A NMN and Mg-based 2+ Synergistic multifunctional hydrogel and its preparation method and application

Abstract

The present invention discloses a method based on NMN and Mg 2+ A synergistic multifunctional hydrogel, preparation method, and application thereof belong to the field of biomedical materials technology. The preparation method comprises the following steps: sequentially adding quaternized chitosan and PEGSD copolymer to deionized water under water bath heating to obtain a precursor solution; mixing magnesium chloride hexahydrate, nicotinamide mononucleotide, and the precursor solution to obtain a mixed solution; adding a hydrogen peroxide / horseradish peroxidase oxidation system to the mixed solution, and vortexing the mixed solution to perform Schiff base crosslinking to form QP / NMN / Mg 2+ Multifunctional hydrogel network, obtain multifunctional hydrogel. Use hydrogel as wound dressing to slowly release NMN; hydrogel carries magnesium ions, and the slowly released magnesium ions enhance the antibacterial effect of hydrogel; through QP / NMN / Mg 2+ The multiple effects of hydrogels will enhance ROS clearance and promote wound angiogenesis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a method based on NMN and Mg 2+ Synergistic multifunctional hydrogel, preparation method and application. Background Art

[0002] Disturbance of sugar metabolism is the fundamental cause of the difficulty in healing diabetic wounds. The tricarboxylic acid cycle (TAC) is the core of the energy metabolism of the three major nutrients. + It is an important coenzyme of tricarboxylic acid cycle. Many studies have shown that NAD in diabetic patients + Levels of NAD + It can maintain cellular NAD through the biosynthesis of tryptophan de novo pathway / nicotinamide (NAM) / nicotinic acid (NA) salvage pathway + level, but the excess nicotinamide in the above process is toxic, and due to the presence of restriction endonucleases, nicotinamide is converted to NAD + The conversion efficiency is limited.

[0003] Compared with NMN, NAD + The direct precursor of NAD + Existing studies have found that NAD + This can be achieved by supplementing NMN (Nicotinamide Mononucleotide). According to the inventor's previous research, supplementing NMN can reduce the ROS level of HUVEC cells caused by high sugar, promote HUVEC cell proliferation, migration and tube formation; further in vivo experiments showed that injecting NMN around the wound can significantly accelerate the healing of the wound in diabetic mice and increase the expression of PI3K and SIRT1.

[0004] However, when injecting NMN to accelerate wound healing, it is difficult to achieve long-term release of NMN to obtain maximum therapeutic effect due to its short half-life. Direct application of NMN to the wound site or intravascular injection may lead to drug extravasation and low efficacy. There are problems such as short NMN half-life, no antibacterial activity, peri-wound swelling, drug leakage, and low drug concentration in the center of the wound. Moreover, NMN has no antibacterial effect, and continuous exposure of the wound will increase the risk of infection and moisture loss in the wound.

[0005] Furthermore, wounds in a high-glucose microenvironment are susceptible to bacterial infection, and macrophages produce more reactive oxygen species (ROS) to defend against foreign pathogens. Excessive ROS damages normal cells and tissues, leading to nutrient deficiencies, impaired angiogenesis, hypoxia, and neuropathy, ultimately inducing persistent inflammation and chronic wounds that do not heal. Wound healing typically progresses through four phases: hemostasis, inflammation, proliferation, and matrix remodeling. Therefore, hemostasis is the primary concern after wound exposure, and blood vessels are required to provide nutrients and oxygen for the healing process to proceed smoothly. Furthermore, microvascular dysfunction caused by hyperglycemia is one of the main causes of diabetic complications, resulting in ischemia and slowed wound healing.

[0006] Therefore, how to further eliminate infection and reduce inflammation such as ROS and improve wound angiogenesis and revascularization in diabetic patients is also crucial for treating chronic diabetic wounds and accelerating wound healing. Summary of the Invention

[0007] In order to overcome the shortcomings of the above-mentioned prior art NMN, such as short half-life of NMN, drug leakage, swelling around the wound, low drug concentration in the wound center, no antibacterial effect of NMN, increased risk of infection due to continuous exposure of the wound, loss of moisture in the wound, and inability to further remove ROS and promote angiogenesis, the present invention aims to provide a novel NMN-based Mg-based 2+ Synergistic multifunctional hydrogel, preparation method and application, using hydrogel as wound dressing to slowly release NMN; hydrogel carries magnesium ions, and the slowly released magnesium ions enhance the antibacterial effect of the hydrogel; through NMN / Mg 2+ The multiple effects of hydrogels will enhance ROS clearance and promote wound angiogenesis.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a method based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel comprises the following steps:

[0010] Quaternized chitosan and PEGSD copolymer were sequentially added to deionized water under water bath heating to obtain a precursor solution;

[0011] Mixing magnesium chloride hexahydrate, nicotinamide mononucleotide, and a precursor solution to obtain a mixed solution;

[0012] Hydrogen peroxide / horseradish peroxidase oxidation system was added to the mixed solution and vortexed to perform Schiff base crosslinking to form QP / NMN / Mg 2+ Multifunctional hydrogel network to obtain multifunctional hydrogel.

[0013] In the specific implementation process, the temperature of the water bath heating condition is 50-60°C, preferably 60°C.

[0014] In a specific implementation process, the mass ratio of the quaternized chitosan to the PEGSD copolymer is (18-20): (100-120).

[0015] In a specific implementation process, the mass ratio of the magnesium chloride hexahydrate to nicotinamide mononucleotide is 1:(1-1.25).

[0016] In a specific implementation process, the concentration of nicotinamide mononucleotide in the mixed solution is 5-6.25 mg / mL.

[0017] In a specific implementation process, the mass ratio of the mixed solution to the hydrogen peroxide / horseradish peroxidase oxidation system is (1158-1185): (100-120).

[0018] In a specific implementation process, the mass ratio of hydrogen peroxide to horseradish peroxidase in the hydrogen peroxide / horseradish peroxidase oxidation system is 1:1.

[0019] In a specific implementation process, the concentration of hydrogen peroxide in the hydrogen peroxide / horseradish peroxidase oxidation system is 280-300 μg / mL; the concentration of horseradish peroxidase in the hydrogen peroxide / horseradish peroxidase oxidation system is 20-25 μg / mL.

[0020] The present invention also provides a method based on NMN and Mg according to any one of the above methods. 2+ Synergistic multifunctional hydrogel prepared by NMN and Mg 2+ Synergistic multifunctional hydrogels.

