Dressing capable of promoting healing of chronic wound

Abstract

The present invention provides a dressing capable of promoting healing of a chronic wound. Specifically, provided is a multifunctional wound dressing, comprising: first active components: β-nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+), or a combination thereof; and a dressing matrix. Experiments of the present invention prove that the multifunctional wound dressing of the present invention has an excellent effect of promoting healing of chronic refractory wounds such as diabetic feet.

Description

A dressing capable of promoting the healing of chronic wounds Technical Field

[0001] The present invention belongs to the field of biomaterials, and in particular relates to a drug-loaded composite thermosensitive hydrogel dressing and an application thereof in promoting the healing of chronic wounds. Background Art

[0002] Chronic wounds are wounds caused by various reasons that have failed to heal or show no signs of healing after more than one month of treatment. They often occur in patients with severe chronic conditions and acute injuries, such as diabetes, trauma, varicose veins, vascular sclerosis, and paraplegia who are bedridden for extended periods. They are characterized by complex pathogenesis, difficult treatment, long treatment cycles, and high costs, severely impacting people's physical and mental health and quality of life. Therefore, researchers are urgently needed to develop safe and effective treatments.

[0003] Biological dressing therapy is a key local treatment for wound healing. Conventional dressings are only an adjunct in the treatment of diabetic foot ulcers, as they generally only provide debridement, moisturizing, and antibacterial properties. With the continuous development of dressings, localized treatments are gradually gaining public recognition. Currently, dressings have evolved from traditional dressings based on gauze and bandages to new dressings that maintain wound moisture, enhance autolytic debridement, and relieve pain (such as foam dressings, hydrocolloid dressings, alginate dressings, and hydrogel dressings), and finally to functional dressings that can load and release small molecule drugs (antibacterial and anti-inflammatory drugs), proteins (cell growth factors, collagen), cells, or exosomes to promote wound healing. However, these current functional dressings typically have a single function and limited effectiveness in promoting wound healing. Chronic wounds often have difficulty healing due to multiple pathological factors, including long-term inflammatory responses, recurrent bacterial infection, biofilm formation, persistent oxidative stress, and limited angiogenesis. These factors can coexist during the wound healing process, thereby delaying wound healing. Therefore, there is a need in the art to provide a dressing that can promote the healing of chronic, refractory wounds such as diabetic foot.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a dressing capable of promoting the healing of chronic refractory wounds such as diabetic foot and its application.

[0006] In a first aspect, the present invention provides a dressing comprising:

[0007] First active ingredient: β-nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+), or a combination thereof; and

[0008] Dressing matrix.

[0009] In another preferred embodiment, the dressing further comprises a second active ingredient: an antibacterial agent.

[0010] In another preferred embodiment, the antibacterial agent is selected from the following group: chitosan, chitosan oligosaccharides, chitosan; polymer guanidine antibacterial agents; metal ions such as silver ions, copper ions and iron ions; or metal oxidase nanozymes such as cerium oxide (CeO2), iron oxide (Fe3O4), manganese oxide (MnO2); metal oxides such as zinc oxide (ZnO), copper oxide (CuO), titanium oxide (TiO2), vanadium oxide (V2O5); antimicrobial peptides; or a combination thereof.

[0011] The second active ingredient is selected from the group consisting of a polymeric guanidine antibacterial agent or a pharmaceutically acceptable salt thereof: polyhexamethylene biguanide (PHMB), polyhexamethylene biguanide, polyaminopropyl biguanide, or a combination thereof. Preferably, the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, stearate, propionate, and phosphate. Preferably, it is polyhexamethylene biguanide or a pharmaceutically acceptable salt thereof.

[0012] In another preferred embodiment, the dressing further comprises a third active ingredient, and the third active ingredient is selected from the following group, or a pharmaceutically acceptable salt thereof: nicotinamide riboside (NR), nicotinamide nucleotide, nicotinamide adenine dinucleotide phosphate (NADP+), ergothioneine, pyrroloquinoline quinone (PQQ), coenzyme Q10 (CoQ10), coenzyme B12, vitamin substances such as vitamin C (Vitamin C), vitamin E (Vitamin E), vitamin A (Vitamin A), vitamin D (Vitamin D), vitamin K (Vitamin K), vitamin B2 (Vitamin B2), folic acid (Vitamin B9), glutathione, α-lipoic acid, resveratrol, carotenoids, proanthocyanidins, catechins, quercetin isoflavones, or a combination thereof.

[0013] In another preferred embodiment, the form of the dressing is selected from the following group: hydrogel dressing, hydrocolloid dressing, fiber dressing, foam dressing, liquid, paste dressing, gauze, cotton swab, cotton ball, cotton pad, adhesive tape, sticker, or a combination thereof.

[0014] In another preferred embodiment, the present invention provides a thermosensitive hydrogel dressing comprising:

[0015] First active ingredient: β-nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+), or a combination thereof; and

[0016] Thermosensitive hydrogel matrix,

[0017] Wherein, the active ingredient is dispersed in the hydrogel matrix.

[0018] In another preferred embodiment, the gelling agent used to form the hydrogel is selected from the group consisting of Pluronic F127, Pluronic F68, chitosan (CS), gelatin, hyaluronic acid, alginate (such as sodium salt), polyethylene glycol, or a combination thereof.

[0019] In another preferred embodiment, the hydrogel dressing is a hydrogel formed by including alginate as a gelling agent, and preferably, the content of alginate is 1-10 wt%, preferably 1-5 wt% or 2-3 wt%.

[0020] In another preferred embodiment, the hydrogel dressing is a thermosensitive hydrogel formed by using Pluronic F127 as a gelling agent, and preferably, the content of Pluronic F127 is 15-25 wt %, preferably 15-20 wt %.

[0021] In another preferred embodiment, the matrix of the hydrocolloid dressing may include one or more of the following components: gelatin, pectin, carboxymethyl cellulose, sodium alginate (SA) and polyisobutylene, or a combination thereof.

[0022] In another preferred embodiment, the fiber dressing is selected from the following group: sheet or strip dressings made of alginate fiber, ethylsulfonate fiber, carboxymethyl cellulose fiber, polyester fiber, etc., or a combination thereof.

[0023] In another preferred embodiment, the foam dressing is selected from the group consisting of silicone foam dressing, polyvinyl alcohol foam dressing, thin foam dressing, polyurethane foam (adhesive) dressing, self-adhesive foam dressing, or a combination thereof.

[0024] In another preferred embodiment, the liquid or creamy dressing is selected from the following group: sterile liquid dressing, sterile spray dressing, sterile wound care ointment, sterile liquid wound dressing, or a combination thereof.

[0025] In another preferred embodiment, the content of the first active ingredient is 0.001-10wt%, preferably 0.01-7wt%, and more preferably 0.02-5wt%, such as 0.03wt%, 0.05wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.8wt%, 1wt%, 2wt% or 3wt%, based on the total weight of the dressing.

[0026] In another preferred embodiment, the content of the second active ingredient is 0.001-10wt%, preferably 0.01-5wt%, and more preferably 0.02-3wt%, such as 0.03wt%, 0.05wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.8wt%, 1wt%, 2wt% or 3wt%, based on the total weight of the dressing.

[0027] In another preferred embodiment, the content of the third active ingredient is 0.001-10wt%, preferably 0.01-7wt%, and more preferably 0.02-5wt%, such as 0.03wt%, 0.05wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.8wt%, 1wt%, 2wt% or 3wt%, based on the total weight of the dressing.

[0028] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10); preferably 1:(0.2-5); more preferably 1:(0.2-2), such as 1:0.3, 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5 or 1:4.

[0029] In another preferred embodiment, the weight ratio of the first active ingredient to the third active ingredient is 1:(0.1-10); preferably 1:(0.2-5); more preferably 1:(0.2-2), such as 1:0.3, 1:0.5, 1:0.8, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5 or 1:4.

[0030] In another preferred embodiment, the first active ingredient is β-nicotinamide mononucleotide (NMN) or a pharmaceutically acceptable salt thereof, and the second active ingredient is chitosan; preferably, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10); preferably 1:(0.2-8); more preferably 1:(0.5-5); preferably, the content of the first active ingredient is 0.01-2wt%, preferably 0.02-1.5wt%, more preferably 0.1-1wt%, based on the total weight of the dressing; preferably, the content of the second active ingredient is 0.05-2wt%, preferably 0.1-1.5wt%, more preferably 0.8-1.2wt%, based on the total weight of the dressing.

[0031] In another preferred embodiment, the first active ingredient is β-nicotinamide mononucleotide (NMN) or a pharmaceutically acceptable salt thereof, and the second active ingredient is polyhexamethylene biguanide or a pharmaceutically acceptable salt thereof; preferably, the weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10); preferably 1:(0.2-3); more preferably 1:(0.5-2); preferably, the content of the first active ingredient is 0.01-2wt%, preferably 0.02-1.5wt%, more preferably 0.1-1wt%, based on the total weight of the dressing; preferably, the content of the second active ingredient is 0.05-0.5wt%, preferably 0.08-0.3wt%, more preferably 0.1-0.2wt%, based on the total weight of the dressing.

[0032] In another preferred example, the first active ingredient is β-nicotinamide mononucleotide (NMN) or a pharmaceutically acceptable salt thereof, and the second active ingredient is CeO2; preferably, the weight ratio of the first active ingredient to the second active ingredient is 1:(1-10); preferably 1:(2-8); more preferably 1:(4-6); preferably, the content of the first active ingredient is 0.01-2wt%, preferably 0.02-1.5wt%, more preferably 0.1-1wt%, based on the total weight of the dressing; preferably, the content of the second active ingredient is 0.5-2wt%, preferably 0.8-1.5wt%, more preferably 1-1.2wt%, based on the total weight of the dressing.

[0033] In another preferred embodiment, the gelling agent used to form the hydrogel includes Pluronic F127 and Pluronic F68.

[0034] In another preferred embodiment, the gelling agent used to form the hydrogel is a combination of Pluronic F127, Pluronic F68 and chitosan (CS).

[0035] In another preferred embodiment, in the gelling agent used to form the hydrogel, the sum of the weights of Pluronic F127 and Pluronic F68 accounts for more than 90 w / w% of the total weight of the gelling agent, preferably more than 95 w / w%, such as more than 96 w / w%, more than 97 w / w%, more than 98 w / w%, or more than 99 w / w%.

[0036] In another preferred embodiment, in the dressing, the content of Pluronic F127 is 16-24 wt / vol% (wt / vol is g / mL), preferably 18-22 wt / vol%, and more preferably 19-21 wt / vol%.

[0037] In another preferred embodiment, in the dressing, the content of Pluronic F68 is 4-8 wt / vol%, preferably 5-7 wt / vol%, and more preferably 4-6 wt / vol%.

[0038] In another preferred embodiment, in the dressing, the content of chitosan (CS) is 0.05-1 wt / vol%, preferably 0.1-0.5 wt / vol%, and more preferably 0.2-0.3 wt / vol%.

[0039] In another preferred example, in the dressing, the contents of F127, F68 and CS are 20 wt / vol%, 6 wt / vol% and 0.2 wt / vol%, respectively.

[0040] In another preferred embodiment, in the gelling agent used to form the hydrogel, the sum of the masses of Pluronic F127 and Pluronic F68 accounts for more than 90 w / w% of the total weight of the gelling agent, preferably more than 95 w / w%, such as more than 96 w / w%, more than 97 w / w%, more than 98 w / w%, more than 99 w / w% or even 100 w / w%.

[0041] In another preferred embodiment, in the dressing, the contents of gelatin, hyaluronic acid and sodium alginate are 15% wt / vol%, 0.5 wt / vol% and 1 wt / vol%, respectively.

[0042] In another preferred embodiment, in the dressing, the content of the active ingredient is independently 0.01 μg / mL to 20 mg / mL, preferably 0.1 μg / mL to 10 mg / mL, preferably 200 μg / mL to 4 mg / mL, such as 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, or 180 μg / mL, 300 μg / mL, 500 μg / mL, 800 μg / mL, 2 mg / mL or 5 mg / mL.

[0043] In another preferred embodiment, the dressing is a hydrogel dressing or its dried form comprising the following components, calculated based on the total weight of the dressing:

[0044] a first active ingredient and a second active ingredient;

[0045] Alginate 1-10wt%, preferably 1-5wt% or 2-3wt%;

[0046] Cross-linking agent 0.1-2 wt%, preferably 0.5-1 wt%; and

[0047] Water, physiological saline or a physiological buffer solution with a pH of 6.5-7.5, preferably a physiological buffer solution with a pH of 7-7.4.

