Metal ion coordinated hyaluronic acid hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a metal ion coordinated hyaluronic acid hydrogel and a preparation method and application thereof.The hydrogel can release nitric oxide and / or oxygen and is prepared by mixing hyaluronic acid, L-arginine and metal ions at room temperature, the molecular weight of the hyaluronic acid is 100-2000 kDa, and the concentration of the hyaluronic acid is 0.5-20.0 wt.%; the content of the L-arginine is 0.01 to 20.0 weight percent; the content of metal ions is 0.01-0.2 wt.%, and the metal ions comprise divalent, trivalent or tetravalent metal ions. The preparation method comprises the following steps: weighing hyaluronic acid, dissolving the hyaluronic acid in deionized water, stirring until the hyaluronic acid is completely dissolved, adding an L-arginine solution, uniformly stirring, slowly adding a metal ion solution, and uniformly stirring. The hydrogel can release nitric oxide and oxygen at the same time, not only can resist bacteria and relieve inflammation, but also can adjust the hypoxic microenvironment, and is used in the fields of wound healing, tissue repair, surface coating and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a metal ion coordinated hyaluronic acid hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] L-arginine is an endogenous nitric oxide donor in organisms and generates nitric oxide under the catalysis of nitric oxide synthase. In the special microenvironment of acute wound inflammation and chronic wound with high levels of reactive oxygen species, the guanidine group of L-arginine provides electrons to free radicals in the wound microenvironment to generate nitric oxide, which functions to eliminate excessive reactive oxygen species and supplement nitric oxide at the wound site, thereby promoting neovascularization and wound healing. Hyaluronic acid is one of the main components of the extracellular matrix. Based on the great potential of hyaluronic acid hydrogels in the field of tissue repair and regeneration, the cumbersome modification steps and purification methods not only increase costs, but also make it difficult to control the quality. Chen Xuesi et al. reported a biomimetic natural dynamic biomineralization process in the literature "A drug-mineralized hydrogel orchestrated by spontaneous dynamic mineralization" and the patent document CN113943430A, in which sodium hyaluronate, alendronate sodium, and CaCl2 were mixed in deionized water to prepare a drug-mineralized hydrogel with spontaneous dynamic biomineralization behavior, and it showed the prospect of accelerating bone repair in a rat cranial defect model. However, there is no report on a hyaluronic acid hydrogel that can simultaneously release nitric oxide and oxygen. Summary of the Invention

[0003] The main object of the present invention is to provide a metal ion coordinated hyaluronic acid hydrogel, which can simultaneously release nitric oxide and / or oxygen, inhibit inflammatory reactions, regulate the hypoxic microenvironment, promote re-epithelialization, angiogenesis, and collagen deposition, accelerate the healing of acute and chronic wounds and tissue remodeling, and can be applied to fields such as wound healing, tissue repair, and biocoatings.

[0004] Another object of the present invention is to provide a preparation method of the metal ion coordinated hyaluronic acid hydrogel, which is rapidly constructed by mixing hyaluronic acid, L-arginine, and metal ions at room temperature.

[0005] Another object of the present invention is to provide an application of the metal ion coordinated hyaluronic acid hydrogel in the preparation of biomedical materials for wound healing, tissue repair, and biocoatings.

[0006] The above objects of the present invention can be achieved by the following technical solutions:

[0007] In a first aspect of the present invention, there is provided a metal ion-coordinated hyaluronic acid hydrogel which can release nitric oxide and / or oxygen and is prepared by mixing hyaluronic acid, L-arginine and metal ions at room temperature.

[0008] Preferably, the metal ion-coordinated hyaluronic acid hydrogel is selected from at least one of the forms of solution, powder, foam, sponge or gel.

[0009] Preferably, the molecular weight of hyaluronic acid is 100 - 2000 kDa and the solution concentration is 0.5 - 20.0 wt.%.

[0010] Preferably, the content of L-arginine is 0.01 - 20.0 wt.% and the content of metal ions is 0.01 - 0.2 wt.%.

[0011] Preferably, the metal ions include divalent, trivalent and tetravalent metal ions and are selected from one or more of cerium(III) ions, cerium(IV) ions, europium ions, gallium ions, zinc ions, magnesium ions, calcium ions, strontium ions, ferrous ions and ferric ions.

[0012] More preferably, the metal ions are selected from one or more of cerium(III), cerium(IV), europium, gallium, zinc and magnesium ions, which are derived from cerium(III) nitrate, europium nitrate, gallium nitrate, zinc acetate and magnesium chloride respectively.

[0013] Preferably, the metal ion-coordinated hyaluronic acid hydrogel can release nitric oxide gas with a concentration of 1.0 - 5.0 μM under the stimulation of reactive oxygen species (ROS) and / or hydrogen peroxide solution.