[0021] The present invention provides a method based on NMN and Mg 2+ Application of synergistic multifunctional hydrogels in the preparation of wound dressings for the treatment of diabetes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a method based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel comprises the following steps: first, quaternized chitosan and PEGSD copolymer are mixed to obtain a precursor solution; the PEGSD copolymer containing catechol groups is used to give the hydrogel dressing significant antioxidant and adhesion capabilities, wherein the complexation between the catechol groups and metal ions realizes Mg 2+Sustained release; quaternized chitosan was used for the synthesis of hydrogel, and quaternized chitosan was superior to traditional chitosan in terms of solubility and antibacterial activity; secondly, NMN and Mg 2+ The hydrogel is loaded in a QP hydrogel obtained by forming a dynamic Schiff base bond between the amino groups of QCS and the catechol groups of PEGSD under a mild catalytic system of HRP / H2O2. The hydrogel has excellent injectability and excellent adhesive properties, which help it adapt to irregular wound sites and provide a physical barrier to keep bacteria out and water in, stop bleeding by physical closure, and based on the electrostatic force between NMN and QCS and Mg 2+ The metal coordination between the catechol group of PEGSD achieves sustained delivery. Using hydrogel as wound dressing to slowly release NMN; hydrogel carries magnesium ions to enhance the antibacterial effect of hydrogel; NMN / Mg 2+ The multiple effects of the hydrogel will enhance ROS clearance and promote wound angiogenesis. Through tissue adhesion, hemostasis, bacterial elimination, ROS clearance, and significant promotion of human umbilical vein endothelial cell (HUVEC) migration, proliferation, and angiogenesis QP / NMN / Mg 2+ The synergistic effect of the hydrogels creates a protective environment for diabetic wound healing by promoting angiogenesis, anti-inflammation, and increasing the number of pro-healing M2 macrophages, avoiding the high cost and time-consuming work required to include growth factors or cell / cell-derived therapies.

[0024] Furthermore, the C=O double bond of the aldehyde group in the precursor solution reacts with the amino group (NH2) through a Schiff base reaction to form a C=N double bond cross-linked into a gel. The o-catechol groups can also react with each other to cross-link into a gel. By adding an oxidizing system (H2O2 / HRP) to the precursor solution, HRP can catalyze the decomposition of H2O2 into free radicals, which oxidize the o-catechol groups into o-benzoquinone. O-benzoquinone can react with the amino groups on QCS to cross-link, quickly forming a gel and forming a polymer network. 2+ There is a coordination effect with the catechol of the PEGSD copolymer, which achieves a sustained release effect. There is a Coulomb attraction between NMN and the positive charge on QCS, which can also achieve a sustained release effect.

[0025] Furthermore, the hydrogel described herein is based on PEGSD and QCS. The catechol groups of PEGSD have high affinity for thiols, imidazoles, amines in proteins, and tissue peptides, forming strong covalent bonds, π-π interactions, and hydrogen bonds with various substances. These principles are responsible for the hydrogel's tissue adhesion mechanism. Furthermore, the positively charged quaternary ammonium groups of QCS form electrostatic interactions with the negatively charged carboxyl groups of tissues, further enhancing the hydrogel's bioadhesion strength. This broad adhesion to target tissues offers the hydrogel the potential for widespread practical medical applications in wound healing.

[0026] Furthermore, the hydrogel of the present invention is used to deliver NMN / Mg 2+ As a dressing repair system to promote diabetic wound healing, when Mg is introduced into the dressing 2+ When the hydrogel is loaded with NMN molecules, the hardness reaches 221Pa, which is beneficial for skin wound repair. At the same time, the hydrogel has the ability to regulate and precisely release therapeutic substances and can promote wound healing by simultaneously loading a large number of bioactive compounds into the hydrogel network, which can achieve long-term release and improve cell behavior. During the preparation of the hydrogel, NMN is introduced into the physical cross-linking of QCS and inhibits the oxidation of PEGSD through its free radical scavenging ability for ·OH, giving the hydrogel excellent ROS scavenging ability, which is beneficial for wound healing. 2+ It has the potential to reduce excessive oxidative stress and protect cells in the diabetic wound microenvironment.

[0027] Furthermore, Mg 2+ The synergistic antibacterial effect of Mg and QCS is responsible for the enhanced antibacterial efficiency, as the strong electrostatic interaction between the positive charge of QCS and the negatively charged components of bacterial phospholipid membranes leads to bacterial death through membrane disintegration. 2+ There are three potential mechanisms for limiting microbial growth. First, metal ions can directly disrupt ATP synthesis and DNA replication by crossing the cell membrane; second, Mg 2+ It may use electrostatic forces to fix the cell membrane, damage cell integrity, and prevent protons and other molecules from effectively passing through the cell membrane; finally, metal ions induce oxidative stress by producing ROS, which can disrupt the normal activities of bacteria and ultimately kill bacteria by destroying the bacterial membrane, DNA and mitochondria. The combination of all the above properties significantly improves the antibacterial activity of the hydrogel.

[0028] QP / NMN / Mg 2+ Hydrogel adhesive is a promising hemostatic barrier for wound closure because it combines the natural hemostatic properties of chitosan, positively charged quaternary ammonium groups, and excellent adhesion, which synergistically enhances the hemostatic activity of NMN and Mg. 2+ The inherent pro-regenerative activity of QCS and PEGSD synergistically accelerated the healing time of diabetic wounds. In addition, the moist environment provided by the hydrogel and the QP / NMN / Mg 2+ The beneficial activities of hydrogels promote wound healing. As a dressing, hydrogels can integrate a variety of active molecules with different functions to promote diabetic wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a macroscopic photograph of the hydrogel of the present invention;

[0030] Figure 2These are photos of the hydrogel of the present invention adhered to a bent and extended finger after complete gelation;

[0031] Figure 3 QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Statistical graph of the hydrogel's pigskin shear adhesion strength test results, expressed as mean ± SD (n = 5);

[0032] Figure 4 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ FT-IR spectrum of hydrogel;

[0033] Figure 5 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Schematic diagram of the rheological properties of hydrogels;

[0034] Figure 6 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Schematic diagram of the degradation curves of hydrogel samples in the physiological microenvironment that characterizes their drug and ion release kinetics, where (A) is QP / NMN and QP / NMN / Mg 2+ Schematic diagram of NMN release (μg / mL) from hydrogels in vitro; Figure (B) is QP / Mg 2+ and QP / NMN / Mg 2+ In vitro Mg of hydrogels 2+ Schematic diagram of release amount (μmol / L);

[0035] Figure 7 The HFB of the present invention and QP, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ Representative schematic diagram of live-dead cell staining after 24 h of hydrogel co-culture;

[0036] Figure 8 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ DPPH free radical scavenging ability test diagram of hydrogel;

[0037] Figure 9 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Figure 2. Test of the hydrogel's ROS scavenging ability.

[0038] Figure 10 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Figure 1 shows the antibacterial activity of the hydrogel. Figure (A) shows the antibacterial activity test image when it comes into contact with Escherichia coli, and Figure (B) shows the antibacterial activity test image when it comes into contact with Staphylococcus aureus.