[0048] In another preferred embodiment, the dressing is a thermosensitive hydrogel dressing or its dried form comprising the following components, calculated based on the total weight of the dressing:

[0049] a first active ingredient and a second active ingredient;

[0050] Pluronic F127 15-25 wt%, preferably 15-20 wt%; and

[0051] Water, physiological saline or a physiological buffer solution with a pH of 6.5-7.5, preferably a physiological buffer solution with a pH of 7-7.4.

[0052] In another preferred embodiment, the dressing further comprises Pluronic F68.

[0053] In another preferred embodiment, the dressing may further include one or more substances selected from the following group: sodium chloride, buffer salts, pH regulators, pigments, moisturizers, preservatives, or a combination thereof.

[0054] In another preferred embodiment, the hydrogel matrix further comprises water or an aqueous solution. Preferably, the aqueous solution is physiological saline or a physiological buffer solution (such as a PBS solution with a pH of 7.35-7.4, a tris-hydrochloric acid solution or a phosphate buffer solution).

[0055] In another preferred embodiment, the dressing may further include other therapeutic agents, such as antibacterial agents and anti-inflammatory agents.

[0056] In another preferred embodiment, the dressing is liquid at a temperature of ≤25°C, preferably 4-25°C, such as 5°C, 10°C, 15°C, or 20°C.

[0057] In another preferred embodiment, the dressing is in a gel-solid state at ≥30°C, preferably 32-38°C, such as 33°C, 34°C, 35°C, 36°C, 37°C or 37.5°C.

[0058] In a second aspect, the present invention provides a method for preparing the dressing according to the first aspect of the present invention, comprising the steps of:

[0059] Mix the active ingredient with the dressing matrix components to prepare the corresponding dressing form (hydrogel dressing, hydrocolloid dressing, foam dressing, liquid, paste dressing); or

[0060] Dispersing the active ingredient in a solvent and then coating the resulting mixture on a dressing matrix or impregnating the dressing matrix (such as a film, fiber, gauze, etc.); or

[0061] The active ingredients are mixed with the dressing matrix components to prepare the corresponding dressing form (such as hydrogel, hydrocolloid, foam, liquid, paste), and the obtained dressing is further loaded on a solid phase carrier such as film, fiber, gauze, etc.

[0062] In another preferred embodiment, a method for preparing the dressing according to the first aspect of the present invention comprises the steps of:

[0063] 1) providing water or an aqueous solution; and

[0064] 2) dissolving the gelling agent and active ingredient in the water or aqueous solution at liquid temperature to form a liquid dressing.

[0065] In another preferred embodiment, the method further comprises the step: 3) forming a solid gel dressing at a solid temperature.

[0066] In another preferred embodiment, step 2 further includes a cross-linking agent, such as calcium chloride.

[0067] In another preferred embodiment, the liquid temperature and the solid temperature are different temperatures.

[0068] In another preferred embodiment, the liquid temperature is ≤25°C, preferably, 4-25°C, such as 5°C, 10°C, 15°C, or 20°C.

[0069] In another preferred embodiment, the solid state temperature is ≥30°C, preferably 32-38°C, such as 33°C, 34°C, 35°C, 36°C, 37°C or 37.5°C.

[0070] In another preferred embodiment, the aqueous solution is selected from the following group: physiological saline or physiological buffer solution (such as PBS solution with a pH of 7.35-7.4, tris-hydrochloric acid solution or phosphate buffer solution).

[0071] The third aspect of the present invention provides a use of the dressing according to the first aspect of the present invention in the preparation of a medicine or medical device for promoting wound healing.

[0072] In another preferred embodiment, the drug or medical device has one or more uses selected from the following groups:

[0073] Promote cell proliferation and / or migration;

[0074] Promote angiogenesis;

[0075] Antioxidant effect;

[0076] Anti-inflammatory;

[0077] Antimicrobial or biofilm-inhibiting; and / or

[0078] Repairs skin and / or mucous membrane damage.

[0079] Preferably, the above functions promote wound healing.

[0080] In another preferred embodiment, the wound is a chronic (difficult-to-heal) wound, such as diabetic foot ulcer, vascular ulcer, pressure sore, fistula, sinus tract and other skin injuries or mucosal (such as oral, digestive tract and uterine mucosa) injuries.

[0081] In another preferred embodiment, the medical device further comprises a solid phase carrier for loading or fixing the hydrogel, such as medical gauze, cotton swabs, cotton balls, cotton pads, adhesive tape, plastic stickers, etc.

[0082] A fourth aspect of the present invention provides a method for promoting wound healing, comprising the steps of:

[0083] The auxiliary material according to the first aspect of the present invention is brought into contact with a wound, thereby promoting the healing of the wound.

[0084] In another preferred embodiment, the method comprises the steps of: injecting or applying the thermosensitive hydrogel dressing described in the first aspect of the present invention on the wound in liquid state, thereby promoting the healing of the wound.

[0085] In another preferred embodiment, the wound is a chronic (difficult-to-heal) wound, such as diabetic foot ulcer, vascular ulcer, pressure sore, fistula, sinus tract and other skin injuries or mucosal injuries.

[0086] In another preferred embodiment, the wound is a wound of a human or other mammal (such as a rat or mouse).

[0087] In another preferred embodiment, the amount of the dressing of the present invention is 2 The wound is about 0.1-0.6 grams of dressing, preferably 0.2-0.3 grams.

[0088] In another preferred embodiment, the dressing is changed once every 1-5 days, preferably once every two days, and more preferably once every other day.

[0089] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, each drawing described below is for some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0091] Figure 1: Optimized ratio of components of composite hydrogel;

[0092] Figure 2: Microscopic morphology of the composite hydrogel under a scanning electron microscope (magnification from left to right: 150x, 250x, and 2500x);

[0093] Figure 3: Thermosensitivity of composite hydrogels;

[0094] Figure 4: Sustained release trend of NMN by thermosensitive hydrogel;

[0095] Figure 5: Colony count reflects the antibacterial effect of F127 / F68 / CS thermosensitive hydrogel at different CS concentrations;

[0096] Figure 6: Effect of composite hydrogel on cell survival (HaCaT cells on the left, EA.hy 926 cells on the right);

[0097] Figure 7: Hemolysis of thermosensitive hydrogel;

[0098] Figure 8: Stimulation response of NMN-loaded thermosensitive hydrogel under rat skin;

[0099] Figure 9: Effects of NMN composite hydrogels loaded with different concentrations on cell activity;

[0100] Figure 10: Effects of NMN composite hydrogels loaded with different concentrations on the migration ability of HaCaT cells;

[0101] Figure 11: Effects of NMN hydrogels loaded with different concentrations on angiogenesis;

[0102] Figure 12: Effects of NMN hydrogels loaded with different concentrations on cell oxidation;

[0103] Figure 13: Healing of the skin of diabetic mice in each group at 1, 4, 7, 10, 14, 18, and 21 days after full-thickness perforation application;

[0104] Figure 14: HE staining analysis of wound tissues in each group of diabetic mice on day 10 after perforation dressing;

[0105] Figure 15: Expression of VEGF / TGF-β1 in wound tissues of diabetic mice in each group on day 10 after perforation dressing;

[0106] Figure 16: Screening of antibacterial activity and cytotoxic concentration of antibacterial substances (A is the minimum inhibitory concentration (MIC) of chitosan (CS), B is the minimum bactericidal concentration (MBC) of chitosan (CS), C is the MIC of polyhexamethylene biguanide hydrochloride (PHMB), D is the MBC of polyhexamethylene biguanide hydrochloride (PHMB), and E is the cytotoxicity of PHMB. The left side is EA.hy 926 cells, and the right side is HaCaT cells);

[0107] Figure 17: Optimized ratio of PHMB and thermosensitive hydrogel components;

[0108] Figure 18: Effects of polymeric guanidine antimicrobial agents on the biological functions of active ingredients (A and B show the effects of PHMB on the migration-promoting activity of NMN and their statistical graphs, C shows the effects of PHMB on the antioxidant activity of NMN, D and E show the effects of PHMB on the angiogenic activity of NMN, and F shows the effects of PHMB on the anti-inflammatory activity of NMN);

[0109] Figure 19: Effect of the first active ingredient on the bactericidal effect of polymeric guanidine antimicrobial agents (A shows the effect of different concentrations of NMN on the bactericidal effect of PHMB, and B shows the statistical graph of Figure A);

[0110] Figure 20: Microscopic morphology of the dressing under a scanning electron microscope (A: alginate hydrogel, the left picture is alginate hydrogel alone, the right picture is alginate hydrogel loaded with active ingredients and antibacterial substances, B: alginate hydrogel loaded on gauze, the left picture is ordinary gauze, the right picture is alginate hydrogel loaded with active ingredients and antibacterial substances loaded on gauze);

[0111] Figure 21: A graph showing the sustained release of NMN from alginate hydrogel;

[0112] Figure 22: Swelling ratio of composite hydrogels (A is the swelling ratio of alginate hydrogel alone and drug-loaded alginate hydrogel, B is the swelling ratio of alginate hydrogel alone and gauze-added hydrogel);

[0113] FIG23 : Inhibitory effect of the dressing matrix with antibacterial properties on biofilms formed by Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (A is a graph showing inhibition of biofilm formation, and B is a statistical graph of A);

[0114] FIG24 : Hemolysis of composite alginate hydrogel;

[0115] Figure 25: In vitro biological activity assay of thermosensitive hydrogels loaded with different concentrations of NAD+ (A and B show the effect on cell migration, and B is its statistical graph; C and D show the angiogenic activity, and D is its statistical graph; E and F show the antioxidant activity);

[0116] Figure 26.1: Effects of different concentrations of NMN as the active ingredient in PHMB / F127 gel on wound healing;

[0117] Figure 26.2: Effect of 0.2% NMN@0.1% PHMB / F127 on wound healing;

[0118] Figure 26.3: Effect of 0.2% NMN@0.1% PHMB / SA hydrogel on wound healing;

[0119] Figure 26.4: Effect of 1% CeO2@0.2% NMN / SA hydrogel on wound healing;

[0120] Figure 26.5: Effect of 0.2% NMN@0.1% PHMB / hydrocolloid on wound healing;

[0121] Figure 26.6: Effect of 0.1% PHMB@0.2% NMN / carboxymethyl cellulose fiber dressing on wound healing;

[0122] Figure 26.7: Effects of different concentrations of NAD+ on wound healing in mixed gels of CS and F127, F68;

[0123] Figure 27.1: Effect of 0.1% PHMB@0.2% NMN / F127 hydrogel on wound healing in patients with diabetic foot;

[0124] Figure 27.2: Effect of 0.1% PHMB@0.2% NMN / F127 hydrogel on sinus wound healing. DETAILED DESCRIPTION

[0125] After extensive and in-depth research, screening, and testing, the present inventors have developed a dressing loaded with NMN or NAD+ and its application. The present inventors unexpectedly discovered that the dressing exhibits excellent technical effects in multiple aspects, making it well-suited for promoting the healing of chronic wounds such as diabetic foot. This invention was completed on this basis.

[0126] the term

[0127] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0128] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0129] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0130] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 10-30°C, preferably, 25±5°C.

[0131] Active ingredient

[0132] The first active ingredient of the present invention is: β-nicotinamide mononucleotide, or a pharmaceutically acceptable salt thereof; and / or nicotinamide adenine dinucleotide, or a pharmaceutically acceptable salt thereof.

[0133] β-Nicotinamide mononucleotide (NMN), chemical formula:

[0134] C 11 H 15 N2O8P; NMN is the synthetic substrate of coenzyme I, and NMN is also used in anti-aging research.

[0135] The nicotinamide adenine dinucleotide (beta-Nicotinamide adenine dinucleotide trihydrate, NAD+), also known as coenzyme I, chemical formula C 21 H 27 N7O 14 P2; NAD+ participates in various physiological activities such as cell metabolism, energy synthesis, and cell DNA repair, and plays an important role in the body's immune ability.

[0136] The present invention is the first to load the above active ingredients into a hydrogel to form a medical dressing. Unexpectedly, the dressing can promote the healing of chronic wounds.

