Marine high-molecular polysaccharide hydrogel wound dressing, preparation method thereof and application of marine high-molecular polysaccharide hydrogel wound dressing in preparation of medical apparatus and instruments

Abstract

The invention discloses a preparation method and application of a marine high-molecular polysaccharide hydrogel wound dressing. The hydrogel is formed by cross-linking the marine polysaccharide composition and the cross-linking agent, and the gelation time is short and is about 15 seconds; pores are uniform and moderate; the self-healing property, the adhesion property and the swelling property are high; the antibacterial effect is strong, and the inhibition rates on escherichia coli and staphylococcus aureus are both higher than 90%; skin irritation and skin allergy are avoided, and the biological safety is high; hemostasis can be rapidly carried out within 10s, and the bleeding amount is obviously reduced; the hydrogel dressing can rapidly shrink and scab a wound surface, has the effect of promoting wound healing, and has the effects of stopping bleeding and promoting wound healing which are obviously superior to those of commercially available hydrogel dressings (German Hydrosorb Gel) (Plt; 0.05), and is a safe and effective high-performance wound dressing. In addition, the hydrogel is simple in formula and high in industrialization prospect, is expected to be developed into novel wound dressings and medical instruments, and breaks through the trapping that domestic hydrogel dressings are mostly dependent on import.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering polymer materials, and specifically relates to a marine polymer polysaccharide hydrogel wound dressing and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The skin is the largest, most exposed, and most vulnerable organ in the human body. It plays an important role in protecting internal tissues from external environmental interference, maintaining a stable internal environment, and regulating body temperature. Once damaged, it often needs to undergo a long self-repair process and cause a series of complex health problems, including inflammation, secondary injury, bacterial infection, etc. Sudden accidents, wars, and surgical operations often cause serious skin trauma, especially full-thickness skin loss. If not treated, chronic wounds will form and are difficult to heal. Moreover, uncontrolled bleeding caused by trauma is the leading cause of death worldwide. The first hour of rescue after trauma is the "golden hour" of life and death. During this hour, rapid hemostasis of the wound is the key to saving the wounded. Rapid and effective hemostasis can improve survival rates and reduce complications of the wounded. Skin injury, bleeding, and bacterial infection often occur together. Therefore, the development of bioactive dressings that can quickly stop bleeding and have antibacterial and wound healing effects has become a research hotspot in the medical field.

[0004] For trauma, traditional bandages are mainly based on cotton yarn. Although they have certain air permeability and wound protection capabilities, they have insufficient absorption capacity for wound fluid, poor packing and compression for severe bleeding, and limited ability to prevent infection and promote wound healing. Studies have shown that wounds heal faster in a moist state, and wet therapy has become the main theoretical basis for the design of new medical dressings. More and more researchers have developed a series of wet wound dressings, such as hydrogel dressings and hydrocolloid dressings. These wet products are more suitable for the special needs of the wound healing process, and can also shorten the wound healing time and improve the treatment effect. The ideal wound dressing should be able to quickly and effectively cover the wound, have excellent mechanical properties, good biosafety, antibacterial and antibacterial functions, and hemostatic effects, thereby promoting wound healing. Existing research, patents or products show that macromolecules with antibacterial and antibacterial functions, promoting wound healing, etc., are combined in various ways to make wet dressings, which can well achieve the treatment and care of trauma, and the market prospects are huge.

[0005] With the development of biotechnology, natural polymer polysaccharides such as chitosan, alginate, fucoidan sulfate, and hyaluronic acid have been gradually applied to the research of hemostasis. They have good biocompatibility, hemostasis, and wound healing ability, and the dressings formed can also be gradually peeled off from the healing site. These characteristics have greatly promoted the progress of the corresponding dressings in the field of hemostasis and wound healing. Chitosan is a natural polymer polysaccharide derived from the ocean, with good biocompatibility, and has been often used as a wound dressing in recent years. However, the hemostatic effect of chitosan itself is limited, and the hemostatic effect on extensive bleeding wounds is not ideal. In clinical applications, it is often used in combination with other hemostatic materials, and its structural characteristics can also be used for chemical modification. For example, carboxymethyl chitosan has better antibacterial activity and hemostatic effect than chitosan. Chitosan quaternary ammonium salt not only inherits the film-forming, antibacterial, flocculating, and biodegradable properties of chitosan, but also has better water solubility, stronger positive charge, and a wider pH range of application. In addition to chitosan, there are many studies on other natural high-molecular polysaccharides for promoting wound healing. Sodium alginate is a natural linear polysaccharide polymer with high safety. Hyaluronic acid has the properties of moisturizing and promoting wound healing, and is often used to make dressings to accelerate wound healing and reduce scar formation. Fucoidan sulfate is a marine sulfated polysaccharide that can accelerate blood coagulation at low concentrations, showing procoagulant activity. Related studies have shown that fucoidan sulfate with procoagulant function can be used as a lead compound for the treatment of hemophilia and the development of new medical hemostatic materials. The above-mentioned marine-derived high-molecular polysaccharides all have good biocompatibility and biosafety.