[0039] Figure 11 QP / NMN / Mg of the present invention 2+ Figure 1 shows the hemostatic performance of the hydrogel in the SD rat liver bleeding model. Figure (A) is a schematic diagram of the rat liver incision model; Figure (B) is a representative photo of bleeding; Figure (C) is a statistical chart of the amount of bleeding.

[0040] Figure 12 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Diagram showing the hydrogel's in vivo diabetic wound healing performance test;

[0041] Figure 13 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Statistical diagram of wound size of hydrogel and control group;

[0042] Figure 14 QP, QP / Mg of the present invention 2+ , QP / NMN and QP / NMN / Mg 2+ Statistical graph of the number of CD31-positive new blood vessels in the hydrogel and control groups;

[0043] Figure 15 Schematic diagram of the synthesis route of PEGSD and QCS used in the present invention. DETAILED DESCRIPTION

[0044] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0045] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0046] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0047] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0048] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0049] The present invention provides a method based on NMN and Mg 2+ Synergistic multifunctional hydrogel, preparation method and application.

[0050] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0051] The following examples were prepared using conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art. In the present specification and the following examples, unless otherwise specified, "%" indicates percentage by mass, "part" indicates parts by mass, and "ratio" indicates mass ratio.

[0052] Hydrogels are polymer materials with a three-dimensional network structure that exhibit excellent biocompatibility, effective adhesion, good antibacterial properties, and a high water content (70-95%). They can absorb wound exudate and have oxygen permeability and hemostasis functions. In addition, the three-dimensional network structure of hydrogels facilitates the loading of active factors and drugs for slow release, thus showing broad application prospects in wound dressings. Hydrogels overcome the shortcomings of conventional dressings and not only provide a physical barrier to secondary infection, but also provide a compatible physiological environment, that is, hydrogels provide wounds with suitable biocompatibility, moisture, and antibacterial interfaces. Hydrogel dressings are widely used in diabetic wound treatment due to their soft properties similar to the extracellular matrix (ECM), their ability to fill any irregularly shaped wound, excellent biocompatibility, effective drug encapsulation, and sustained drug delivery. In particular, adhesive hydrogels can adhere to and bond damaged tissues, act as hemostats or sealants to stop bleeding, block the flow of liquid or gas in the wound, and act as a barrier to protect microorganisms.

[0053] The precursor solution of the hydrogel contains two components, A and B. Component A is quaternized chitosan (QCS), which exerts cationic antibacterial effects, seals wounds, stops bleeding (cations react with negative charges on platelets to aggregate platelets), and promotes wound repair (QCS promotes macrophage secretion of PDGF and IL-1, promoting angiogenesis and fibroblast proliferation). Component B is a PEGSD copolymer (poly(glycerol sebacate)-co-polyethylene glycol-g-catechol) formed by the reaction of sebacic acid-terminated polyethylene glycol, glycerol, and dihydrocaffeic acid. The carboxyl group (-COOH) of sebacic acid and the hydroxyl group (-OH, grafted dihydrocaffeic acid) of PEG undergo dehydration esterification. The catechol group of this component provides adhesion and antioxidant properties. The catechol group chemically cross-links with amino groups on the skin surface and can also form hydrogen bonds with the skin to exert adhesion properties. The antioxidant effect is that the catechol group has reducing properties, reacts with free radicals, and consumes free radicals to achieve the antioxidant effect. The C=O double bond of the aldehyde group in components A and B reacts with the amino group (NH2) through a Schiff base reaction to form a C=N double bond cross-linked into a gel. The o-catechol groups can also react with each other to cross-link into a gel. By adding an oxidizing system (H2O2 / HRP) to the precursor solution, HRP can catalyze the decomposition of H2O2 into free radicals. The free radicals oxidize the o-catechol groups, oxidizing them into o-benzoquinone. O-benzoquinone can react with the amino groups on QCS to cross-link, quickly forming a gel and forming a polymer network. 2+ There is a coordination effect with catechol to achieve a sustained release effect. There is a Coulomb attraction between NMN and the positive charge on QCS, which can also achieve a sustained release effect.

[0054] Magnesium ion, also known as Mg 2+ , is the fourth most abundant metal cation in the human body, along with Ag + and Cu 2+Compared with other potentially toxic ions such as Mg, Mg has good biocompatibility. 2+ It plays an important role in the energy metabolism and regeneration of damaged tissues. In addition, it has been shown to have excellent antibacterial activity against two common bacteria, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and has the ability to effectively eliminate bacteria and prevent infection during diabetic wound repair. 2+ It can help macrophage phenotype change from pro-inflammatory M1 to pro-healing M2 and increase angiogenesis to alleviate microvascular damage induced by hyperglycemia, while the adhesion and migration of human dermal fibroblasts (HSFs) are affected by Mg. 2+ Effect of concentration, in order to promote wound healing, a controlled concentration of Mg at the wound site 2+ It is crucial to administer NMN / Mg simultaneously in diabetic wound dressings 2+ , NMN and Mg 2+ loaded in QP hydrogel and based on the electrostatic force between NMN and QCS and Mg 2+ The metal coordination between the catechol groups of PEGSD enables sustained delivery, allowing Mg to be deposited at the wound site. 2+ The concentration of Mg is controlled to avoid localized excess and explosion. 2+ Causes damage to cells.

[0055] QCS and PEGSD were synthesized based on previous studies. PEGSD was prepared based on X. Zhao, Y. Liang, Y. Huang, J. He, Y. Han, and B. Guo. Physical double-network hydrogel adhesives with rapid shape adaptability, fast self-healing, antioxidant, and NIR / pH stimulus-responsiveness for multidrug-resistant bacterial infection and removable wound dressing, Advanced Functional Materials 30(17)(2020)1910748.

[0056] PEGSD polymer was prepared by mixing PEG (polyethylene glycol, Mn=2000), glycerol, sebacic acid and 3,4-dihydroxyphenylpropionic acid by melt polycondensation reaction in the absence of a catalyst to prepare PEGSD copolymer (poly(glyceryl sebacate)-co-polyethylene glycol-g-catechol).

[0057] The specific preparation process is as follows Figure 15 As shown, the steps are as follows: sebacic acid (202.0 mg) and PEG (500 mg) are evacuated in an oil bath at 140° C. for 1 h, and then nitrogen is passed through at 140° C. for 24 h to generate sebacic acid-terminated PEG, and then glycerol (70 mg) and 3,4-dihydroxyphenylpropionic acid (95.6 mg) are added to the reaction system, and the reaction is evacuated in an oil bath at 140° C. for 1 h, and then nitrogen is passed through at 140° C. for 24 h to prepare PEGSD copolymer (poly(glycerol sebacate)-co-polyethylene glycol-g-catechol).