[0137] Furthermore, the present invention also includes a second active ingredient, which is primarily used as an antimicrobial agent. Preferably, the antimicrobial agents that can be used in the present invention include (but are not limited to): chitosan, chitosan oligosaccharides, chitin; polymeric guanidine antimicrobial agents; metal ions such as silver ions, copper ions, and iron ions; or metal oxidase nanozymes such as cerium oxide (CeO2), iron oxide (Fe3O4), and manganese oxide (MnO2); metal oxides such as zinc oxide (ZnO), copper oxide (CuO), titanium oxide (TiO2), and vanadium oxide (V2O5); antimicrobial peptides; or combinations thereof.

[0138] The polymer guanidine antimicrobial agents that can be used in the present invention include polyhexamethylene biguanide (PHMB), polyhexamethylene biguanide, polyaminopropyl biguanide, or a combination thereof.

[0139] Surprisingly, the present inventors discovered that the first active ingredient and the second active ingredient have a significant synergistic effect in promoting the healing of chronic wounds, and can significantly shorten the healing time after chronic wounds.

[0140] In addition, the present invention may further include a third active ingredient, which is selected from the following group, or a pharmaceutically acceptable salt thereof: nicotinamide riboside (NR), nicotinamide nucleotide, nicotinamide adenine dinucleotide phosphate (NADP+), ergothioneine, pyrroloquinoline quinone (PQQ), coenzyme Q10 (CoQ10), coenzyme B12, vitamin substances such as vitamin C (Vitamin C), vitamin E (Vitamin E), vitamin A (Vitamin A), vitamin D (Vitamin D), vitamin K (Vitamin K), vitamin B2 (Vitamin B2), folic acid (Vitamin B9), glutathione, α-lipoic acid, resveratrol, carotenoids, proanthocyanidins, catechins, quercetin isoflavones, or a combination thereof.

[0141] The active ingredients of the present invention are intended to include pharmaceutically acceptable salts, crystal forms and the like.

[0142] As used herein, the term "pharmaceutically acceptable salt" refers to a salt suitable for pharmaceutical use formed by any active ingredient of the present invention, optionally independently and optionally with an acid or base. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is the salt formed by the active ingredient of the present invention and an acid. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0143] Another preferred salt refers to a salt of any active ingredient of the present invention optionally formed independently with a base, such as an alkali metal salt (such as a sodium salt or a potassium salt), an alkaline earth metal salt (such as a magnesium salt or a calcium salt), an ammonium salt such as a methylamine salt, an ethylamine salt, a propylamine salt, a dimethylamine salt, a trimethylamine salt, a diethylamine salt, a triethylamine salt, a tert-butylamine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trihydroxyethylamine salt, and an amine salt formed from morpholine, piperazine, or lysine, respectively.

[0144] dressing

[0145] The present invention provides a dressing comprising the active ingredient of the present invention and a dressing matrix as a drug carrier.

[0146] The present invention has no special requirements for the dressing matrix, and various materials commonly used in the art for preparing medical dressings can be used.

[0147] Common forms of dressings include (but are not limited to): hydrogel dressings, hydrocolloid dressings, fiber dressings, foam dressings, liquid dressings, paste dressings, film dressings, gauze, Band-Aids, etc.

[0148] The above-mentioned active materials can be dispersed in the dressing matrix, such as dispersed in a hydrogel, hydrocolloid, foam, liquid, or paste dressing matrix; or coated on the dressing matrix or impregnated on a solid phase carrier such as a film, fiber or gauze; or the above-mentioned active ingredients are first dispersed in the dressing matrix and then coated or impregnated on the solid phase carrier.

[0149] Gelling agents that can be used to form hydrogels may include, but are not limited to, Pluronic F127, Pluronic F68, chitosan (CS), gelatin, hyaluronic acid, alginate, polyethylene glycol, or combinations thereof.

[0150] Hydrocolloid dressings are made by mixing water-soluble polymer particles, such as carboxymethyl cellulose, pectin, gelatin, and sodium alginate, with rubber adhesives and / or hydrophobic polymers such as polyisobutylene. Matrices that can be used to form hydrocolloids include (but are not limited to): gelatin, pectin, carboxymethyl cellulose, sodium alginate (SA), polyisobutylene, or combinations thereof.

[0151] A fiber dressing is a sheet or strip dressing made of hydrophilic fibers, such as alginate fibers, ethylsulfonate fibers, carboxymethyl cellulose fibers, polyamide fibers, polyester fibers, etc. The matrix that can be used to form the fiber dressing may include (but is not limited to): a sheet or strip dressing made of alginate fibers, ethylsulfonate fibers, carboxymethyl cellulose fibers, polyester fibers, etc., or a combination thereof.

[0152] Foam dressings include silicone foam dressings, polyvinyl alcohol foam dressings, thin foam dressings, polyurethane (adhesive) foam dressings, self-adhesive foam dressings, or combinations thereof. Substrates that can be used to form foam dressings include (but are not limited to): polyurethane particles or liquids, silicone resins, polyvinyl alcohol (PVA), isocyanate compounds, tin compounds or titanium compounds, water, inorganic foaming agents such as carbon dioxide, silicone oil, polyvinyl alcohol, or combinations thereof.

[0153] The base material that can be used to form the liquid, cream-like dressing may include, but is not limited to, a sterile liquid dressing, a sterile spray dressing, a sterile wound care ointment, a sterile liquid wound dressing, or a combination thereof.

[0154] The dressing may further include one or more substances selected from the group consisting of sodium chloride, buffer salts, pH regulators, pigments, moisturizers, preservatives, or combinations thereof.

[0155] In particular, a thermosensitive hydrogel dressing is provided, characterized in that it comprises:

[0156] Active ingredient: β-nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+), or a combination thereof; and a hydrogel matrix, wherein the active ingredient is dispersed in the hydrogel matrix.

[0157] Preferably, the hydrogel matrix is ​​the above-mentioned thermosensitive hydrogel.

[0158] Preferably, the dressing of the present invention is in liquid state (stored or handled at liquid temperature (e.g., 4-25°C)) before contacting the wound. After contacting the wound, it transforms into a gel solid state at room temperature or body temperature (e.g., 34-40°C) and slowly releases the active ingredient.

[0159] In another preferred embodiment, the dressing is an alginate-based hydrogel, comprising alginate (such as sodium salt) as a gelling agent, a calcium salt such as CaCl2 as a cross-linking agent, and water or an aqueous solvent.

[0160] Based on the mass percentage of the alginate-based hydrogel, the content of sodium alginate is 2-3wt%, and the content of the cross-linking agent calcium salt, such as CaCl2, is 0.5-1wt%. Preferably, the pH value of the aqueous solvent is 5-7.5, preferably 6.5-7. Preferably, the gelling temperature is preferably 4°C-30°C, preferably 15°C-25°C. In another preferred embodiment, the dressing is a thermosensitive hydrogel and includes Pluronic F127 as a gelling agent. Preferably, based on the mass percentage of the thermosensitive hydrogel, the content of Pluronic F127 is 15-25wt%, preferably 15-20wt%.

[0161] Preferably, the hydrogel dressing can be in a dried (eg freeze-dried) form for easy storage and transportation, and can be rehydrated before use.

[0162] Thermosensitive hydrogel

[0163] Preferably, the hydrogel used in the present invention is a temperature-sensitive gel (referred to as thermosensitive hydrogel or thermosensitive gel), which is a temperature-responsive in situ gel whose state is closely related to temperature. When used at room temperature, it is in a flowable sol state, which is convenient for medical personnel to administer the drug. After injection or application to the wound, it can achieve an in situ transformation of gel-sol through physical cross-linking under the action of body temperature, covering the wound with a non-flowable gel state and having good adhesion. After administration, the entrapped drug can be slowly released to maintain the efficacy. Its in situ thermosensitive gelation method can more fully fill irregular wounds and achieve a perfect match between the gel and the shape of the wound defect. In another preferred embodiment, the gelling agent used to form the hydrogel is selected from the following group: Pluronic F127, Pluronic F68, chitosan (CS), gelatin, hyaluronic acid, sodium alginate, polyethylene glycol, or a combination.

[0164] In another preferred embodiment, the gelling agent used to form the thermosensitive hydrogel includes Pluronic F127.

[0165] In particular, the hydrogel of the present invention is a thermosensitive hydrogel formed based on a combination of Pluronic F127, Pluronic F68 and chitosan (CS).

[0166] As used herein, Pluronic F127 or F127 is a polyoxyethylene polyoxypropylene ether block copolymer with the chemical formula: HO(C2H4O) m (C3H6O) n H (average Mn: 12600), mainly used as a thickener and colloid former, sustained-release material, adhesive, coating material, etc. to prepare tablets, capsules, gels, etc. The Pluronic F127 is commercially available.

[0167] As used herein, Pluronic F68 or F68 is a polyoxyethylene polyoxypropylene ether block copolymer with the chemical formula: HO(C2H4O) m (C3H6O) n Pluronic F68 (average Mn: 8400) is a synthetic oil-in-water emulsifier with stable physical properties and the ability to withstand hot pressing and low-temperature freezing. The Pluronic F68 is commercially available.

[0168] As used herein, chitosan (CS) is the product of N-deacetylation of chitin, with the chemical formula: (C6H 11 NO4)n, has a variety of physiological functions such as broad-spectrum antibacterial, biodegradability, biocompatibility, non-toxicity, anti-cancer, lipid-lowering, and immune-enhancing. Chitosan can be used as a gelling agent to form a gel, but the present invention mainly utilizes its antibacterial properties, which basically does not affect the temperature sensitivity and other properties of the hydrogel.

[0169] As used herein, gelatin is a protein extracted from animal connective tissue, the main component of which is collagen, with the chemical formula: C6H9O4(X)n. It can be used to make food, medicine, cosmetics, collagen patches, etc.

[0170] As used herein, hyaluronic acid is a polysaccharide naturally present in the human body, also known as hyaluronic acid, with a chemical formula of: (C 14 H 21 NO 11 )n, widely used in the field of medical cosmetology, as a filler to fill wrinkles and depressions in facial skin, with good moisturizing and lubricating effects.

[0171] As used herein, alginate or alginates are polysaccharides extracted from the cell walls of seaweeds such as brown algae, with the chemical formula: (MC6H7O6) n, It is commonly used as a functional additive for thickening, emulsification, stabilization, etc., and is used in the production of medical dressings and artificial blood vessels. Alginate is preferably sodium or potassium salt.

[0172] In particular, the hybrid hydrogel based on F127, F68, and CS provided by the present invention exhibits thermosensitivity and sustained-release properties. It is liquid at low temperatures and solid at body temperature. It is easy to use, rapidly solidifying upon contact with a wound and slowly releasing the drug it contains. Furthermore, the hybrid hydrogel composed of F127, F68, and CS is biocompatible, has no adverse effects on cell activity, is non-irritating to the skin, and does not cause hemolysis, making it ideal for use as a medical dressing.

[0173] Preparation method

[0174] The present invention also provides a method for preparing the dressing according to the present invention, comprising the steps of:

[0175] Mix the active ingredient with the dressing matrix components to prepare the corresponding dressing form (hydrogel dressing, hydrocolloid dressing, foam dressing, liquid, paste dressing); or

[0176] Dispersing the active ingredient in a solvent and then coating the resulting mixture on a dressing matrix or impregnating the dressing matrix (such as a film, fiber, gauze, etc.); or

[0177] The active ingredient is mixed with the dressing matrix components to prepare a corresponding dressing form (such as hydrogel, hydrocolloid, foam, liquid, paste), and the obtained dressing is further loaded on a solid phase carrier such as a film, fiber, gauze, etc., to form the dressing.

[0178] The present invention has no special requirements for the preparation method of various forms of dressings, and they can be prepared using methods commonly used in the art, or by referring to the methods in the embodiments of the present invention.

[0179] In particular, the present invention provides a method for preparing the thermosensitive hydrogel, comprising the following steps:

[0180] 1) providing water or an aqueous solution; and

[0181] 2) dissolving the gelling agent and active ingredient in the water or aqueous solution at liquid temperature to form a liquid dressing.

[0182] In another preferred embodiment, the method further comprises the step: 3) forming a solid gel dressing at a solid temperature.

[0183] In another preferred embodiment, the liquid temperature and the solid temperature are different temperatures.

[0184] In another preferred embodiment, the liquid temperature is ≤25°C, preferably, 4-25°C, such as 5°C, 10°C, 15°C, or 20°C.

[0185] In another preferred embodiment, the solid state temperature is ≥30°C, preferably 32-38°C, such as 33°C, 34°C, 35°C, 36°C, 37°C or 37.5°C.

