donor is synthesized by chemically coupling keratin with thiomalic acid and then reacting it with tert-butyl nitrite. Under certain conditions, the nitric oxide donor provided by this invention can sustainably release nitric oxide. Compared with existing keratin-based biomacromolecule nitric oxide donors (KSNO, application number ZL 201710423554.6), the keratin-based biomacromolecule nitric oxide donor (KM) provided by this invention exhibits higher nitric oxide release capacity and efficiency. This donor can be used alone as a NO-releasing material or blended with other polymers to further prepare biologically functional NO sustained-release materials, thereby playing an important role in fields such as vascular tissue engineering, wound healing, and antibacterial applications.

[0025] The first aspect of this invention provides a method for preparing a keratin-based nitric oxide donor, the method comprising the following steps: Step S1: React activated thiomalic acid with keratin to obtain a conjugate; Step S2: The conjugate obtained in step S1 is reacted with tert-butyl nitrite to obtain the keratin-based nitric oxide donor.

[0026] In some embodiments, in step S1, the reaction is controlled to proceed at room temperature.

[0027] In some implementations, the reaction time in step S1 is 10 to 30 hours, for example, 10 hours, 15 hours, 20 hours, 25 hours, or 30 hours.

[0028] In some embodiments, step S1 is carried out under a nitrogen atmosphere.

[0029] In some embodiments, the molar ratio of the thiomalic acid to the keratin is 1 to 10:1, for example, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, and 10:1.

[0030] In some embodiments, the molar ratio of the thiomalic acid to the keratin is 1 to 5:1.

[0031] In some embodiments, the molar ratio of the thiomalic acid to the keratin is 1 to 3:1.

[0032] In a specific and preferred embodiment, the molar ratio of the thiomalic acid to the keratin is 2:1.

[0033] In some embodiments, the tert-butyl nitrite is added to the reaction system by dropwise addition.

[0034] In some embodiments, step S1 includes a post-processing step, which includes purifying the reaction product and freeze-drying it to obtain the conjugate.

[0035] In some embodiments, the activated thiomalic acid is activated using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) as an activator.

[0036] In some embodiments, the activation condition is an ice bath, and the activation time is 0.5 to 2 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, etc.

[0037] In some embodiments, the reaction in step S2 is carried out in the presence of a solvent selected from diethyl ether and / or methanol.

[0038] In a specific and preferred embodiment, the solvent is diethyl ether.

[0039] In some implementations, the reaction in step S2 is carried out under dark conditions.

[0040] In some embodiments, the reaction in step S2 is carried out under a nitrogen atmosphere.

[0041] In some embodiments, in step S2, the stoichiometric ratio of the mercapto group to tert-butyl nitrite in the coupling compound is 1:1 to 3, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, or 1:3.0.

[0042] In some embodiments, the stoichiometric ratio of the thiol group to tert-butyl nitrite in the coupling compound is 1:1 to 2.

[0043] In some embodiments, the stoichiometric ratio of the mercapto group to tert-butyl nitrite in the coupling compound is 1:1.2~1.6.

[0044] In some embodiments, step S2 further includes a post-processing step, which includes concentration and drying. In some embodiments, the concentration includes evaporative concentration and / or vacuum cryogenic concentration. In some embodiments, the drying includes vacuum drying.

[0045] In this invention, conventional methods are used to obtain keratin, including but not limited to keratin extraction by reduction.

[0046] A second aspect of the present invention provides a keratin-based nitric oxide donor prepared according to any one of the methods described above.

[0047] A third aspect of the present invention provides the use of the keratin-based nitric oxide donor described above in the preparation of biomedical materials.

[0048] In some embodiments, the biomedical material is selected from blood-contact materials; further, the biomedical material is selected from one or more of artificial blood vessels, vascular stents, and wound dressings.

[0049] In some embodiments, keratin-based nitric oxide donors are blended with polymers 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.

[0050] In some embodiments, keratin biomacromolecule nitric oxide donor / polymer nanofibers are prepared by electrospinning and used to prepare artificial blood vessels, vascular stents, or wound dressings.