[0058] Preparation of QCS refers to 9(1)(2018)2784. And J.Qu,

[0059] Chitosan (1 g, Sigma-Aldrich) was reacted with GTMAC (glycidyl trimethylammonium chloride) at 55 °C for 18 h to generate quaternary chitosan (QCS), which was then separated, dialyzed (or precipitated), and freeze-dried. Figure 15 As shown in the synthesis route for QCS, 0.5 g of chitosan was suspended in 18 mL of 0.5% v / v glacial acetic acid. Subsequently, 773 μL of GTMAC was added dropwise to the solution, followed by stirring at 55°C for 18 hours. Finally, the solution was centrifuged (6500 rpm, 8 minutes). The supernatant was precipitated with 5-8 volumes of acetone. The entire purification process was repeated three times, and the final product was dried in a vacuum oven.

[0060] 1. Hydrogel Preparation Method

[0061] 1.1QP / NMN / Mg2+ The hydrogel preparation process is as follows:

[0062] Step 1: adding quaternized chitosan and PEGSD copolymer to deionized water in sequence under water bath heating conditions to obtain a precursor solution; wherein the water bath heating temperature is 50-60° C.; and the mass ratio of quaternized chitosan to PEGSD copolymer is (18-20): (100-120).

[0063] Specifically, 18-20 mg of freeze-dried quaternized chitosan (QCS) was weighed and placed in a 5 mL centrifuge tube, 1 mL of deionized water was added, and the mixture was dissolved at 50-60°C for 4-6 hours. After complete dissolution, 100-120 mg of PEGSD was added and heated at 50-60°C for 2-5 minutes. After complete dissolution, a precursor solution was obtained. The most preferred water bath heating temperature was 60°C.

[0064] Step 2: Mixing magnesium chloride hexahydrate, nicotinamide mononucleotide, and a precursor solution to obtain a mixed solution; wherein the mass ratio of magnesium chloride hexahydrate to nicotinamide mononucleotide is 1:(1-1.25); and the concentration of nicotinamide mononucleotide in the mixed solution is 5-6.25 mg / mL.

[0065] Specifically, 20 μl of magnesium chloride hexahydrate (3 mg / mL) and 20 to 25 μl of nicotinamide mononucleotide (NMN, 200 to 250 mg / mL is the concentration of the mother solution) were added to the precursor solution to obtain a mixed solution.

[0066] Step 3: Add hydrogen peroxide / horseradish peroxidase oxidation system to the mixed solution and vortex to perform Schiff base crosslinking to form QP / NMN / Mg 2+ A multifunctional hydrogel network is obtained, wherein the mass ratio of the mixed solution to the hydrogen peroxide / horseradish peroxidase oxidation system is (1158-1185):

[0067] (100-120); the mass ratio of hydrogen peroxide to horseradish peroxidase in the hydrogen peroxide / horseradish peroxidase oxidation system was 1:1; the concentrations of hydrogen peroxide and horseradish peroxidase in the hydrogen peroxide / horseradish peroxidase oxidation system were 280-300 μg / mL and 20-25 μg / mL, respectively. The vortexing treatments were performed by the first vortexing after adding hydrogen peroxide to the hydrogen peroxide / horseradish peroxidase oxidation system and the second vortexing after adding horseradish peroxidase.

[0068] Specifically, add 50-60 μl of hydrogen peroxide (0.6% H2O2, concentration of 280-300 μg / mL) to the mixed solution, vortex for 5-15 seconds, and then add horseradish peroxidase (HRP, concentration of 20-25 μg / mL).

[0069] 50-60ul, vortex for 10-20s, and then the gel solidifies to obtain QP / NMN / Mg 2+ Among them, polyester PEGSD was selected as the main component of the multifunctional hydrogel because the catechol group has significant antioxidant, adhesion and Mg 2+ Complexing ability; QCS is an ingredient with good solubility and antibacterial activity.

[0070] The present invention provides a novel biocompatible, adhesive, antibacterial, sustained-release NMN and Mg 2+ , scavenging ROS and promoting angiogenesis QP / NMN / Mg 2+ Multifunctional hydrogels provide new therapeutic targets and ideas for the treatment of diabetic wounds, laying the foundation for the clinical application of new hydrogels. 2+ Synergistic QP / NMN / Mg 2+ The multifunctional hydrogel can be used in the preparation of wound dressings for treating diabetes.

[0071] 1.2 The preparation process of QP hydrogel is as follows:

[0072] Weigh 18-20 mg of freeze-dried quaternized chitosan (QCS) and place it in a 5 mL centrifuge tube. Add 1 mL of deionized water and dissolve it at 50-60°C for 4-6 hours. After complete dissolution, add 100-120 mg of PEGSD and heat it at 50-60°C for 2-5 minutes. After complete dissolution, add 50-60 μl of hydrogen peroxide (0.6% H2O2, concentration of 280-300 μg / mL) and vortex for 5-15 seconds. Then add 50-60 μl of horseradish peroxidase (HRP, concentration of 20-25 μg / mL) and vortex for 10-20 seconds to solidify the gel to obtain QP hydrogel.

[0073] 1.3QP / Mg 2+ The preparation process of the hydrogel is as follows:

[0074] 18-20 mg of freeze-dried quaternized chitosan (QCS) was weighed and placed in a 5 mL centrifuge tube. 1 mL of deionized water was added and dissolved at 50-60 ° C for 4-6 h. After complete dissolution, 100-120 mg of PEGSD was added and heated at 50-60 ° C for 2-5 min. After complete dissolution, 20 μl of magnesium chloride hexahydrate (3 mg / mL) was added. Subsequently, 50-60 μl of hydrogen peroxide (0.6% H2O2, concentration of 280-300 μg / mL) was added and vortexed for 5-15 s. Then, 50-60 μl of horseradish peroxidase (HRP, concentration of 20-25 μg / mL) was added and vortexed for 10-20 s. After the gel solidified, QP / Mg was obtained. 2+ hydrogel.

[0075] The preparation process of 1.4QP / NMN hydrogel is as follows:

[0076] 18-20 mg of freeze-dried quaternized chitosan (QCS) was weighed and placed in a 5 mL centrifuge tube. 1 mL of deionized water was added and dissolved at 50-60 ° C for 4-6 h. After complete dissolution, 100-120 mg of PEGSD was added and heated at 50-60 ° C for 2-5 min. After complete dissolution, 20-25 μl of nicotinamide mononucleotide (NMN, 200-250 mg / mL) was added. Then, 50-60 μl of hydrogen peroxide (0.6% H2O2, concentration of 280-300 μg / mL) was added and vortexed for 5-15 seconds. Then, 50-60 μl of horseradish peroxidase (HRP, concentration of 20-25 μg / mL) was added and vortexed for 10-20 seconds. After the gel solidified, QP / NMN hydrogel was obtained.