[0186] In another preferred embodiment, the aqueous solution is selected from the following group: physiological saline or physiological buffer solution (such as PBS solution with a pH of 7.35-7.4, tris-hydrochloric acid solution or phosphate buffer solution).

[0187] use

[0188] The present invention provides use of the dressing in preparing medicines or medical devices for promoting wound healing.

[0189] In the present invention, wound refers to the place where the injury is broken, mostly referring to the skin, muscles, mucous membranes, etc. of humans or other animals. Wounds can usually be divided into acute wounds and chronic wounds according to the healing time. Acute wounds mainly refer to sudden injuries (such as wounds within 7 days after injury), such as abrasions, grinds, scratches, punctures, etc.; chronic wounds (i.e., refractory wounds) refer to various reasons that cause the acute wound to be unable to heal according to the normal physiological mechanism of the body, resulting in prolonged wound healing. Generally, from a medical point of view, wounds that cannot heal for more than one month are called chronic wounds or refractory wounds. The causes of chronic wounds include (but are not limited to): injuries caused by pressure (such as bedsores), ulcers caused by varicose veins or insufficient arterial blood supply, diabetic ulcers, etc.; or atypical wounds caused by various diseases such as skin and soft tissue infections, vascular inflammation, connective tissue diseases, tumors, etc.; wounds with poor prognosis due to long-term radiation exposure leading to necrosis and infection of wound tissue, etc.

[0190] Preferably, the wound can be (but is not limited to): chronic non-healing wounds caused by traumatic ulcers, chronic non-healing wounds caused by pressure ulcers (such as pressure sores), and chronic non-healing wounds caused by arterial ulcers (such as diabetic ulcers), vascular (such as venous) ulcers, pressure sores, fistulas and sinus tracts and other skin injuries or mucosal injuries.

[0191] In another preferred embodiment, the repair / healing of refractory wounds includes improving the wound healing rate and / or shortening the wound healing time to achieve an anatomically and functionally complete state. For example, compared with a dressing not using the dressing of the present invention or a dressing not containing the active ingredient of the present invention, the healing rate of the wound using the dressing of the present invention is improved and / or the healing time is shortened by 10%, 20%, 50% or even 100%; or the dressing of the present invention repairs wounds that cannot be repaired by other conventional wound repair drugs.

[0192] Experiments have demonstrated that the dressing of the present invention, or the medicine or medical device prepared therefrom, has the following uses: promoting cell proliferation and / or migration; promoting angiogenesis; providing antioxidant effects; combating inflammation; providing antibacterial and biofilm inhibition; and / or repairing skin and / or mucosal damage. These diverse activities and functions can significantly promote wound healing.

[0193] In another preferred embodiment, the medical device further comprises a solid phase carrier for loading or fixing the hydrogel, such as medical gauze, cotton swabs, cotton balls, cotton pads, adhesive tape, plastic stickers, etc.

[0194] The present invention also provides a method for promoting wound healing, comprising the steps of: contacting the dressing according to the first aspect of the present invention with a wound, thereby promoting wound healing.

[0195] The present invention also provides a method for promoting wound healing, comprising the steps of injecting or applying the thermosensitive hydrogel dressing described in the first aspect of the present invention in liquid state onto a wound, thereby promoting wound healing.

[0196] Beneficial effects of the present invention

[0197] 1. The present invention is the first to discover that NMN and / or NAD+ can promote chronic wound healing;

[0198] 2. The present invention further discovered that the combination of NMN and / or NAD+ with specific antimicrobial agents has a significant synergistic effect in promoting the healing of chronic wounds;

[0199] 3. The present invention provides a multifunctional dressing with excellent anti-inflammatory, antioxidant, angiogenesis-promoting, antibacterial and biofilm-inhibiting biological activities. It can also absorb wound exudate and slowly release active ingredients. It is easy to use and has good biocompatibility. It can significantly promote wound healing and is very suitable for clinical use.

[0200] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by mass / volume percentage.

[0201] Example 1

[0202] The preparation and screening method of the dressing comprises the following steps:

[0203] 1. Preparation of physiological saline: Weigh 0.45 g of sodium chloride powder and dissolve it in 50 mL of distilled water to obtain a 0.9% sodium chloride solution.

[0204] 2. Dissolution of Thermosensitive Hydrogel: Thermosensitive hydrogel was prepared by a low-temperature dissolution method. F127, F68, and CS were weighed on a precision electronic balance and placed in three 50-mL centrifuge tubes. An appropriate amount of physiological saline was added to ensure full solid-liquid contact. The solution was then allowed to stand at 4°C for 3–5 days until completely dissolved, yielding a clear gel solution. The solution was then diluted to the specified volume with physiological saline. The higher concentrations of F127, F68, and CS were placed in a 4°C refrigerator for later use.

[0205] 3. Based on the gelation temperature (T m) as the index (ordinate), and the mass fractions of F127 and F68 as the variable influencing factors (abscissa). Different mass fractions of F127 and F68 were mixed in a 1.5mL centrifuge tube. The tube was then placed in a 37°C water bath. The gelation temperature and time were observed and recorded. Gelation was considered complete when the liquid stopped flowing within 1 minute when the tube was inverted. Based on the results of previous single-factor experiments, the F127 values ​​were 16%, 18%, 20%, 22%, and 24%, and the F68 values ​​were 2%, 4%, 6%, and 8%. The two sets of values ​​were paired and the gelation temperature and time were measured and recorded. Each sample was tested three times, and the results were averaged.

[0206] 4. Determination of gelation temperature: The gelation temperature refers to the phase transition temperature when the thermosensitive hydrogel changes from liquid to semi-solid. The gelation temperature is determined by the centrifuge tube inversion method. Take out the prepared thermosensitive hydrogel solution with mass fraction gradient from the 4°C refrigerator, and place 1 mL of each gradient in a 1.5 mL centrifuge tube. The centrifuge tube is completely placed in a constant temperature water bath, ensuring that the hydrogel liquid level is 3 cm below the water bath liquid level. The initial temperature of the water bath is 20°C, the set temperature is 60°C, and the temperature is continuously increased. An instant thermometer is inserted under the water bath liquid level to detect the temperature in real time, while observing the flow of the hydrogel in the centrifuge tube. When the hydrogel in the centrifuge tube stops flowing and can be maintained for 1 minute, record the temperature on the thermometer at this moment and record it as the gelation temperature. Each sample is measured three times, and the results are averaged.

[0207] 5. Determination of gelation time: Gelation time refers to the time required for a thermosensitive hydrogel to transform from a liquid to a semisolid state. The gelation time is determined using the centrifuge tube inversion method. Remove the prepared thermosensitive hydrogel solution with a mass fraction gradient from a 4°C refrigerator. Place 1 mL of each gradient solution in a 1.5 mL centrifuge tube. Place the centrifuge tube completely in a constant temperature water bath, ensuring that the hydrogel liquid level is 3 cm below the water bath level. The water bath temperature is 34°C. Start timing when the centrifuge tube containing the hydrogel enters the water bath. Invert the centrifuge tube every few seconds as needed to observe the flow of the hydrogel in the centrifuge tube. When the hydrogel in the centrifuge tube stops flowing and can be maintained for 1 minute, record the time on the timer at this moment and record it as the gelation time. Repeat the measurement three times for each sample, and take the average result.

[0208] 6. Add NMN to the F127 / F68 / CS mixed hydrogel.

[0209] Example 2. Optimized ratio of components of composite hydrogel

[0210] The composite hydrogel was prepared by a low-temperature dissolution method. F127, F68, and CS were dissolved separately and then mixed according to the proportion after each was completely dissolved. As shown in Tables 1 and 2, with the increase of F127 concentration, the initial gelation temperature of the gel decreased. The higher the concentration of F68 and F127, the shorter the gelation time. Since the skin administration temperature is around 34°C, the concentration of F127 is 20% and the concentration of F68 is 4%-6%. In order to shorten the gelation time and facilitate use, the concentration of F68 is 6%. The composite hydrogel is in a gel state within the temperature range of 32°C to 37°C, and its physical state is stable. At a constant F127 / F68 concentration, adding varying amounts of chitosan (0.2, 0.4, and 0.8%) revealed that gelation time gradually decreased with increasing chitosan concentration. However, at a low chitosan concentration (0.2%), no significant effect on temperature sensitivity was observed. Furthermore, considering chitosan's antibacterial properties and cytotoxicity, a chitosan concentration of 0.2% was determined. In summary, the optimized F127 / F68 / CS ratio (20 / 6 / 0.2 wt / vol%) produced a composite hydrogel that met the desired temperature for skin administration, exhibited a short gelation time, and was easy to use (see Figure 1).

[0211] Table 1 Gelation temperature of F127 / F68 with different concentration ratios

[0212] Table 2 Gelation time of F127 / F68 with different concentration ratios at 34°C

[0213] Example 3. Microscopic morphology of composite hydrogel under scanning electron microscope

[0214] 10 mL of the prepared composite hydrogel was pre-cooled at -80°C for 2 hours, freeze-dried, sprayed with gold, and photographed using a scanning electron microscope. Scanning electron microscopy revealed that the composite hydrogel dressing had a three-dimensional, porous, and well-defined reticular structure with a pore size of approximately 20 μm, allowing for drug loading and controlled release (see Figure 2, magnified 150x, 250x, and 2500x, respectively, from left to right).

[0215] Example 4. Thermosensitivity of the composite hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0216] The composite hydrogel will undergo a phase change at a specific temperature, changing from a liquid state at a lower temperature to a solid state at a higher temperature, or from a solid state to a liquid state, with a reversible phase change as the temperature changes. At 25°C, it is in liquid state, the centrifuge tube is tilted, and a picture is taken. At 37°C, it is in solid state, the centrifuge tube is inverted, and a picture is taken. The results show that at 25°C, it is in liquid state, the centrifuge tube is tilted, and the fluidity is good, the liquid level changes, and it is horizontal, the texture is uniform, and it is colorless and transparent. At 37°C, it is in solid state, the centrifuge tube is inverted, the liquid level does not change, there is no fluidity, and it is maintained for more than 1 minute (see Figure 3 for the results). Moreover, the hydrogel system of the present invention is a viscous solution before forming a gel, and has good viscoelasticity after forming a solid hydrogel. The dressing can better adhere to the wound and reduce the impact of external forces on the wound.

[0217] Example 5. Sustained release experiment of NMN loaded on the composite thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0218] At a detection wavelength of 235 nm on a UV spectrophotometer, the relative concentration values ​​of NMN at different absolute concentrations were detected to determine the relationship between absolute and relative concentrations. NMN (1 mg / mL) was placed in a carrier thermosensitive hydrogel (F127 / F68 / CS), mixed at 4°C to form an NMN-loaded composite hydrogel system, and placed in a 34°C incubator. After 24 hours, the NMN-loaded thermosensitive hydrogel was solid. 1 mL of normal saline was added, and the BSA-loaded thermosensitive hydrogel was immersed in normal saline. The two were placed together on a shaker at 34°C and 100 rpm. The supernatant was collected at predetermined time points (0h, 4h, 8h, 12h, 24h, 36h, 48h) and replaced with fresh normal saline to maintain a constant volume for the system. The experiment was repeated three times. The supernatant collected at each time point was measured on a UV spectrophotometer at a detection wavelength of 235 nm to measure the concentration of NMN, and the cumulative release was calculated using the formula: Release = (mass of released NMN / mass of administered NMN) × 100%. The results showed that before 24 hours, the release rate was relatively fast, with a cumulative release of 73.1%. After 24 hours, the release rate slowed down, and at 48 hours, the cumulative release reached 96.9%, indicating that the thermosensitive hydrogel system (F127 / F68 / CS) has a sustained release effect on NMN and can be used as a dressing loaded with the drug to slowly release it to the wound, achieving controlled release of NMN (see Figure 4).