[0014] Preferably, the metal ion-coordinated hyaluronic acid hydrogel is a hyaluronic acid hydrogel coordinated with cerium(III) and / or cerium(IV) ions and can release oxygen with a concentration of 1 - 10 mg / L under the stimulation of ROS and / or hydrogen peroxide solution.

[0015] In a second aspect of the present invention, there is provided a method for preparing the metal ion-coordinated hyaluronic acid hydrogel, which comprises the following steps:

[0016] (1) Weigh hyaluronic acid and dissolve it in deionized water, and stir until completely dissolved to obtain a hyaluronic acid solution;

[0017] (2) Add an L-arginine solution to the hyaluronic acid solution and stir evenly to obtain a mixed solution;

[0018] (3) Slowly add a metal ion solution to the mixed solution and stir evenly to obtain the product.

[0019] Preferably, the concentration of the hyaluronic acid solution is 0.5-20.0 wt.%, the content of L-arginine is 0.01-20.0 wt.%, and the content of metal ions is 0.01-0.2 wt.%.

[0020] In the third aspect of the present invention, there is provided an application of the metal ion-coordinated hyaluronic acid hydrogel in the preparation of biomedical materials for wound healing, tissue repair, and surface coating.

[0021] Preferably, the biomedical materials include wound dressings, antibacterial materials, anti-inflammatory materials, and surface coating materials for medical devices.

[0022] More preferably, the metal ion-coordinated hyaluronic acid hydrogel is in the form of a hydrogel and is used for the preparation of acute and chronic wound healing dressings.

[0023] Compared with the prior art, the present invention provides a hyaluronic acid hydrogel crosslinked based on salt bridge and metal ion coordination, which can release nitric oxide and oxygen. It is prepared from hyaluronic acid, L-arginine, and metal ions at room temperature, and can simultaneously release nitric oxide and oxygen, which can not only antibacterial and relieve inflammation, but also regulate the hypoxic microenvironment, and is used for the surface coating of medical devices or biomaterials, such as wound dressings, antibacterial, anti-inflammatory and other medical materials or surface coatings of medical devices. The beneficial effects are as follows:

[0024] (1) The metal ion-coordinated hyaluronic acid hydrogel in the present invention does not require chemical modification, and has the advantages of simple preparation process, fast gelation rate, and adjustable biological functions.

[0025] (2) The metal ion-coordinated hyaluronic acid hydrogel of the present invention can respond to reactive oxygen species at the wound site and externally added hydrogen peroxide solution, and release nitric oxide and (or) oxygen, and has biological functions such as antibacterial, inhibiting inflammatory reactions, and regulating the hypoxic microenvironment, promoting re-epithelialization, angiogenesis, and collagen deposition, accelerating the healing rate of acute and chronic wounds and tissue remodeling rate, and achieving 100% healing of chronic skin wounds in diabetic mice in 14 days.

[0026] (3) The metal ion-coordinated hyaluronic acid hydrogel in the present invention has excellent biocompatibility, degradability, and in vivo biosafety, and can be used in the fields of wound healing, tissue repair, biomaterial coating, etc. Description of the Drawings

[0027] Figure 1 It is a picture of the appearance of the cerium ion-coordinated hyaluronic acid hydrogel prepared in Example 1.

[0028] Figure 2 It is the nitric oxide release curve of the cerium, europium, and gallium ion-coordinated hyaluronic acid hydrogels prepared in Examples 1, 2, and 3.

[0029] Figure 3 Oxygen release curve of the cerium ion-coordinated hyaluronic acid hydrogel prepared in Example 1.

[0030] Figure 4 Picture showing the diabetic wound healing effect of the cerium ion-coordinated hyaluronic acid hydrogel prepared in Example 1.

[0031] Figure 5 Picture showing the mechanism of action of the cerium ion-coordinated hyaluronic acid hydrogel prepared in Example 1. Detailed implementation manners

[0032] The technical solutions in the embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all embodiments. Based on the claims and embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1: Preparation of cerium ion-coordinated hyaluronic acid hydrogel

[0034] (1) Weigh 20.0 mg of hyaluronic acid and dissolve it in 900 μL of deionized water, and stir until completely dissolved;

[0035] (2) Add 50 μL of 100 mg / mL L-arginine solution to the solution obtained in (1), and stir evenly;

[0036] (3) Slowly add 50 μL of 5 mol / L cerium(III) nitrate solution to the solution obtained in (2), and stir evenly to obtain a cerium(III) ion-coordinated hyaluronic acid hydrogel, which is named HA / Arg / Ce.