[0077] 1.5 Example

[0078] Example 1

[0079] Step 1: Weigh 20 mg of freeze-dried quaternized chitosan (QCS) into a 5 mL centrifuge tube, add 1 mL of deionized water, and dissolve at 50°C for 4 h. After complete dissolution, add 100 mg of PEGSD and heat at 50°C for 2 min. After complete dissolution, obtain a precursor solution.

[0080] Step 2: Add 20 μl of magnesium chloride hexahydrate (3 mg / mL) and 20 μl of nicotinamide mononucleotide (NMN, 200 mg / mL) to the precursor solution to obtain a mixed solution.

[0081] Step 3: Add 50 μl of hydrogen peroxide (0.6% H2O2, concentration of 280 μg / mL) to the mixed solution, vortex for 5 seconds, then add 50 μl of horseradish peroxidase (HRP, concentration of 20 μg / mL), vortex for 10 seconds, and then solidify the gel to obtain QP / NMN / Mg 2+ hydrogel.

[0082] Example 2

[0083] Step 1: Weigh 18 mg of freeze-dried quaternized chitosan (QCS) into a 5 mL centrifuge tube, add 1 mL of deionized water, and dissolve at 60°C for 6 h. After complete dissolution, add 120 mg of PEGSD and heat at 60°C for 5 min. After complete dissolution, obtain a precursor solution.

[0084] Step 2: Add 20 μl of magnesium chloride hexahydrate (3 mg / mL) and 25 μl of nicotinamide mononucleotide (NMN, 250 mg / mL) to the precursor solution to obtain a mixed solution.

[0085] Step 3: Add 60 μl of hydrogen peroxide (0.6% H2O2, concentration of 300 μg / mL) to the mixed solution, vortex for 15 seconds, then add 60 μl of horseradish peroxidase (HRP, concentration of 25 μg / mL), vortex for 20 seconds, and then solidify the gel to obtain QP / NMN / Mg 2+ hydrogel.

[0086] Example 3

[0087] Step 1: Weigh 19 mg of freeze-dried quaternized chitosan (QCS) into a 5 mL centrifuge tube, add 1 mL of deionized water, and dissolve at 55°C for 5 h. After complete dissolution, add 110 mg of PEGSD and heat at 55°C for 4 min. After complete dissolution, obtain a precursor solution.

[0088] Step 2: Add 20 μl of magnesium chloride hexahydrate (3 mg / mL) and 24 μl of nicotinamide mononucleotide (NMN, 250 mg / mL) to the precursor solution to obtain a mixed solution.

[0089] Step 3: Add 55ul of hydrogen peroxide (0.6% H2O2, concentration of 290μg / mL) to the mixed solution, vortex for 10s, then add 55ul of horseradish peroxidase (HRP, concentration of 24μg / mL), vortex for 15s, and then solidify the gel to obtain QP / NMN / Mg 2+ hydrogel.

[0090] Example 4

[0091] The difference from Example 1 is that the nicotinamide mononucleotide (NMN, 200 mg / mL) in step 2 is replaced with nicotinamide mononucleotide (NMN, 230 mg / mL) to obtain QP / NMN / Mg 2+ hydrogel.

[0092] Example 5

[0093] The difference from Example 2 is that the 18 mg quaternized chitosan (QCS) in step 1 is replaced with 20 mg quaternized chitosan (QCS) to obtain QP / NMN / Mg 2+ hydrogel.

[0094] Example 6

[0095] The difference from Example 3 is that the hydrogen peroxide concentration in step 3 is replaced by 290 μg / mL to 300 μg / mL to obtain QP / NMN / Mg 2+ hydrogel.

[0096] Comparative Example 1QP hydrogel:

[0097] The preparation method of QP hydrogel is as follows:

[0098] QP hydrogels were prepared by sequentially dissolving QCS (20 mg / mL) and PEGSD (100 mg / mL) in deionized water in a heating water bath and then mixing. QCS / PEGSD (QP) hydrogels were formed by chemical crosslinking between the amino groups of QCS and the catechol groups of PEGSD, and oxidative coupling between the catechol groups of PEGSD, under the oxidation of the solution in a 0.6% H2O2 (300 μg / mL) / HRP (horseradish peroxidase, Sigma, 25 μg / mL) system.

[0099] Comparative Example 2QP / Mg 2+ Hydrogel, comparative example 3QP / NMN hydrogel:

[0100] For Mg doping 2+ and / or NMN QP hydrogel (QP / NMN / Mg 2+ ), magnesium ions (MgCl2·6H2O, containing 58.82μg / mL Mg 2+ 1 mL hydrogel) and / or nicotinamide mononucleotide (NMN, 5 mg / mL) were premixed in the QP hydrogel precursor solution and vortexed for homogeneity. Schiff base crosslinking was performed to obtain QP / Mg 2+ hydrogel and QP / NMN hydrogel.

[0101] 2. Performance testing and results

[0102] PEG (polyethylene glycol, Mn = 2000), glycerol, sebacic acid, and 3,4-dihydroxyphenylpropionic acid were mixed by melt polycondensation in the absence of a catalyst to prepare the PEGSD copolymer precursor. Chitosan (1 g, Sigma-Aldrich) was reacted with GTMAC (glycidyltrimethylammonium chloride) at 55° for 15 hours to produce quaternary chitosan (QCS), which was then isolated, dialyzed (or precipitated), and freeze-dried.

[0103] Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), energy dispersive spectrometer (EDS), nuclear magnetic resonance (1H NMR), degradation analysis, adhesion strength, NMN and Mg 2+ Release test to explore comparative example 1QP and comparative example 2QP / Mg 2+ , Comparative Example 3 QP / NMN and QP / NMN / Mg 2+ Properties of hydrogels.

[0104] 2.1 Viscoelasticity test:

[0105] like Figure 1As shown, QCS, PEGSD, NMN and Mg 2+ Mixed solution and QP / NMN / Mg 2+ Macroscopic image of a hydrogel demonstrating good tissue adhesion for use as a wound dressing.

[0106] like Figure 2 As shown in the figure, the hydrogel adheres to the bent and stretched fingers after complete gelation. The hydrogel shows good viscoelasticity after 2 minutes of cross-linking and can be well stretched between two fingers. The hydrogel can firmly adhere to the bent fingers without shrinkage or rupture, indicating that stable and close contact can be established between the hydrogel and the dynamic wound with good viscoelasticity.

[0107] 2.2 Adhesion strength test:

[0108] QP, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ The adhesion properties of the hydrogel were tested using fresh pigskin. Pigskin was selected to simulate human tissue and was cut into rectangular strips (10 mm × 30 mm) and washed with PBS before the adhesion test. Then, 200 μL of the test hydrogel was applied and sandwiched between two pieces of pigskin with a bonding area of ​​10 × 10 mm. The adhesion properties were then measured by a lap shear test at a rate of 5 mm / min-1 on an Instron materials testing system (MTS standard 43) equipped with a 50N load cell. In addition, QP / NMN / Mg was tested on eight organs (heart, liver, lung, spleen, kidney, bladder, colon, and stomach) removed from C57 mice. 2+ In vitro adhesion of hydrogel. Specifically, 200 μL QP / NMN / Mg 2+ The hydrogels were attached to stainless steel forceps before being attached to the moist surfaces of the organs, and all of the above tests were used more than three times.