[0219] Example 6. Antibacterial performance test of composite thermosensitive hydrogel

[0220] Bacterial culture: Take out the frozen Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) from the -80℃ freezer, quickly put them into a 37℃ water bath and shake to thaw. After the bacterial liquid melts, take 100μL of each bacterial liquid and transfer it to a shaking tube containing 5mL liquid culture medium. Puff evenly and place it in a constant temperature shaker at 37℃, 220r / min. Shake and culture for 18h. The bacterial expansion culture enters the logarithmic growth phase. Antibacterial efficiency was determined: After expanding Escherichia coli and Staphylococcus aureus cultures, 80 μL of each culture was aspirated and 20 μL of the thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.1-1 wt / vol%) was added to each culture. 100 μL of the culture-hydrogel mixture was thoroughly mixed. The S. aureus-hydrogel mixture was then added dropwise to a BHIS culture plate, while the E. coli-hydrogel mixture was added dropwise to an LB culture plate. The plates were then incubated at 37°C for 12 hours, and the colony counts were recorded. The inhibition rate = 1 - (colony count on the hydrogel plate / colony count on the non-hydrogel plate) × 100%. The results showed that as the molecular weight of the chitosan in the hydrogel increased, the colony count decreased, indicating a significant inhibitory effect. The inhibition rate against S. aureus ranged from 30% to 82%, and against E. coli from 19% to 70% (see Figure 5).

[0221] Example 7. Effect of thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%) on cell viability

[0222] The CCK-8 method was used to test the EA.hy.926 and HaCaT cell lines. First, cell culture media containing 20%, 1%, and 0.5% hydrogel were prepared. EA.hy926 cells were cultured in DMEM medium, and HaCaT cells were cultured in MEM medium. The two cell lines were trypsinized, and cells in the logarithmic growth phase were collected by centrifugation. The cell concentration was adjusted to 10 5 cells / mL, 100 μL of cell solution was plated in each well of a 96-well plate, i.e. 10 4The cells were placed in a 37°C incubator with a 5% CO2 concentration. After 12 hours, the cells adhered to the wall. The old culture medium in each well was discarded, and 100 μL of pre-prepared culture medium containing the hydrogel was added. The cells were incubated with the culture medium for 24 hours, 48 ​​hours, and 72 hours. The culture medium was then aspirated, and complete culture medium containing 10% CC-K8 was added to each well. The cells were incubated at 37°C for 2 hours. The absorbance of the cells was measured at a wavelength of 450nm on a microplate reader, and the relative cell proliferation rate (RGR) was calculated. Each group had three replicate wells. The relative cell proliferation rate = (average absorbance of the experimental group / average absorbance of the control group) × 100%. The results showed that for HaCaT cells or EA.hy926 cells, there was no significant difference in the absorbance values ​​of the hydrogel group and the non-gel group at different time points. The two different cell types also demonstrated that the thermosensitive hydrogel was non-toxic to cells and had good biocompatibility (see Figure 6).

[0223] Example 8. Hemolytic compatibility experiment of thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0224] Blood was collected from rats via the eye sockets. Fresh blood was collected using EDTA anticoagulant tubes and placed in a centrifuge at 2000 rpm for 5 minutes. After centrifugation, the blood was separated into layers, and the white blood cells and plasma in the middle and upper layers were discarded. The blood was then washed with normal saline three times, and the supernatant was discarded each time the supernatant was centrifuged until the supernatant was no longer red. The resulting precipitate was red blood cells. 2 mL of the resulting red blood cells was taken and added to 98 mL of normal saline to prepare a 2% red blood cell suspension. Three 1.5 mL EP centrifuge tubes were used. One centrifuge tube was filled with 0.5 mL of 2% red blood cell suspension and 0.5 mL of normal saline as the negative control group; one centrifuge tube was filled with 0.5 mL of 2% red blood cell suspension and 0.5 mL of thermosensitive hydrogel as the experimental group; and one centrifuge tube was filled with 0.5 mL of 2% red blood cell suspension and 0.5 mL of distilled water as the positive control group. After mixing, the mixture was placed in a constant temperature water bath at 37°C for warming. After 1 hour and 24 hours, the hemolysis of each tube was observed and recorded. After taking a picture, the mixture was placed in a centrifuge and centrifuged at 2000 rpm for 5 minutes at room temperature. The supernatant was collected and the absorbance was measured using a spectrophotometer at a wavelength of 545 nm to calculate the hemolysis rate. This experiment was repeated three times and the average value was taken. Hemolysis rate = (absorbance of experimental group / absorbance of positive control group) × 100%. The results showed that the supernatant of the composite thermosensitive hydrogel experimental group, its negative control group (normal saline), and the positive control group (bovine serum albumin) at 1 hour and 24 hours was clear and transparent, without reddening or becoming turbid, while the solution of the positive control group had already undergone hemolysis, with the entire solution uniformly reddening and becoming turbid. In addition, the hemolysis rates of the composite hydrogel experimental group and the negative control group were less than 2%, indicating no hemolysis, and had good blood compatibility (see Figure 7).

[0225] Example 9. Intradermal stimulation experiment of NMN-loaded thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0226] SD rats were selected as experimental subjects, and both sides of the dorsal spine were used as injection sites. The injection area was first shaved, and then the hair was removed with a depilatory cream to expose the back skin to ensure that there was no edema, erythema, etc. in the skin. The experimental group was injected with 0.2 mL of NMN-loaded thermosensitive hydrogel (0.04 mg / rat, 0.4 mg / rat, 2 mg / rat). There were three groups, and the negative control group was injected with 0.2 mL of sterile saline. The status of the dorsal injection site was observed at 0 h, 24 h, and 48 h after injection, and the edema and erythema were accurately recorded and photographed. The results showed that there was no obvious erythema, edema or other conditions on the back of the rats (see Figure 8 for the results), indicating the safety of the NMN-loaded thermosensitive hydrogel.

[0227] Example 10. Cell proliferation assay of EA.hy926 / HaCaT cells using NMN-loaded thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0228] Remove EA.hy926 or HaCaT cells from the incubator, wash and digest, count, and transfer the cells quantitatively into a 96-well plate. Add 10 4 cells, with 3 parallel wells in each group. 24 hours after plating, the cells were attached to the bottom of the wells, the supernatant was removed, and replaced with complete culture medium containing different concentrations of NMN (0, 500ng / mL, 10μg / mL, 200μg / mL) and 1μg / mL PDGF, and cultured in an incubator at 37°C and 5% CO2. After culturing for 24h and 48h, the culture medium was aspirated, and complete culture medium containing 10% CC-K8 was added to each well, placed at 37°C, incubated for 2h, and the absorbance of the cells was measured at a wavelength of 450nm on a microplate reader to calculate the relative proliferation rate (RGR). Relative cell proliferation rate (RGR) = (absorbance value of the experimental group / absorbance value of the control group) × 100%. The results showed that compared with the control group, NMN not only did not affect the cell viability of the two cell types, but also significantly improved the cell viability. Compared with the cell viability measured at 24 hours, the cell viability at 48 hours was more significantly improved. This shows that a certain concentration of NMN can improve cell viability and promote cell proliferation under certain conditions (see Figure 9 for the results).

[0229] Example 11. Cell migration assay of HaCaT cells using NMN-loaded thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0230] Take a new six-well plate, use a ruler to measure the bottom of the plate, and use a pen to draw lines so that two parallel straight lines can be seen at the bottom of each well. Then inoculate 10 cells per well of the six-well plate. 6HaCaT cells were cultured in a 37°C, 5% CO2 incubator for 24 h until all the cells attached to the bottom of the plate and grew to an appropriate density. The HaCaT cells were scratched with the tip of a 200 μL pipette. The scratch was perpendicular to the line on the outer bottom. The cells were washed twice with PBS solution until the cells detached from the scratch were clean. Then, MEM complete medium (containing 2% FBS) containing 500 ng / mL, 10 μg / mL and 200 μg / mL NMN and MEM complete medium (containing 2% FBS) without NMN were added, respectively. The cells were cultured in a 37°C, 5% CO2 incubator. The migration of cells at the scratch was observed under a microscope at 0 h, 12 h, 24 h and 48 h, and photos were taken to calculate the area of ​​the scratch pores. The data were analyzed using Image J. The results showed that within the same period of time, as the NMN concentration increased, the smaller the pore area, the stronger the cell migration ability. Compared with the control group, the NMN group with the highest concentration showed the best cell migration performance. NMN can significantly enhance the cell migration ability. The cell migration speed of the highest concentration NMN group was nearly twice that of the control group (see Figure 10).

[0231] Example 12. Evaluation of the angiogenic properties of the NMN-loaded thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0232] Angiogenesis assays The Matrigel basement membrane matrix is ​​ready. The day before the experiment, place the Matrigel in an ice box and refrigerator at 4°C to slowly thaw overnight. Pre-chill a 96-well plate and pipette tips. Prepare two pre-chilled 1.5 mL centrifuge tubes for diluting the Matrigel. Dilute the Matrigel with MEM medium at a ratio of 2:1. Add 50 μL of the diluted Matrigel to each well, perpendicularly above the inner well. Gently shake the 96-well plate to spread the gel evenly across the bottom, avoiding bubbles. Place in a 37°C incubator for 45 min-1 h. When EA.hy.926 cells are approximately 80% confluent, digest them and resuspend them in serum-free DMEM without, with 500 ng / mL, 10 μg / mL, and 200 μg / mL NMN. Count the cells and add 50 μL of the resuspended cell solution to each well at a concentration of 30,000 cells / well. Each set of wells should be replicated in triplicate. The cells were placed in an incubator at 37°C and 5% CO2 for incubation. The number of blood vessels formed was observed under a microscope at 12h and 24h, and photographs were taken. The number of blood vessels formed was counted and analyzed using Image J software. The results showed that in the control group without NMN, the number of new blood vessels was small, and almost no obvious new blood vessels were observed at 12h. In the experimental group containing NMN, more new blood vessels were observed, especially in the high-dose group of 200μg / mL NMN. More tubular structures and more blood vessels were observed under the microscope, and the number of new blood vessels was 3.5 times that of the control group (see Figure 11).

[0233] Example 13. Antioxidant performance test of NMN-loaded thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0234] According to the trend graph of HaCaT cell survival rate versus hydrogen peroxide concentration (Figure 12A), the median lethal dose of hydrogen peroxide to HaCaT cells was obtained. 4Cells were plated into 96-well plates and incubated at 37°C, 5% CO₂ for 24 hours, until the cells adhered to the plate bottom. The remaining MEM medium was discarded and replaced with MEM medium containing the same concentration of hydrogen peroxide at a median lethal dose. 0, 0.5 μg / mL, 10 μg / mL, and 200 μg / mL NMN, along with 10 μg / mL vitamin C, were added, with 100 μL per well. The group without hydrogen peroxide and NMN served as the blank control, the group with hydrogen peroxide but not NMN served as the negative control, and the group with hydrogen peroxide and vitamin C served as the positive control. The remaining experimental groups with varying concentrations of NMN were replicated in triplicate. After incubation at 37°C, 5% CO₂ for 12 hours, the cells were measured using a CCK-8 assay with absorbance at 450 nm using a microplate reader. Relative cell viability was calculated by comparing the experimental and positive control groups with the negative control, with the negative control as 100%. The results showed that the average cell viability in the control group without NMN was 47.16%. In the experimental group containing NMN, the higher the concentration of NMN, the higher the cell viability. The average cell viability of the high-dose group of 200 μg / mL NMN could reach 77.71% (see Figure 12B for the results).

[0235] Example 14. Treatment of diabetic mouse skin wounds with NMN-loaded thermosensitive hydrogel F127 / F68 / CS (20 / 6 / 0.2 wt / vol%)

[0236] After two weeks of stable blood sugar levels in the diabetic mouse model, the mice were anesthetized and placed on their backs. The hair on the back was shaved with an electric shaver, and then further depilatory cream was applied to the area and disinfected. A hole was punched on the mouse's back with a punch. A 1.0 cm diameter circular incision was then made along the circular mark created with sterile surgical scissors, and the full-thickness skin tissue in this area was excised. Afterwards, different dressings were added to the wounds of each group of mice. The group with normal saline was used as the blank negative control group, the group with thermosensitive hydrogel without NMN was used as the hydrogel control group, the group with thermosensitive hydrogel containing NMN (3 concentrations: 0.5μg / mL, 10μg / mL and 200μg / mL) was used as the experimental group, the group with thermosensitive hydrogel without NMN (10μg / mL) alone, and the group with Fujifu (National Medicine Standard No. S20020023, Beijing Shuanglu Pharmaceutical) were used as the positive control group. After adding the dressings, the wound was wrapped with a 3M wound patch. There were 6 mice in each group, and they were housed in separate cages. They were observed, photographed, measured, recorded, and the wound area was calculated at the prescribed time. On the 10th day, one mouse was randomly selected from each group, and the wound and surrounding tissues were taken and divided into two, one for histological analysis and the other for qPCR detection.