[0037] Example 2: Preparation of europium ion-coordinated hyaluronic acid hydrogel

[0038] (1) Weigh 25.0 mg of hyaluronic acid and dissolve it in 1000 μL of deionized water, and stir until completely dissolved;

[0039] (2) Add 50 μL of 100 mg / mL L-arginine solution to the solution obtained in (1), and stir evenly;

[0040] (3) Slowly add 50 μL of 5 mol / L europium nitrate solution to the solution obtained in (2), and stir evenly to obtain a europium ion-coordinated hyaluronic acid hydrogel, which is named HA / Arg / Eu.

[0041] Example 3: Preparation of gallium ion-coordinated hyaluronic acid hydrogel

[0042] (1) Weigh 40.0 mg of hyaluronic acid, dissolve it in 1500 μL of deionized water, and stir until completely dissolved;

[0043] (2) Add 200 μL of 100 mg / mL L-arginine solution to the solution obtained in (1), and stir evenly;

[0044] (3) Slowly add 200 μL of 10 mol / L gallium nitrate solution to the solution obtained in (2), stir evenly to prepare a gallium ion-coordinated hyaluronic acid hydrogel, and name it HA / Arg / Ga.

[0045] Example 4: Preparation of zinc ion and cerium ion co-coordinated hyaluronic acid hydrogel

[0046] (1) Weigh 20.0 mg of hyaluronic acid, dissolve it in 800 μL of deionized water, and stir until completely dissolved;

[0047] (2) Add 100 μL of 50 mg / mL L-arginine solution to the solution obtained in (1), and stir evenly;

[0048] (3) Slowly add 50 μL of 2.5 mol / L zinc acetate solution to the solution obtained in (2), and stir evenly;

[0049] (4) Slowly add 50 μL of 2.5 mol / L cerium(III) nitrate solution to the solution obtained in (3), stir evenly to prepare a zinc ion and cerium ion co-coordinated hyaluronic acid hydrogel, named HA / Arg / Zn / Ce.

[0050] Example 5: Preparation of zinc ion and gallium ion co-coordinated hyaluronic acid hydrogel

[0051] (1) Weigh 30.0 mg of hyaluronic acid, dissolve it in 900 μL of deionized water, and stir until completely dissolved;

[0052] (2) Add 50 μL of 100 mg / mL L-arginine solution to the solution obtained in (1), and stir evenly;

[0053] (3) Slowly add 50 μL of 2.5 mol / L zinc acetate solution to the solution obtained in (2), and stir evenly;

[0054] (4) Slowly add 50 μL of 2.5 mol / L gallium nitrate solution to the solution obtained in (3), stir evenly to prepare a zinc ion and gallium ion co-coordinated hyaluronic acid hydrogel, named HA / Arg / Zn / Ga.

[0055] Example 6: Preparation of zinc ion, magnesium ion and europium ion co-coordinated hyaluronic acid hydrogel

[0056] (1) Weigh 0.2 g of hyaluronic acid and dissolve it in 9 mL of deionized water, and stir until completely dissolved;

[0057] (2) Add 500 μL of 100 mg / mL L-arginine solution to the solution obtained in (1), and stir evenly;

[0058] (3) Slowly add 500 μL of 2.0 mol / L magnesium chloride solution to the solution obtained in (2), and stir evenly;

[0059] (4) Slowly add 500 μL of 2.0 mol / L zinc acetate solution to the solution obtained in (3), and stir evenly;

[0060] (5) Slowly add 500 μL of 2.0 mol / L europium nitrate solution to the solution obtained in (4), and stir evenly to prepare a zinc ion, magnesium ion and europium ion co-coordinated hyaluronic acid hydrogel, named HA / Arg / Zn / Mg / Eu.

[0061] Example 7: Diabetic wound healing experiment

[0062] Use a full-thickness skin defect model of diabetic rats to evaluate the effect of metal ion-coordinated hyaluronic acid hydrogel on diabetic wound healing. The steps are as follows: Anesthetize SD rats by intraperitoneal injection of pentobarbital sodium, and cut a circular wound with a diameter of 10 mm and full-thickness skin defect on the back. Different treatments are performed on the wounds according to the experimental design: The PBS group is used as the negative control group, the commercial Tegaderm is used as the positive control group, and the metal ion-coordinated hyaluronic acid hydrogel is used as the experimental group. Each group of samples is repeated 4 times. After taking skin tissue samples from the wound site, the healing conditions of inflammation, blood vessels, collagen, hair follicles, etc. are judged by pathological analysis of tissue sections.