[0109] The results are as follows Figure 3 As shown, according to the pig skin lap shear test, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ The average adhesive strength of the hydrogel increased by about 2 kPa. 2+ The hydrogel adhered to pig skin with an adhesive strength of approximately 7 kPa, which is higher than that of commercial dressings (approximately 5 kPa) and an effective value for staying in place on diabetic wounds despite natural skin movement.

[0110] QP / NMN / Mg 2+The excellent adhesion behavior exhibited by the hydrogel suggests its great potential for wound repair. The reasons for the hydrogel's tissue adhesion mechanism are as follows: the catechol groups of PEGSD have high affinity for thiols, imidazoles, amines in proteins, and tissue peptides, forming strong covalent bonds, π-π interactions, and hydrogen bonds with various substances. Furthermore, the positively charged quaternary ammonium groups of QCS may form electrostatic interactions with the negatively charged carboxyl groups of tissues, which may further enhance the bioadhesion strength of the hydrogel. This broad adhesion property on target tissues offers the hydrogel the potential for widespread practical medical applications in wound healing.

[0111] 2.3 Fourier transform infrared spectroscopy (FT-IR) test:

[0112] QP, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ FT-IR spectra of freeze-dried hydrogels were obtained on a Nicolet 6700, Thermo Scientific, at 550–4000 cm -1 was performed to study the chemical composition of the hydrogel.

[0113] The test results are as follows Figure 4 As shown, the four hydrogels have a peak at 1735 cm -1 and 2891cm -1 The absorption peaks in the FT-IR spectrum at 1475 cm-1 and 1475 cm-2 correspond to the ester group and the methylene group, respectively. -1 The characteristic absorption peak representing the methyl group in GTMAC is shown at .

[0114] 2.4 Rheological properties test:

[0115] Evaluation of QP, QP / Mg by using a rheometer (DHR-2) 2+ , QP / NMN and QP / NMN / Mg 2+ Rheological properties of hydrogels. The test temperature was 37°C, and the storage modulus (G′) and loss modulus (G″) of different hydrogels were evaluated by placing the hydrogel solution on a 20 mm parallel plate with a 1 mm gap. Time sweep tests were performed at a strain amplitude of 1% and a frequency of 10 rad / s. The rheological behavior of the hydrogel was analyzed, and the storage modulus (G′) and loss modulus (G″) were determined using a rheometer in time sweep mode.

[0116] Rheological test results are as follows Figure 5 As shown, QP, QP Mg 2+ , QP / NMN and QP / NMN / Mg 2+ The G′ value of the hydrogel is always 5-10 times the G″ value, showing elastic properties.2+ Hydrogel and QP / NMN / Mg 2+ The G' value of the hydrogels is similar, about 230Pa, indicating that the cross-linked network is stronger than that of QP / NMN (98Pa). The introduction of NMN reduces the hardness of the QP hydrogel because NMN has a certain degree of reducing property when reacting with H2O2, which affects the cross-linking strength of the QP hydrogel. In contrast, when Mg is introduced at the same time, the cross-linked network is stronger than that of QP / NMN (98Pa). 2+ When NMN molecules were added, the hardness of the hydrogel increased to 221 Pa, which is sufficient for the application of hydrogels because G′ near 100 Pa is beneficial for the healing of skin wounds.

[0117] 2.5 In vitro NMN and Mg 2+ Release test:

[0118] For NMN release assay, a standard curve was established. Specifically, NMN was dissolved in PBS at 15 to 1000 μg / mL to prepare a solution and tested at a wavelength of 280 nm (because UV-vis spectroscopy showed that the maximum absorption spectrum of NMN was 280 nm). QP, QP / NMN and QP / NMN / Mg 2+ Hydrogels (1 mL, n = 3) were immersed in PBS (9 mL) at 37 ° C (shaking at 100 rpm) and analyzed by subtracting the QP hydrogel solution at a wavelength of 280 nm. At the predetermined time point, 300 μL of release buffer was removed for analysis and 300 μL of fresh PBS was added to maintain the same volume. This method is similar to that of QP / Mg 2+ and QP / NMN / Mg 2+ Mg 2+ Release test, while using blood Mg 2+ Mg concentration was detected by a kit (Solaibao, Beijing, China) 2+ .

[0119] QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ The hydrogel was immersed in a physiological microenvironment that characterized the release kinetics of its drugs and ions, and the physiological microenvironment that characterized the release kinetics of its drugs and ions was a PBS environment with a temperature of 37°C and a pH of 7.4. Figure 6 As shown in Figure A, QP / NMN / Mg 2+ The cumulative release of NMN is higher than QP / NMN, indicating that Mg 2+ The incorporation of slightly increased the release of NMN, and the average daily release concentration of NMN in the two hydrogels could be maintained at nearly 200 μg / mL. Figure 6 As shown in Figure B, in the first 4 days, Mg 2+The average release concentration of QP / NMN / Mg is more than 160μmol / L, which will decrease later but still be higher than 120μmol / L. 2+ Can provide NMN and Mg at the same time 2+ Sustained release for diabetic wound repair.

[0120] 2.6 In vitro cell compatibility and proliferation test:

[0121] Human fibroblasts (HFB) were cultured in DMEM medium (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin (Hyclone) at 37°C in a 5% CO2 incubator. The cytotoxicity of the hydrogels was studied using an activity / cytotoxicity kit (YESAN, Shanghai, China). HFB cells were seeded in a 96-well plate at a density of 10,000 cells / well, and hydrogel slices (5 mm in diameter, 0.5 mm in thickness) were prepared and co-cultured with HFB for 24 h. The kit was used to assess the viability of cells co-cultured with the hydrogel disc. After 24 hours of co-culture, the hydrogel disc and culture medium were removed and the culture medium was added according to the instructions. After incubation for another 45 minutes, images were taken using a fluorescence microscope (Nikon, DS-Ri2, Japan).

[0122] HFB (human fibroblasts) were co-cultured with the hydrogels, and when a live / dead assay was used to show cell morphology, the results were as follows: Figure 7 As shown, most HFB cells were stained green (live cells), and only a few cells were stained red (dead cells), showing QP, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ High cell compatibility of the hydrogel.