[0051] In this application, room temperature refers to 20℃~30℃, such as 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, and 30℃.

[0052] 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.

[0053] 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.

[0054] Example 1

[0055] Thiomalic acid (1 eq) was dissolved in water, and carbodiimide hydrochloride (5 eq) was added to activate the carboxyl groups on the thiomalic acid. The mixture was stirred in an ice-water bath for 1 h. Keratin (0.5 eq) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 24 h. After dialyzing and lyophilization, the keratin-thiomalic acid conjugate (pre-KM) was obtained. The keratin-thiomalic acid conjugate (pre-KM) was dispersed in diethyl ether and mixed dropwise with a tert-butyl nitrite solution (catalog number 540-80-7) (the stoichiometric ratio of thiol groups to tert-butyl nitrite in pre-KM was 1:1.5, and the volume ratio of diethyl ether to tert-butyl nitrite was 5:1). The mixture was stirred at room temperature under a nitrogen atmosphere for 24 h in the dark. Diethyl ether, unreacted tert-butyl nitrite, and the byproduct tert-butanol were removed by rotary evaporation at room temperature. The mixture was then vacuum dried and stored in the dark to obtain the keratin-based biomacromolecule nitric oxide donor (KM).

[0056] The keratin-based biomacromolecule nitric oxide donor (KM) prepared in this embodiment and keratin as a control were respectively prepared into solutions of a certain concentration and scanned using a UV-Vis spectrophotometer (wavelength range of 200-600 nm). The results are as follows. Figure 2 As shown. Figure 2 As shown, a new characteristic peak for nitrosothiol compounds appeared at 334 nm, while the characteristic absorption peak of thiol groups within keratin was located at 276 nm. These spectral changes indicate that the thiol groups on the keratin-thiomalic acid conjugate have reacted with NO groups to form SNO groups, thus confirming that NO groups have been successfully chemically bonded to the keratin-thiomalic acid conjugate backbone, meaning that the keratin-based nitric oxide donor (KM) has been successfully synthesized.

[0057] Detection Example 1 Five mg each of the novel keratin-based biomacromolecule nitric oxide donor (KM) prepared in Example 1 of this invention and the keratin-based biomacromolecule nitric oxide donor (KSNO) prepared according to the method in Example 1 of patent document CN107141345A were dissolved in 5 mL of phosphate-buffered saline (PBS). Ascorbic acid (250 μg / mL) was then added to each solution to catalyze NO release. Samples were taken every 1 hour, and the nitrite content in the solution was determined using the Griess reagent method to quantitatively characterize the NO release of the two donors over time. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that both keratin-based NO donors can release nitric oxide under ascorbic acid catalysis. Under the same catalytic conditions and time range, the NO release of the novel keratin-based biomacromolecule nitric oxide donor (KM) prepared in this invention is significantly higher than that of the KSNO control group.

[0058] Example 2

[0059] Keratin-based biomacromolecule nitric oxide donor (KM) / polymer nanofibers were prepared by electrospinning, and the preparation method is as follows: Polycaprolactone (PCL) and the novel keratin-based biomacromolecule nitric oxide donor (KM) prepared in this invention were weighed and blended at a mass ratio of 7:3. 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 an applied voltage of 20 kV and a receiving distance of 15-20 cm to finally obtain KM / PCL nanofiber samples.

[0060] The morphology of KM / PCL nanofiber samples was characterized using scanning electron microscopy (SEM). Figure 4 As shown, the results indicate that the prepared nanofibers have continuous morphology, uniform diameter distribution, smooth surface, and good morphology, and can be used for artificial blood vessels, vascular stents, or wound dressings.

[0061] Detection Example 2 The KM / PCL nanofiber samples prepared in Example 2 of this invention were dissolved in 4 mL of phosphate-buffered saline (PBS), and then ascorbic acid (250 μg / mL) was added to the solution to catalyze NO release. Samples were taken every 1 hour, and the nitrite content in the solution was determined using the Griess reagent method to quantitatively characterize the NO release of the KM / PCL nanofiber samples over time. The results are as follows. Figure 5 As shown. By Figure 5 It is evident that the KM / PCL nanofiber sample can continuously and stably release nitric oxide for over 12 hours under ascorbic acid catalysis.