[0063] Results

[0064] As Figure 1 shown, the hyaluronic acid hydrogel HA / Arg / Ce was successfully synthesized according to the method of Example 1, showing excellent formability, injectability, adhesiveness and self-healing ability. Skin wounds usually show an irregularly defective shape. A hydrogel wound dressing with formability and injectability can effectively fill the irregular wound surface area and improve the treatment effect.

[0065] As Figure 2As shown, the release behavior of nitric oxide in the hyaluronic acid hydrogels prepared in Examples 1, 2, and 3 was monitored under the condition of a 100 μM hydrogen peroxide solution. Under the condition of 100 μM hydrogen peroxide, the concentration of NO reached 1.4 - 2.0 μM, indicating that L-arginine successfully consumed some of the reactive oxygen species and was converted into nitric oxide beneficial for angiogenesis, which helped to promote the wound healing process.

[0066] As Figure 3 shown, the release behavior of oxygen in the HA / Arg / Ce hydrogel prepared in Example 1 was monitored under the condition of a 100 μM hydrogen peroxide solution. Reactive oxygen species represented by hydrogen peroxide oxidized Ce 3+ to Ce 4+ , and the unique Ce 3+ / Ce 4+ metal ion pair helped to convert hydrogen peroxide into water and oxygen, and the oxygen release concentration reached 9.44 mg / L at 24 h.

[0067] As Figure 4 shown, the application of the HA / Arg / Ce hydrogel of Example 1 in Example 7 showed that the wound healing rate at 14 days after surgery was 100%, achieving the purpose of curing the chronic wounds of diabetic mice.

[0068] In summary, the present invention prepared a series of metal ion-coordinated hyaluronic acid hydrogels that can release nitric oxide and oxygen, and applied them to acute and chronic wound healing dressings, with significant effects and broad application prospects.

[0069] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A metal ion-coordinated hyaluronic acid hydrogel, characterized in that, It can release nitric oxide and / or oxygen, and is prepared by mixing hyaluronic acid, L-arginine and metal ions at room temperature.

2. The metal ion-coordinated hyaluronic acid hydrogel according to claim 1, wherein The molecular weight of hyaluronic acid is 100 - 2000 kDa, the solution concentration is 0.5 - 20.0 wt.%, the content of L-arginine is 0.01 - 20.0 wt.%, and the content of metal ions is 0.01 - 0.2 wt.%.

3. The metal ion-coordinated hyaluronic acid hydrogel according to claim 1, wherein The metal ions include divalent, trivalent or tetravalent metal ions, and are selected from one or more of cerium(III) ions, cerium(IV) ions, europium ions, gallium ions, zinc ions, magnesium ions, calcium ions, strontium ions, ferrous ions, ferric ions.

4. The metal ion-coordinated hyaluronic acid hydrogel according to claim 3, characterized in that, The metal ions are selected from one or more of cerium(III), cerium(IV), europium, gallium, zinc, magnesium ions, and are respectively derived from cerium(III) nitrate, europium nitrate, gallium nitrate, zinc acetate, magnesium chloride.

5. The metal ion-coordinated hyaluronic acid hydrogel according to claim 1, wherein The metal ion coordinated hyaluronic acid hydrogel can release nitric oxide gas with a concentration of 1.0 - 5.0 μM under the stimulation of ROS and / or hydrogen peroxide solution.

6. The metal ion coordinated hyaluronic acid hydrogel according to claim 1, wherein The metal ion coordinated hyaluronic acid hydrogel is a hyaluronic acid hydrogel coordinated with cerium(III) and / or cerium(IV) ions, and can release oxygen with a concentration of 1 - 10 mg / L under the stimulation of ROS and / or hydrogen peroxide solution.

7. The preparation method of the metal ion-coordinated hyaluronic acid hydrogel according to any one of claims 1 to 6, characterized in that, It includes the following steps: (1) Weigh hyaluronic acid and dissolve it in deionized water, and stir until completely dissolved to obtain a hyaluronic acid solution; (2) Add an L-arginine solution to the hyaluronic acid solution and stir evenly to obtain a mixed solution; (3) Slowly add a metal ion solution to the mixed solution and stir evenly to obtain it. The concentration of the hyaluronic acid solution is 0.5 - 20.0 wt.%, the content of L-arginine is 0.01 - 20.0 wt.%, and the content of metal ions is 0.01 - 0.2 wt.%.

8. Use of the metal ion coordinated hyaluronic acid hydrogel according to any one of claims 1 to 6 in the preparation of biomedicinal materials for wound healing, tissue repair, and surface coating.

9. The application according to claim 8, wherein The biomedicinal materials include wound dressings, antibacterial materials, anti-inflammatory materials, and surface coating materials for medical devices; and / or the metal ion coordinated hyaluronic acid hydrogel is in the form of a hydrogel and is used for the preparation of acute and chronic wound healing dressings.

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