[0123] 2.7 In vitro ROS scavenging ability test:

[0124] 2',7'-dichlorofluorescein diacetate (DCFH-DA, Maokang, Shanghai, China) probe was used to evaluate the intracellular ROS scavenging ability of hydrogels. Briefly, HUVECs were seeded on 6-well plates, and ROS were induced by ROS up (MKbio, Shanghai, China) and co-cultured with hydrogel samples for 12 hours. A negative control (PBS) was set up. Afterwards, the hydrogels were removed and DCFH-DA was added for staining at 37°C for 20 minutes. The cells were analyzed using a flow cytometer (BD Biosciences). C6, USA) were used to determine the ROS scavenging ability of each group, with each group repeated three times.

[0125] The antioxidant properties of the hydrogels were determined using a ROS-specific probe, and 1,1-diphenyl-2-picrylhydrazyl (DPPH·) was used as the model free radical for detection. 2+ , QP / NMN and QP / NMN / Mg 2+ The hydrogels reduced the production of active ROS when HUVECs were subjected to intracellular oxidative stress by stimulation with ROSup reagent (ROS inducer).

[0126] The results are as follows Figure 8 and 9 As shown, ROS-positive cells labeled with DCFH-DA and ROS inducers significantly promoted ROS production, while the hydrogels effectively alleviated ROS production, with the most significant groups being QP / NMN and QP / NMN / Mg. 2+ , which may be due to the introduction of NMN, a potent antioxidant that acts as an agonist of sirtuins. Compared with QP, Mg 2+ The incorporation of did not significantly enhance the QP / Mg 2+ The ROS scavenging ability of Figure 9 In addition, QP / NMN and QP / NMN / Mg 2+ The hydrogel-treated group showed almost complete free radical scavenging ability (more than 90% Figure 8 ), while QP and QP / Mg 2+ The group showed only 50-60% DPPH clearance, indicating that the addition of NMN can significantly improve the DPPH scavenging ability. Figure 8 . The free radical scavenging ability of pure QP may be due to the interaction between DPPH radicals and the residual catechol groups of QP hydrogel and the residual free amino groups of QCS. During the preparation of the hydrogel, NMN was introduced into the physical cross-linked quaternary ammonium of QCS and partially inhibited the oxidation of PEGSD through its ability to scavenge ·OH radicals generated by hydrogen peroxide; this gave the hydrogel excellent ROS scavenging ability and contributed to wound healing. In summary, the above findings indicate that QP / NMN / Mg 2+ Potential to reduce excessive oxidative stress and protect cells in the diabetic wound microenvironment.

[0127] 2.8 In vitro antibacterial performance test:

[0128] The antibacterial properties of the hydrogels were determined by plate count method. Briefly, 10 μL of Escherichia coli (E.coil) and Staphylococcus aureus (S.aureus) suspension (108 CFU / mL) was added to QP, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+The cells were plated on the surface of a hydrogel disc (size: 10 mm × 10 mm × 4 mm) and incubated in a 48-well plate at 37°C for 2 hours. 1 mL of PBS was then introduced into each well to resuspend any bacterial survivors. Finally, 10 μL of the above resuspension was added to an agar plate and colonies were counted after incubation at 37°C for 24 hours. Antibacterial rate (%) = (bacterial count of the control - surviving count on the hydrogel) / number of bacteria in the control) × 100%.

[0129] Figure 10 The middle figure (A) and figure (B) are PBS, QP(I), QP / Mg 2+ (II), QP / NMN(III) and QP / NMN / Mg 2+ Images of the contact antimicrobial activity of the treated hydrogel against (A) Escherichia coli and (B) Staphylococcus aureus. Dressings with antimicrobial activity can minimize infection by harmful microorganisms and improve delayed wound healing, which is one of the key challenges in the healing process of diabetic wounds. The intrinsic antimicrobial activity of QCS has been widely demonstrated before, but it is still unknown in QP. Here, Figure 10 As shown, the antibacterial ability of the hydrogels against Escherichia coli and Staphylococcus aureus was tested using surface antibacterial activity assays, and all Escherichia coli (100%) were inactivated after exposure to these hydrogels at 37°C for 2 hours (see Figure 10 A), Compared with the PBS group, most of the Staphylococcus aureus (>90%) were infected by QP, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ Hydrogel kills (see Figure 10 B), showing effective antibacterial ability. Mg 2+ The synergistic antibacterial effect of Mg and QCS is responsible for the enhanced antibacterial efficiency. The strong electrostatic interaction between the positive charge of QCS and the negatively charged bacterial phospholipid membrane components leads to bacterial death through membrane disintegration. 2+ There are three potential mechanisms for limiting microbial growth: first, metal ions can directly disrupt ATP synthesis and DNA replication by crossing the cell membrane; second, Mg 2+ The researchers found that the metal ions may immobilize the cell membrane with electrostatic forces, impair cell integrity, and prevent protons and other molecules from effectively passing through the cell membrane. Finally, the metal ions may induce oxidative stress by generating ROS, which can disrupt the normal activities of bacteria and ultimately kill the bacteria by damaging their membranes, DNA, and mitochondria. The combination of all the above properties significantly enhanced the antibacterial activity of the hydrogel, indicating that the hydrogel has great potential in protecting diabetic wounds from contamination.

[0130] 2.9 In vivo hemostasis assay:

[0131] In the rat hemorrhagic liver model, the rat was anesthetized and then fixed to a surgical operating table. The rat liver was exposed through an abdominal incision. Filter paper was weighed and placed under the liver. Then, a 5 mm deep bleeding wound was introduced into the liver of the SD rat using a blade, and 200-250 μL of QP / NMN / Mg was immediately administered. 2+ The hydrogel was applied to the bleeding site. A control group was left untreated. The filter paper was then weighed and blood loss was calculated.

[0132] The first step in wound healing is hemostasis. It is hypothesized that the adhesive hydrogel can adhere to the surrounding tissue and act as a physical barrier to promote the hemostasis process due to its excellent tissue adhesion ability. 2+ The hydrogel was selected for in vivo studies due to its good adhesion, high antibacterial efficacy and excellent biocompatibility. Figure 11 A) was used to evaluate the hemostatic effect of the prepared hydrogels. The blood loss was recorded after application or not of the hydrogel. In all cases of bleeding, the hydrogel-treated group had a greater hemostatic effect, as almost no obvious blood was seen, while the untreated group showed obvious bleeding ( Figure 11 B). Figure 11 C showed that when applied to a bleeding liver, the sticky hydrogel significantly reduced blood loss (356 mg) compared to the untreated group (1181 mg), demonstrating the hydrogel's excellent hemostatic ability. The strong adhesion between the hydrogel and the wound tissue surrounding the bleeding site is the reason why the hydrogel has good hemostatic ability, and the hydrogel can act as a physical barrier to hemostasis. In addition to superficial bleeding, intraoperative or inadvertent bleeding from deep blood vessels is also a problem that cannot be ignored. QP / NMN / Mg 2+ The hydrogel adhesive is a promising hemostatic barrier for wound closure because it combines the natural hemostatic properties of chitosan, positively charged quaternary ammonium groups, and excellent adhesion, which synergistically enhances hemostatic activity. These findings provide evidence for the hydrogel's excellent hemostatic capacity in vivo and point to its potential applicability in repairing infected and / or bleeding diabetic wounds.