[0237] The results in Table 3 show that on the 4th day, the wound of the blank negative control group had almost no change, and the wound still maintained its original shape, while the wound area of ​​the high-dose experimental group had already shown a trend of shrinking, and the wound shape had changed; on the 7th day, the average relative wound area of ​​the experimental group was 59.74% of the initial value, while that of the blank negative control group was 85.04%. On the 14th day, the wound surface of the high-dose experimental group had completely healed, while the wound of the positive control group was basically healed on the 14th day, and the wound of the blank negative control group was basically healed on the 21st day (see Figure 13 for the results). This shows that the NMN-loaded thermosensitive hydrogel accelerates the healing of chronic wounds.

[0238] Table 3 Relative wound areas of diabetic mice in each group at 1d, 4d, 7d, 10d, 14d, 18d and 21d after perforation and dressing

[0239] All experimental results are the mean values ​​obtained from 6 diabetic mice. Error bars represent standard deviations. Statistical differences were detected by t-test. Compared with the blank negative control group and other groups, *p<0.05, **p<0.01, ***p<0.001; compared with the marketed drug positive control group and the high-dose experimental group and the medium-dose experimental group, #p<0.05, ##p<0.01, ###p<0.001; (n=6).

[0240] Example 15. HE staining of skin wound tissue sections

[0241] To further evaluate wound healing in diabetic mice, histological analysis was performed. The figure shows that compared to the listed drug positive control group, the high-dose and medium-dose experimental groups showed significantly more pronounced wound healing, with the epidermal structure of the newly formed granulation tissue intact and continuous. The epidermal layer thickness in the high-dose experimental group was approximately 68.13 μm, while the blank negative control group did not form a confluent epidermal layer (see Figure 14).

[0242] Example 16. Expression of VEGF and TGF-β1 in skin wound tissue

[0243] TGF-β1 and VEGF are two important growth factors in the wound healing process. In order to further explore the mechanism by which NMN-loaded thermosensitive hydrogel promotes wound healing, we detected the relative expression of TGF-β1 and VEGF in the skin tissue of the wound site of each group. On the 10th day after modeling and dressing, qPCR detection was performed on the skin tissue structure of the wound site of each group of mice. The results showed that the expression levels of VEGF and TGF-β1 mRNA in the experimental group were upregulated, with statistical differences (see Figure 15 for the results).

[0244] Example 17. Optimized ratio of each component of alginate hydrogel

[0245] 2.1 Material Preparation: Prepare sodium alginate (SA) solutions at predetermined concentrations of 1%, 2%, 3%, 4%, and 5% (wt / wt). Prepare crosslinker solutions (e.g., calcium chloride solutions) at predetermined concentrations of 0.1%, 0.5%, 1%, and 2% (wt / wt).

[0246] 2.2 Preparation steps

[0247] (1) Dissolving sodium alginate: Dissolve alginate in deionized water and stir until completely dissolved to form a uniform sodium alginate solution.

[0248] (2) Debubbling: The sodium alginate solution is placed in a vacuum environment to debubble to ensure that the prepared hydrogel is uniform and free of bubbles.

[0249] (3) Cross-linking reaction: Sodium alginate solution was added dropwise to the cross-linking agent solution to form a hydrogel. The pH values ​​were adjusted to 5, 6, 7, and 8 to screen for the optimal pH value. At the same time, different cross-linking temperatures were set to 4°C, 20°C, 25°C, 30°C, and 35°C to screen for the optimal cross-linking temperature.

[0250] (4) Gelation and post-treatment: Allow the gel to stand in the crosslinker solution for a certain period of time to ensure sufficient crosslinking. Remove the gel and wash it with deionized water or an appropriate buffer to remove unreacted crosslinker and other residues.

[0251] (5) Drying and storage: The hydrogel can be freeze-dried or subjected to other drying treatments as needed to prepare a dry hydrogel. The prepared hydrogel is stored under appropriate conditions to maintain its physicochemical properties and biological activity. The results are shown in (Table 4)

[0252] 3. Optimized ratio of the components of thermosensitive hydrogel

[0253] 3.1 Material Preparation: Prepare F127 solutions at predetermined concentrations of 15%, 20%, 25%, and 30% (wt / wt). Preparation of physiological saline: Weigh 0.45 g of sodium chloride powder and dissolve it in 50 mL of distilled water to obtain a 0.9% sodium chloride solution.

[0254] 3.2 Preparation steps

[0255] (1) Sample preparation: Thermosensitive hydrogels were prepared by the low-temperature dissolution method. F127 was weighed on a precision electronic balance and placed in three 50 mL centrifuge tubes. An appropriate amount of physiological saline was added to allow full solid-liquid contact. The solution was then placed at 4°C for 3 to 5 days to completely dissolve and obtain a clear gel solution. The solution was then diluted to the specified volume with physiological saline. The higher concentration of F127 was placed in a 4°C refrigerator for later use.

[0256] (2) Performance testing:

[0257] Determination of gelation temperature: The gelation temperature refers to the phase transition temperature when the thermosensitive hydrogel changes from liquid to semi-solid. The gelation temperature is determined by the centrifuge tube inversion method. The prepared thermosensitive hydrogel solution with mass fraction gradient is taken out from the 4°C refrigerator. 1 mL of each gradient is placed in a 1.5 mL centrifuge tube. The centrifuge tube is completely placed in a constant temperature water bath to ensure that the hydrogel liquid level is 3 cm below the water bath liquid level. The initial temperature of the water bath is 20°C, the set temperature is 60°C, and the temperature continues to rise. An instant thermometer is inserted under the liquid surface of the water bath to detect the temperature in real time, and the flow of the hydrogel in the centrifuge tube is observed at the same time. When the hydrogel in the centrifuge tube no longer flows and can be maintained for 1 minute, the temperature on the thermometer corresponding to this moment is recorded and recorded as the gelation temperature. Each sample is measured 3 times, and the results are averaged.

[0258] Determination of gelation time: gelation time refers to the time required for thermosensitive hydrogel to transform from liquid to semi-solid. The gelation time was determined by centrifuge tube inversion method. The prepared mass fraction gradient thermosensitive hydrogel was taken out from the 4℃ refrigerator.

[0259] For gel solution, add 1 mL of each gradient into a 1.5 mL centrifuge tube. Place the tube completely in a constant-temperature water bath, ensuring the hydrogel level is 3 cm below the bath level. Maintain the water bath at 34°C. Start timing when the tube containing the hydrogel enters the bath. Invert the tube every few seconds as needed to observe the flow of the hydrogel. Record the time on the timer when the hydrogel in the tube stops flowing and remains stationary for 1 minute. This is the gelation time. Repeat the measurement three times for each sample, and average the results.

[0260] (3) Data analysis: Determine the optimal concentration based on the test results.

[0261] (4) Verification: Repeat the experiment at the optimal concentration to ensure stable performance. The results are shown in Table 5.

[0262] Table 4 Optimized ratio of each component of sodium alginate hydrogel

[0263] Table 5 Optimized ratio of each component of thermosensitive hydrogel

[0264] Example 18: Screening of antibacterial activity and cytotoxicity concentration of antibacterial substances, comprising the following steps:

[0265] MIC Assay: Sterilize instruments by placing the prepared culture medium, PBS buffer, sterile water, pipette tips, and centrifuge tubes in an autoclave at 121°C for 25 minutes. Sterilize the 96-well plate under UV light for 30 minutes in a clean bench or biosafety cabinet. Prepare the sample working solution plate: Add 100 μL of liquid culture medium to wells A2-A12, including 200 μL of the highest concentration of the drug to be tested in well A1. Then, pipette 100 μL from well A1 and add it to well A2. Mix thoroughly, then pipette 100 μL from well A2 and add it to well A3. Continue this dilution series to well A12, discarding the last 100 μL of diluted solution. Prepare a new 96-well plate and add 50 μL of sample solution to each well corresponding to the sample working solution plate. Well 12 (i.e., A12-H12) remains untreated as a growth control. Prepare a culture solution with a concentration equivalent to 1.5×108 CFU / mL and dilute it 1:1000 with culture medium to a concentration of 1.5×105 CFU / mL. Add 50 μL of this solution to each well and incubate in a 35°C air-cooled incubator for 16–20 hours. The test is only meaningful when there is noticeable bacterial growth in the negative control wells (i.e., wells without sample solution). A clear, distinct appearance with no colonies is the MIC for that group. When reading the plate, easily overlooked bacterial plaques may appear in the corners of the wells. Therefore, hold the plate steady overhead and carefully observe each well for bacterial growth. The MIC is determined by the wells that are completely clear. Alternatively, the OD600 value can be used for determination (the lowest drug concentration closest to the measured OD value of the negative control well without bacteria is the MIC). However, the lid should be kept open to avoid contamination.

[0266] MBC Determination: Pipette 100 μl of bacterial solution at the MIC concentration onto a solid culture medium plate. After incubation for an appropriate period of time, determine the MBC concentration based on the number of colonies on the plate. Incubate the plate in a 35°C air incubator overnight. The next day, examine each plate for bacterial growth. The minimum bactericidal concentration (MBC) is the minimum bactericidal concentration (MBC) of the drug in the corresponding culture medium tube when the number of colonies growing is less than 0.1% of the inoculum volume (no bacterial growth).

[0267] The CCK-8 method was used to simultaneously detect the cytotoxicity of PHMB on EA.hy.926 and HaCaT cell lines. The detailed steps are shown in Example 7.

[0268] The results showed that by setting different concentrations of chitosan (0.15%, 0.075%, 0.0375%, 0.01875%, 0.009375%, 0.004688%, 0.002344%, 0.001172%, 0.000586%, 0.000293%, 0.000146%, 0%) and PHMB (0.8%, 0.4%, 0.2%, 0.1%, 0.05%, 0.025%) , 0.0125%, 0.00625%, 0.003125%, and 0%), respectively, showed that chitosan had a minimum inhibitory concentration (MIC) of 0.075% against E. coli and a minimum bactericidal concentration (MIC) of 0.0023% against Staphylococcus aureus. PHMB had a MIC of 0.003125% against E. coli and Staphylococcus aureus, respectively. Both exhibited excellent bactericidal activity, particularly PHMB. Based on this, PHMB concentrations of 0.8%, 0.4%, 0.2%, 0.1%, 0.05%, and 0.025% were selected, and CCK8 assays were used to screen for PHMB concentrations with good biocompatibility. PHMB concentrations below 0.3% exhibited excellent biocompatibility and showed little cytotoxicity, with lower concentrations exhibiting reduced cytotoxicity (see Figure 16).

[0269] Example 19

[0270] The optimized ratio of the dressing matrix components with antimicrobial properties includes the following steps:

[0271] 1. Optimized ratio of components of antibacterial alginate hydrogel

[0272] Material preparation: Prepare 20% PHMB stock solution, set the PHMB working solution concentration to 0.02%, 0.05%, 0.1%, 0.2%, 0.3% (wt / wt) PHMB; prepare alginate solution according to the predetermined concentration of 2%, 3%, 4% (wt / wt)

[0273] Preparation steps:

[0274] (1) Dissolving alginate: Dissolve alginate in deionized water and stir until completely dissolved to form a uniform alginate solution.

[0275] (2) Debubbling: The alginate solution is placed in a vacuum environment to debubble to ensure that the prepared hydrogel is uniform and free of bubbles.

[0276] (3) Electrostatic adsorption of PHMB: 20% PHMB stock solution was diluted to the required working solution concentration and then added to alginate solutions of different concentrations.

[0277] (4) Cross-linking reaction: Add the alginate solution dropwise into the cross-linker solution to form a hydrogel.

[0278] (5) Gelation and post-treatment: Allow the gel to stand in the crosslinker solution for a certain period of time to ensure sufficient crosslinking. Remove the gel and wash it with deionized water or an appropriate buffer to remove unreacted crosslinker and other residues.

[0279] (6) Observe the bactericidal effect, physical properties and biocompatibility of the gels in different concentration groups.

[0280] (7) Data analysis: Determine the optimal concentration based on the test results.

[0281] (8) Verification: Repeat the experiment for the optimal concentration to ensure stable performance.

[0282] 2. Optimized ratio of components of antibacterial thermosensitive hydrogel

[0283] Materials Preparation: Prepare a 20% PHMB stock solution and set the PHMB working solution concentrations to 0.02%, 0.05%, 0.1%, 0.2%, and 0.3% (wt / wt) PHMB. Prepare F127 solutions at predetermined concentrations of 16%, 18%, and 20% (wt / wt). Preparation of physiological saline: Weigh 0.45g of sodium chloride powder and dissolve it in 50mL of distilled water to obtain a 0.9% sodium chloride solution.