[0062] Detection Example 3 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 KM / PCL nanofiber sample prepared in Example 2. The steps include: Prepare a 2% (v / v) red blood cell suspension using physiological saline. Immerse PCL and KM / PCL fiber membranes in a mixture of 2.5 mL of red blood cell suspension and 2.5 mL of physiological saline, respectively. Figure 6 As shown in Figure a, the hemolysis rates of PCL and KM / PCL were 2.44% and 1.07%, respectively, both far below 5%. This indicates that both materials have good blood compatibility, and KM / PCL has a lower hemolysis rate.

[0063] The antiplatelet adhesion properties of KM / PCL nanofiber samples were evaluated using platelet-rich plasma (PRP, derived from anticoagulated rabbit blood). The steps included: PCL and KM / PCL nanofiber samples were statically incubated with PRP at 37℃ for 3 h. After incubation, excess PRP was removed, and the samples were gently washed three times with PBS. Fixation was performed with 2.5% glutaraldehyde aqueous solution at 4℃ for 2 h. Dehydration was then performed sequentially with 25%, 50%, 75%, 90%, and 100% ethanol aqueous solutions for 20 min each. Finally, the samples were freeze-dried, and platelet adhesion on the fiber surface was observed under SEM. Figure 6 Figure b shows that, compared to PCL, the number of platelets adhering and aggregating on the surface of KM / PCL nanofibers is significantly reduced, demonstrating excellent anti-platelet activation and adhesion properties.

[0064] Detection Example 4 This test example evaluates the effect of the KM / PCL nanofiber sample prepared in Example 2 on cell growth behavior. The steps include: PCL and KM / PCL nanofiber membranes were co-cultured with human umbilical vein endothelial cells for 72 h, respectively. In the KM / PCL experimental group, ascorbic acid was added to the culture system to catalyze the continuous release of NO from the nitric oxide donor loaded in the catalytic material. After culture, the proliferation activity of cells in each group was detected using the CCK-8 assay.

[0065] The results are as follows Figure 7 As shown, compared with the PCL group alone, the KM / PCL group exhibited higher cell proliferation activity under the condition of catalytic NO release. This indicates that the KM / PCL composite material not only has good cell compatibility, but its controlled release of nitric oxide can also effectively promote endothelial cell proliferation.

[0066] 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 keratin-based nitric oxide donor, characterized in that, The method includes the following steps: Step S1: React activated thiomalic acid with keratin to obtain a conjugate; Step S2: The conjugate obtained in step S1 is reacted with tert-butyl nitrite to obtain the keratin-based nitric oxide donor.

2. The method according to claim 1, characterized in that, In step S1, the reaction is controlled to be carried out at room temperature, and the reaction time is 10-30 hours. And / or, step S1 is carried out under a nitrogen atmosphere.

3. The method according to claim 1, characterized in that, The molar ratio of the thiomalic acid to the keratin is 1~10:

1.

4. The method according to claim 1, characterized in that, The reaction in step S1 is followed by a post-processing step, which includes purifying the reaction product and freeze-drying it to obtain the conjugate.

5. The method according to claim 1, characterized in that, The reaction in step S2 is carried out in the presence of a solvent selected from diethyl ether and / or methanol.

6. The method according to claim 1, characterized in that, The reaction in step S2 is carried out under dark conditions; And / or, the reaction in step S2 is carried out under a nitrogen atmosphere.

7. The method according to claim 1, characterized in that, In step S2, the stoichiometric ratio of the thiol group to tert-butyl nitrite in the coupling compound is 1:1~3.

8. The method according to claim 1, characterized in that, The reaction in step S2 further includes a post-processing step, which includes concentration and drying steps; the concentration includes evaporation concentration and / or vacuum low-temperature concentration; the drying includes vacuum drying.

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

10. The application of the keratin-based nitric oxide donor according to claim 9 in the preparation of biomedical materials, characterized in that, The biomedical material is selected from one or more of artificial blood vessels, vascular stents, and wound dressings.

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