[0133] 2.10 Diabetes Wound Healing Test:

[0134] First, C57 mice were fed a high-fat and high-sugar diet for four weeks and then given streptozotocin (STZ, 10 mg / kg; Meilunbio, China) for five days. Blood glucose levels above 16.7 mM are considered type 2 diabetes. After two weeks, the mice were randomly divided into five groups (n = 12): control (Tegaderm Film, 3M, USA), QP, QP / Mg, and 10 mg / kg of QP. 2+ , QP / NMN and QP / NMN / Mg 2+The gel was then injected. A full-thickness wound was then created in the center of the back using an 8 mm biopsy punch. Next, 150 μL of hydrogel or PBS (control) was injected into the wound site and covered with a Tegaderm membrane (3M, USA). The wound area was measured and photographed using a digital camera on days 0, 3 (inflammatory phase), 7 (proliferation phase), and 14 (remodeling phase).

[0135] A wound with a diameter of 8 mm was made on the dorsal side of the mouse using a skin punch, and then QP, QP / Mg 2+ , QP / NMN and QP / NMN / Mg 2+ hydrogel treatment, and Tegaderm dressing was set as a control. Figure 12 Macroscopic pictures of wounds at different time points and wound closure marks of the wound area are shown. 2+ The wounds in the hydrogel group contracted more than those in the other treatment groups ( Figure 13 , P<0.05). On the 7th day, QP / NMN / Mg 2+ The wound area of ​​the group (19%) was significantly reduced, while the control group, QP group, QP / / Mg 2+ The wound sizes in the hydrogel and QP / NMN groups were up to 59%, 43%, 36%, and 32%, respectively. All wounds treated with hydrogel had greater contraction than those treated with Tegaderm dressing ( Figure 13 control group, P<0.05). On the 14th day, QP / NMN / Mg 2+ The wound closure rate of the group was 98.2%, indicating that the diabetic wounds were almost completely healed. In contrast, the control group and the QP group still had significant wound areas, while the QP / / Mg 2+ and the QP / NMN group healed faster than these two groups (e.g. Figure 13 NMN and Mg 2+ The inherent pro-regenerative activity of QCS and PEGSD synergistically accelerated the healing time of diabetic wounds. In addition, the moist environment provided by the hydrogel and the QP / NMN / Mg 2+ The beneficial activities of these substances promote wound healing.

[0136] Given that hydrogels enhance wound healing, the number of newly formed blood vessels is a key factor in the effectiveness of the healing process. Immunofluorescence was used to measure the expression of CD31, a marker of vascular structure, to estimate the number of blood vessels in the newly formed skin tissue on day 7. The maximum CD31 expression was observed in the QP / NMN / Mg 2+ In hydrogels (such as Figure 14 The number of blood vessels in the hydrogel-treated wounds was significantly higher than that in the control group ( Figure 14, P < 0.05), indicating that the hydrogel has a stronger angiogenesis-promoting potential, proving that QP / NMN / Mg 2+ The results showed that QP / NMN / Mg had the greatest angiogenic effect. 2+ Directly promote the formation of more blood vessels and prevent excessive oxidative stress and inflammation of vascular endothelial cells through the combined action of hydrogels and release of NMN and Mg 2+ Therefore, QP / NMN / Mg 2+ Hydrogels can accelerate the healing of diabetic wounds by removing ROS, reducing inflammation, and promoting angiogenesis.

[0137] Unlike most hydrogel dressings currently available that only target one stage of diabetic wound healing, the QP / NMN / Mg 2+ It has been shown to exhibit pro-healing effects in the hemostasis, inflammation, and proliferation stages of wound repair. Thanks to NMN, the hydrogel can significantly reduce ROS to achieve anti-inflammatory effects. In addition, QCS and Mg 2+ The inherent antibacterial properties of QP / NMN / Mg significantly enhance the antibacterial efficacy of the hydrogel in a synergistic manner. This composite hydrogel can significantly promote the survival of fibroblasts and greatly accelerate the healing of diabetic wounds by scavenging ROS, enhancing angiogenesis, and reducing inflammation. The above research results show that QP / NMN / Mg 2+ The hydrogel is easy to prepare and apply, and shows great promise in future clinical applications as diabetic wound dressings.

[0138] In summary, the present invention provides a method based on NMN and Mg 2+ The synergistic multifunctional hydrogel has good tissue adhesion, excellent biocompatibility, hemostasis, ROS scavenging, angiogenesis-promoting and antibacterial capabilities, and can effectively accelerate the healing of diabetic wounds by continuously delivering drugs to the wound area.

[0139] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The following steps are involved: Quaternized chitosan and PEGSD copolymer were sequentially added to deionized water under water bath heating to obtain a precursor solution; Mixing magnesium chloride hexahydrate, nicotinamide mononucleotide, and a precursor solution to obtain a mixed solution; Hydrogen peroxide / horseradish peroxidase oxidation system was added to the mixed solution and vortexed to perform Schiff base crosslinking to form QP / NMN / Mg 2+ Multifunctional hydrogel network to obtain multifunctional hydrogel.

2. The method according to claim 1 based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The water bath heating condition has a temperature of 50-60°C.

3. The method according to claim 1 based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The mass ratio of the quaternized chitosan to the PEGSD copolymer is (18-20): (100-120).

4. The method according to claim 1 based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The mass ratio of the magnesium chloride hexahydrate to nicotinamide mononucleotide is 1:(1-1.25).

5. The method according to claim 1 based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The concentration of nicotinamide mononucleotide in the mixed solution is 5 to 6.25 mg / mL.

6. The method according to claim 1 based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The mass ratio of the mixed solution to the hydrogen peroxide / horseradish peroxidase oxidation system is (1158-1185): (100-120).

7. The method according to claim 1 based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The mass ratio of hydrogen peroxide to horseradish peroxidase in the hydrogen peroxide / horseradish peroxidase oxidation system is 1:

1.

8. The method according to claim 1 based on NMN and Mg 2+ The preparation method of the synergistic multifunctional hydrogel is characterized in that: The concentration of hydrogen peroxide in the hydrogen peroxide / horseradish peroxidase oxidation system is 280-300 μg / mL; the concentration of horseradish peroxidase in the hydrogen peroxide / horseradish peroxidase oxidation system is 20-25 μg / mL.

9. A method based on NMN and Mg according to any one of claims 1 to 8 2+ Synergistic multifunctional hydrogel prepared by NMN and Mg 2+ Synergistic multifunctional hydrogels.

10. A method according to claim 9 based on NMN and Mg 2+ Application of synergistic multifunctional hydrogels in the preparation of wound dressings for the treatment of diabetes.

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