[0284] Preparation steps

[0285] (1) Sample preparation: Thermosensitive hydrogels were prepared by the low-temperature dissolution method. F127 was weighed on a precision electronic balance and placed in three 50 mL centrifuge tubes. An appropriate amount of physiological saline was added to allow full solid-liquid contact. The solution was then placed at 4°C for 3 to 5 days to completely dissolve and obtain a clear gel solution. The solution was then diluted to the specified volume with physiological saline. The higher concentration of F127 was placed in a 4°C refrigerator for later use.

[0286] (2) Electrostatic adsorption of PHMB: 20% PHMB stock solution was diluted to the required working solution concentration and then added to F127 solutions of different concentrations.

[0287] (3) Observe the bactericidal effect, physical properties and biocompatibility of the gels in different concentration groups. Determine the gelation temperature and time.

[0288] (4) Data analysis: Determine the optimal concentration based on the test results.

[0289] (5) Verification: Repeat the experiment for the optimal concentration to ensure stable performance.

[0290] The results showed that when PHMB was selected at five concentrations (0.02%, 0.05%, 0.1%, 0.2%, and 0.3%) and paired with 16%, 18%, and 20% F127, or 2%, 3%, and 4% alginate, the higher the F127 concentration, the greater the effect of PHMB on the gelation temperature. PHMB at 0.1% had the least effect on the three F127 concentrations. Furthermore, the combination of 0.1% PHMB and 3% alginate did not affect gelation and antibacterial properties (Figure 17, Table 6).

[0291] Table 6 Optimized ratio of antibacterial alginate hydrogel dressing matrix components

[0292] Example 20. Effects of polymeric guanidine antimicrobial agents on the biological functions of active ingredients, comprising the following steps:

[0293] (I) 1. Effect of polymeric guanidine antimicrobial agents on the proliferation-promoting activity of active ingredients The cell proliferation test of EA.hy.926 / HaCaT cells was conducted according to the method of Example 10.

[0294] (II) 2. Effect of polymeric guanidine antimicrobial agents on the migration-promoting activity of active ingredients The cell migration test of the thermosensitive hydrogel on HaCaT was conducted according to the method of Example 11.

[0295] 3. Effect of polymeric guanidine antimicrobial agents on the angiogenic activity of the active ingredient The angiogenic performance of the thermosensitive hydrogel was evaluated according to the method of Example 12.

[0296] 4. Effect of polymeric guanidine antimicrobial agents on the antioxidant activity of active ingredients

[0297] Evaluation of the antioxidant properties of the thermosensitive hydrogel was tested according to the method of Example 13

[0298] 5. Effect of polymeric guanidine antimicrobial agents on the anti-inflammatory properties of active ingredients

[0299] Thp-1 cells were cultured at a rate of 4 × 10 4 Cells were seeded at a density of 180 μL / well in a 96-well plate. Different active ingredients and / or polymeric guanidine antimicrobial agents were added to the plates and incubated at 37°C, 5% CO₂ for 24 hours. Cell supernatants were collected at 1000 g and assayed for TNF-α, IL-6, and IL-10 levels using ELISA.

[0300] The results showed that 0.1% PHMB in the polymeric guanidine antimicrobial agent did not affect the antioxidant, anti-inflammatory, angiogenic, and pro-proliferative effects of the active ingredient. (A and B show the effect of PHMB on the pro-migratory activity of NMN and their statistical graphs; C shows the effect of PHMB on the antioxidant activity of NMN; D and E show the effect of PHMB on the angiogenic activity of NMN; and F shows the effect of PHMB on the anti-inflammatory activity of NMN.) (See Figure 18 for the results.)

[0301] Example 21: Effect of active ingredients on the bactericidal effect of polymeric guanidine antimicrobial agents, comprising the following steps:

[0302] Select PHMB concentration of 0.1%, NMN concentration of 0.05%, 0.1%, 0.2% respectively

[0303] Determination of antibacterial rate: After expanding the culture of Escherichia coli and Staphylococcus aureus, 80 μL of each bacterial solution was aspirated, and 20 μL of the polymer guanidine antimicrobial agent and / or active ingredient was added to each. 100 μL of the bacterial solution-hydrogel mixture was thoroughly mixed. The S. aureus-hydrogel mixture was added dropwise to a BHIS culture medium plate, and the E. coli-hydrogel mixture was added dropwise to an LB culture medium plate. The plates were placed in a 37°C constant temperature incubator. After 12 hours, the plates were observed and the number of colonies was recorded. Inhibition rate = 1-(number of colonies on the plate with hydrogel / number of colonies on the plate without hydrogel) * 100%

[0304] The results showed that 0.1% of the polymer guanidine antimicrobial agent (PHMB) had an antibacterial rate of 100%, and NMN alone had no antibacterial effect. Different concentrations of the active ingredient, NMN (0.05%, 0.1%, 0.2%), did not affect the bactericidal effect of the polymer guanidine antimicrobial agent (see Figure 19).

[0305] Example 22. Microscopic morphology of alginate hydrogel under scanning electron microscope

[0306] 10 mL of the prepared composite hydrogel was pre-cooled at -80°C for 2 hours, freeze-dried, sprayed with gold, and photographed using a scanning electron microscope. The results (see Figure 20) show (A) alginate hydrogel, the left image shows alginate hydrogel alone, the right image shows alginate hydrogel loaded with active ingredients and antimicrobial substances, and (B) alginate hydrogel loaded on gauze (the left image shows ordinary gauze, the right image shows alginate hydrogel loaded with active ingredients and antimicrobial substances loaded on gauze). Scanning electron microscopy revealed that the composite hydrogel dressing had a three-dimensional, porous, and well-defined reticular structure with a pore size of approximately 20 μm, capable of loading and controlled release of drugs.

[0307] Example 23

[0308] Sustained release experiment of NMN loaded in alginate hydrogel

[0309] The assay was performed according to the method of Example 5. The experiment demonstrated that the alginate hydrogel (SA / PHMB) exhibited a sustained release effect on NMN. Within 24 hours, the release rate was relatively fast, with a cumulative release of 61.7%. After 24 hours, the release rate slowed down, reaching a cumulative release of 97.2% at 48 hours. This suggests that the alginate hydrogel exhibits a sustained release effect on NMN and can be used as a dressing loaded with the drug, slowly releasing it into wounds and achieving controlled release of NMN (see Figure 21).

[0310] Example 24 Swelling rate of composite hydrogel

[0311] The freeze-dried hydrogel was cut into small equal slices (1 cm x 1 cm) and repeated three times. The slices were immersed in 4 mL of phosphate-buffered saline (PBS) solution. Samples were taken out at specific time intervals (0.5 h, 2 h, 4 h, 8 h, 16 h, and 24 h) and weighed immediately. The following formula was used to evaluate the cell membrane expansion rate:

[0312] W2 weight after immersion, W1 weight before immersion

[0313] The results showed that the alginate hydrogel could swell to 2-3 times its original size, and drug loading had no effect on the hydrogel's permeability. Gauze-loaded alginate hydrogel could swell to 5-6 times its original size, and drug loading had no effect on the hydrogel's swelling rate (Figure 22).

[0314] Example 25. Verification of the inhibitory effect of a dressing matrix with antibacterial properties on biofilms formed by Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), comprising the following steps:

[0315] A blank control group, a simple dressing matrix group, and a PHMB plus dressing matrix group were set up.

[0316] The mixed bacteria cultured overnight (500 μL, 5×10 8 CFU mL -1 ) 250uL each of Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) were added to a 24-well plate and cultured at 37°C to form a biofilm. After 36h, the top culture medium was discarded and the plate was rinsed three times with PBS. Then, 100μL of each group of samples was added to the wells and incubated at 37°C for 24h. Next, the supernatant was discarded and 500μL of methanol was added to each well for fixation. After 20min, the methanol was removed and the plate was air-dried. Then, 200μL of crystal violet dye was added to stain the biofilm, and after 10min, the wells were rinsed twice to remove unbound crystal violet. Each well was then incubated with 200μL of 33% glacial acetic acid for 30min, and the absorbance of all samples at 590nm was measured using a microplate reader.

[0317] The results showed that the dressing matrix alone could not inhibit biofilm formation, but the dressing matrix containing 0.1% of the previously screened PHMB, which has antimicrobial properties, did inhibit biofilm formation by Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (see Figure 23).

[0318] Example 26. Alginate hydrogel hemolytic compatibility experiment

[0319] According to the method of Example 8, the hemolysis rates of the alginate hydrogel experimental group and the negative control group were both less than 2%, indicating no hemolysis and good blood compatibility (A is the hemolysis state of the drug-loaded alginate hydrogel after 1 hour and 24 hours; B is the hemolysis rate of A after 1 hour and 24 hours) (see Figure 24 for the results).

[0320] Example 27. The in vitro biological effects of composite hydrogels loaded with different concentrations of NAD+ include the following steps:

[0321] 1. Test the cell proliferation of EA.hy.926 / HaCaT cells by the method of Example 10

[0322] 2. Test the cell migration of HaCaT by thermosensitive hydrogel according to the method of Example 11

[0323] 3. Evaluation of the angiogenic performance of thermosensitive hydrogels according to the method of Example 12

[0324] 4. Evaluation of the Antioxidant Performance of Thermosensitive Hydrogels According to the Method of Example 13

[0325] Results: Figure 25 shows the in vitro biological effects of thermosensitive hydrogels loaded with different concentrations of NAD+. Over time, as NAD+ concentration increased, the smaller the pore area, the stronger the cell migration ability. Compared with the control group, the group with the highest NAD+ concentration exhibited the best cell migration performance. NAD+ significantly enhanced cell migration, with the highest concentration group migrating at a rate nearly twice that of the control group (see Figures 25A, B). In the control group without NAD+, the number of newly formed blood vessels was low, while in the experimental groups containing NAD+, a greater number of newly formed blood vessels was observed. In particular, in the high-dose group (200 μg / mL NMN), numerous tubular structures were observed under the microscope, with the number of newly formed blood vessels reaching 2.05 times that of the control group (see Figures 25C, D). The average cell viability in the control group without NMN was 50%. In the experimental groups containing NAD+, higher NAD+ concentrations increased cell viability, reaching an average cell viability of 74.95% in the high-dose group (200 μg / mL NAD+) (see Figure 25).

[0326] Example 28. Screening the concentration of active ingredients and the healing of skin wounds in diabetic mice treated with various multifunctional dressings in an in vivo animal model, comprising the following steps:

[0327] After two weeks of stable blood sugar levels in the diabetic mouse model, the mice were anesthetized and placed on their backs. The hair on the back was shaved with an electric shaver, and then further depilatory cream was applied to the area and disinfected. A hole was punched on the mouse's back with a punch. A 1.0 cm diameter circular wound was then cut along the circular mark created with sterile surgical scissors. The full-thickness skin tissue in this area was then excised, or bacteria were applied. Afterwards, different dressings were applied to the wounds of each group of mice. A blank negative control group received normal saline, while experimental groups received antimicrobial dressings with different ingredients. A simple dressing without active ingredients served as a dressing control group. Marketed drugs such as silver ion fiber dressings, Prandtl liquid wound dressings, and Fujifu (National Medicine Standard No. S20020023, Beijing Shuanglu Pharmaceutical) served as positive controls. After the dressings were applied, the wounds were wrapped with a 3M wound patch. Six mice per group were housed separately and observed, photographed, measured, recorded, and wound area calculated at prescribed intervals. On day 10, one mouse from each group was randomly selected, and the wound site and surrounding tissues were removed for histological analysis. Table 7.1 and Figure 26.1 show the effects of different concentrations of NMN in a PHMB / F127 mixed gel on wound healing. On day 7, the average relative wound area in the experimental group was 28.56% of the initial value, while that in the blank negative control group was 70.32%. On the 14th day, the wounds of the experimental group had completely healed, while the wounds of the positive control group had basically healed on the 14th day, and the wounds of the blank negative control group had basically healed on the 21st day. This shows that the NMN / PHMB-loaded thermosensitive hydrogel accelerates the healing of chronic wounds.

[0328] Table 7.2 and Figure 26.2 show the effects of 0.2% NMN@0.1% PHMB / F127 on wound healing. On day 7, the average relative wound area in the experimental group was 20.3% of the initial value, while that in the blank negative control group was 70.32%. By day 14, the wounds in the experimental group were completely healed, while those in the positive control group were largely healed by day 14 and the blank negative control group were largely healed by day 21. This demonstrates that the NMN / PHMB-loaded thermosensitive hydrogel accelerates the healing of chronic wounds.

[0329] Table 7.3 and Figure 26.3 show the effects of 0.2% NMN@0.1% PHMB / SA on wound healing. On day 7, the average relative wound area in the experimental group was 10.6% of the initial value, while that in the blank negative control group was 80.23%. By day 10, the wounds in the experimental group were completely healed, while those in the positive control group were largely healed by day 14 and the blank negative control group were largely healed by day 21. This demonstrates that the NMN / PHMB-loaded alginate hydrogel accelerates the healing of chronic wounds.

[0330] Table 7.4 and Figure 26.4 show the effects of 1% CeO2@0.2% NMN / SA hydrogel on wound healing. On day 7, the average relative wound area in the experimental group was 30.6% of the initial value, while that in the blank negative control group was 80.23%. By day 10, the wounds in the experimental group were completely healed, while those in the positive control group were largely healed by day 14 and the blank negative control group were largely healed by day 21. This demonstrates that the NMN / CeO2-loaded alginate hydrogel accelerates the healing of chronic wounds.

[0331] Table 7.5 and Figure 26.5 show the effects of 0.2% NMN@0.1% PHMB / SA hydrocolloid on wound healing. On day 7, the average relative wound area in the experimental group was 20.96% of the initial value, while that in the blank negative control group was 80.23%. By day 14, the wounds in the experimental group were completely healed, while those in the positive control group were essentially healed by day 14 and those in the blank negative control group were essentially healed by day 21. This demonstrates that NMN / PHMB-loaded alginate hydrocolloid accelerates the healing of chronic wounds.

[0332] Table 7.6 and Figure 26.6 show the effects of 0.1% PHMB@0.2% NMN / carboxymethyl cellulose fiber dressing on wound healing. On day 7, the average relative wound area in the experimental group was 20.3% of the initial value, while that in the blank negative control group was 81%. By day 14, the wounds in the experimental group were completely healed, while those in the positive control group were essentially healed by day 14 and those in the blank negative control group were essentially healed by day 21. This demonstrates that NMN / PHMB-loaded carboxymethyl cellulose fiber accelerates the healing of chronic wounds.

[0333] Figure 26.7 shows the effect of different concentrations of NAD+ on wound healing in a CS / F127 / F68 mixed gel. Compared to the control group, the high-dose and medium-dose experimental groups showed a trend of shrinking wound area on day 4. By day 7, the high-, medium-, and low-dose experimental groups all showed a faster wound healing rate than the negative control group. In particular, the average wound area of ​​the high-dose group was significantly reduced by approximately 21.4% on day 10 compared to the negative control group. By day 14, the wounds in the high-dose experimental group were almost completely healed, with a wound healing rate of up to 96.9%. In contrast, although the wounds of the medium-, low-dose experimental groups and the positive control group had healed, the epidermal coverage was thin; and the wounds of the negative control group still had scabs and a small amount of blood oozing, and the epidermal coverage was not complete.

[0334] Table 7.1 shows the effects of different concentrations of NMN active ingredients in PHMB and F127 gels on wound healing

[0335] Table 7.2 Effects of 0.2% NMN@0.1% PHMB / F127 on wound healing

[0336] Table 7.3 Effects of 0.2% NMN@0.1% PHMB / SA hydrogel on wound healing

[0337] Table 7.4 Effects of 1% CeO2@0.2% NMN / SA hydrogel on wound healing

[0338] Table 7.5 Effects of 0.2% NMN@0.1% PHMB / hydrocolloid on wound healing

[0339] Table 7.6 Effects of 0.1% PHMB@0.2% NMN / carboxymethyl cellulose fiber dressing on wound healing

[0340] All experimental results are the mean values ​​obtained from five diabetic mice, with error bars representing standard deviations. Figures 26.1 and 26.2 are from the same batch of experiments, shared by the positive and negative groups; and Figures 26.3 and 26.4 are from the same batch of experiments, shared by the positive, negative, and gel groups (SA group).

[0341] Example 29. Effect of 0.1% PHMB@0.2% NMN / F127 Dressing on Chronic Wound Healing in Various Clinical Patients (Results See Table 8, Figures 27.1 and 27.2)

[0342] Dosage regimen: Every 1 cm of clinical wound 2Use approximately 0.1-0.6g of 0.1% PHMB@0.2% NMN / F127 dressing, apply evenly, and change the dressing every 1-5 days.

[0343] Table 8

[0344] As can be seen from Table 8, in clinical trials, patients with various types of chronic wounds achieved complete wound healing, and the effect was very significant.

[0345] discuss

[0346] Nicotinamide mononucleotide (NMN) is a bioactive nucleotide and an intermediate in NAD+ biosynthesis. With its widespread availability and abundant reserves, NMN has opened up new horizons in modern therapeutics, demonstrating numerous promising pharmacological activities in several preclinical disease models, including those for myocardial ischemia, cerebral ischemia, and neurodegenerative diseases such as Alzheimer's disease. β-NMN is a key precursor for NAD+ production, and its functions are mediated through NAD+, providing antioxidant and oxidative stress reduction benefits, particularly in anti-aging settings.

[0347] This application prepares a composite thermosensitive hydrogel dressing loaded with NMN, and unexpectedly finds that it can promote the healing of chronic wounds such as diabetic foot ulcers.

[0348] In particular, the present invention provides a thermosensitive composite hydrogel (F127 / F68 / CS) prepared with poloxamer Pluronic F127 and Pluronic F68 as the matrix and chitosan (CS) added as an antibacterial material. The thermosensitive composite hydrogel has the characteristics of stable structure, suitable solid-liquid phase transition, loose porosity and sustained release performance, as well as adhesion, good antibacterial and biocompatibility. The thermosensitive composite hydrogel loaded with NMN is further prepared, which has the properties of promoting cell proliferation and migration, angiogenesis, antioxidant and anti-inflammatory properties, and has been verified in vivo and in vitro to have good biocompatibility and the effect of accelerating wound healing in diabetic mouse models.

[0349] More preferably, the present invention also provides an alginate-based gel dressing, wherein the gel has an excellent ability to absorb exudate. In addition to exerting the efficacy of the active ingredients, the gel matrix itself can absorb wound exudate, keep the wound dry and clean, and further facilitate wound healing.

[0350] The present invention further verifies the effect of NMN in combination with other antimicrobial agents, as well as its effect on chronic wound healing when loaded on other dressing matrices. Experiments have shown that the combination of NMN and antimicrobial agents (such as PHMB and CeO2) has a significant synergistic effect in the treatment of chronic wound healing, and has similar excellent technical effects on various dressing matrices, which is of great significance for providing new medical excipients for the treatment of chronic wound healing.

[0351] The embodiments described above are merely specific implementations of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be imagined by any person skilled in the art within the technical scope disclosed in the present application without resorting to creative effort should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims in the present application.

Claims

1. A dressing comprising: A first active ingredient: β-nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+), or a combination thereof; and Dressing base.

2. The dressing according to claim 1, characterized in that The dressing also includes a second active ingredient: an antibacterial agent; preferably, the antibacterial agent is selected from the following group: chitosan, chitosan oligosaccharides, chitin, polymer guanidine antibacterial agents, silver ions, copper ions and iron ions, cerium oxide (CeO2), cerium oxide (CeO2), iron oxide (Fe3O4), manganese oxide (MnO2), zinc oxide (ZnO), copper oxide (CuO), titanium oxide (TiO2), vanadium oxide (V2O5), antimicrobial peptides, or a combination thereof.

3. The dressing according to claim 2, characterized in that The second active ingredient is selected from the following group of polymer guanidine antibacterial agents, or pharmaceutically acceptable salts thereof: polyhexamethylene biguanide (PHMB), polyhexamethylene biguanide, polyaminopropyl biguanide, or a combination thereof.

4. The dressing according to claim 2, characterized in that The dressing also includes a third active ingredient, which is selected from the following group, or a pharmaceutically acceptable salt thereof: nicotinamide riboside (NR), nicotinamide nucleotide, nicotinamide adenine dinucleotide phosphate (NADP+), ergothioneine, pyrroloquinoline quinone (PQQ), coenzyme Q10 (CoQ10), coenzyme B12, vitamin substances such as vitamin C (Vitamin C), vitamin E (Vitamin E), vitamin A (Vitamin A), vitamin D (Vitamin D), vitamin K (Vitamin K), vitamin B2 (Vitamin B2), folic acid (Vitamin B9), metal oxide nanozymes such as cerium oxide (CeO2), iron oxide (Fe3O4), manganese oxide (MnO2), etc., glutathione, α-lipoic acid, resveratrol, carotenoids, proanthocyanidins, catechins, quercetin isoflavones, or a combination thereof.

5. The dressing according to claim 2, characterized in that The dressing is in a form selected from the group consisting of a hydrogel dressing, a hydrocolloid dressing, a fiber dressing, a foam dressing, a liquid, a paste dressing, a gauze, a cotton swab, a cotton ball, a cotton pad, an adhesive tape, a sticker, or a combination thereof.

6. The dressing according to claim 1, characterized in that include: Active ingredients: β-nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+), or a combination thereof; and Thermosensitive hydrogel matrix, Wherein, the active ingredient is dispersed in the hydrogel matrix.

7. The dressing according to claim 1, characterized in that The gelling agent used to form the hydrogel is selected from the group consisting of Pluronic F127, Pluronic F68, chitosan (CS), gelatin, hyaluronic acid, alginate, polyethylene glycol, or a combination thereof.

8. The dressing according to claim 2, characterized in that The weight ratio of the first active ingredient to the second active ingredient is 1:(0.1-10).

9. The dressing according to claim 4, characterized in that The weight ratio of the first active ingredient to the third active ingredient is 1:(0.1-10).

10. The dressing according to claim 2, characterized in that The first active ingredient is β-nicotinamide mononucleotide (NMN) or a pharmaceutically acceptable salt thereof, and the second active ingredient is polyhexamethylene biguanide or a pharmaceutically acceptable salt thereof.

11. The dressing according to claim 2, characterized in that The first active ingredient is β-nicotinamide mononucleotide (NMN) or a pharmaceutically acceptable salt thereof, and the second active ingredient is CeO2.

12. The dressing according to claim 2, characterized in that The first active ingredient is β-nicotinamide mononucleotide (NMN) or a pharmaceutically acceptable salt thereof, and the second active ingredient is chitosan.

13. The dressing according to claim 2, characterized in that The dressing is a hydrogel dressing or a dried form thereof comprising the following components, based on the total weight of the dressing: a first active ingredient and a second active ingredient; Alginate 1-10wt%; Crosslinking agent 0.1-2wt%; and Water, saline or physiological buffer solution with a pH of 6.5-7.

5.

14. The dressing according to claim 2, characterized in that The dressing is a thermosensitive hydrogel dressing or its dried form comprising the following components, calculated based on the total weight of the dressing: a first active ingredient and a second active ingredient; Pluronic F127 15-25 wt%; and Water, saline or physiological buffer solution with a pH of 6.5-7.

5.

15. The method for preparing the dressing according to claim 1, comprising the steps of: The active ingredient is mixed with the dressing matrix component to prepare a corresponding dressing form (hydrogel dressing, hydrocolloid dressing, foam dressing, liquid, paste dressing); or Dispersing the active ingredient in a solvent, and then coating the resulting mixture on a dressing matrix or impregnating the dressing matrix (such as a solid carrier such as a film, fiber, gauze, etc.); or The active ingredient is mixed with the dressing matrix component to prepare a corresponding dressing form (such as hydrogel, hydrocolloid, foam, liquid, paste), and the obtained dressing is further loaded on a solid carrier such as a film, fiber, gauze, etc., to form the dressing.

16. Use of the dressing according to claim 1 in preparing a medicine or medical device for promoting wound healing.

17. The use according to claim 16, characterized in that The wound is an acute wound or a chronic wound.

18. The use according to claim 16, characterized in that The wound may be selected from the following group: chronic non-healing wounds caused by traumatic ulcers, chronic non-healing wounds caused by pressure ulcers, chronic non-healing wounds caused by arterial ulcers, vascular ulcers, fistulas, sinus tracts and other skin injuries or mucosal injuries.

19. The use according to claim 16, characterized in that The wound is a chronic, refractory wound caused by diabetes.

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