[0006] At present, there are many literature reports related to hydrogels, but they all have certain defects: the gelation time is long, ranging from several minutes to tens of minutes (Wang H, Huang R, Bai L, et al. Extracellular Matrix-MimeticImmunomodulatory Hydrogel for Accelerating Wound Healing. Adv Healthc Mater. 2023, 12 (27), e2301264; Guo S, Ren Y, Chang R, et al. Injectable Self-Healing Adhesive Chitosan Hydrogel with Antioxidative, Antibacterial, and Hemostatic Activities for Rapid Hemostasis and Skin Wound Healing. ACS Appl MaterInterfaces.2022,14(30):34455-34469, etc.); there are certain limitations in the use method and weak mechanical properties, such as the dressing does not fit the wound, the adhesion is not high, and the ability to absorb wound fluid is limited (Yang Yongyan. Preparation and performance study of multifunctional polymer hydrogel wound dressing [D]. Jilin University, 2024.); the formula is complex, the reagents used are expensive and highly toxic, and the industrialization prospects are low. For example, the commonly used cross-linking agent four-arm polyethylene glycol derivatives (four-arm polyethylene glycol succinimidyl succinate, etc.) are expensive, and the matrix materials used, such as acrylic acid, acrylamide, and cross-linking agent methylene bisacrylamide, are highly toxic (A barnacle biomimetic water-touch adhesion hydrogel and its preparation method and application, 202311079058.5); Existing products, especially domestic products, are mostly targeted at superficial skin injuries, and have poor hemostasis and wound healing performance. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a marine polymer polysaccharide hydrogel wound dressing. The high-performance marine polymer polysaccharide hydrogel dressing provided by the present invention is formed by cross-linking a polysaccharide composition and a cross-linking agent. The coagulation ability of the polysaccharide composition is better than that of Yunnan Baiyao and commercial chitosan hemostatic powder; the hydrogel gelation time is short, about 15s; the pores are uniform and moderate; it has self-healing properties, and can be placed at room temperature and reassembled to form a complete hydrogel without external stimulation, and does not separate under the action of external force; it has adhesion and can adhere to plastics, rubber, metal, glass, skin, and paper; it has high swelling properties, and the swelling rate in 20 minutes can reach about 2000%; it has an inhibitory effect on Escherichia coli and Staphylococcus aureus, and the inhibition rate is higher than 90%; it has no skin irritation and skin allergy, and has high biological safety; it can stop bleeding quickly within 10s, and the amount of bleeding is significantly reduced, and the effect is better than that of commercially available hydrogel dressings (Germany Buchmann Hydrosorb Gel)(P<0.05); it can make the wound shrink and form scabs quickly, which can effectively protect the wound and reduce the risk of infection. At the same time, it has an excellent wound healing effect. The wound healing rate in the early stage of the wound (1 day) can reach 68%, which is much higher than the 29% of the commercially available hydrogel group. After 7 days, the wound healing rate is 98%, which is much higher than the 76% of the commercially available hydrogel group. The wound healing effect is significantly better than the commercially available hydrogel group (P<0.05). Therefore, this hydrogel dressing is a safe and effective high-performance wound dressing with a short gelation time, high fit with the wound, strong adhesion, high swelling, infection prevention, rapid hemostasis, and wound healing.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] The present invention also provides a marine polymer polysaccharide hydrogel wound dressing, which is composed of a polysaccharide composition and a cross-linking agent, and the mass ratio of the two is 1-3:1-2:

[0010] The polysaccharide composition is selected from at least three of modified carboxymethyl chitosan, carboxymethyl chitosan, sodium alginate, fucoidan, chitosan quaternary ammonium salt, and hyaluronic acid.

[0011] Based on this, in order to solve the problems of complex formula, insufficient performance, insufficient and poor safety evaluation, and low industrialization prospects of traditional hydrogels, the present invention provides a marine polymer polysaccharide hydrogel with short gelation time, high fit with wounds, strong adhesion, high swelling ability, infection prevention, rapid hemostasis, and wound healing. It is safe and effective, has a simple formula, and has high industrialization prospects. The hydrogel can be used to prepare wound dressings and medical devices.

[0012] The hydrogel dressing of the present invention is formed by cross-linking a polysaccharide composition and a cross-linking agent. The coagulation ability of the polysaccharide composition is better than that of Yunnan Baiyao and commercial chitosan hemostatic powder. The gelation time of the hydrogel is short, about 15 seconds. The pores are evenly distributed and of moderate size. The hydrogel has self-healing properties. When placed at room temperature, it can be reassembled to form a complete hydrogel without being stimulated by the outside world, and will not separate under the action of external forces. The hydrogel has adhesion properties and can adhere to plastics, rubbers, metals, glass, skins, and paper. The hydrogel has high swelling properties, and the swelling rate can reach about 2000% in 20 minutes. The hydrogel has an inhibitory effect on Escherichia coli and Staphylococcus aureus, and the inhibition rate is higher than 90%. The hydrogel has no skin irritation and skin allergy. The hydrogel can stop bleeding quickly within 10 seconds, and the amount of bleeding is significantly reduced. The effect is better than that of commercially available hydrogel dressings (Hydrosorb from Germany). Gel)(P<0.05); it can make the wound shrink and form scab quickly, and it has excellent wound healing effect. The wound healing rate in the early stage of the wound (1 day) can reach 68%, which is much higher than the 29% of the commercially available hydrogel group. The wound healing rate after 7 days is 98%, which is much higher than the 76% of the commercially available hydrogel group. The wound healing effect is significantly better than the commercially available hydrogel group (P<0.05). It is a safe and effective high-performance wound dressing with short gelation time, high fit with the wound, strong adhesion, high swelling ability, prevention of infection, rapid hemostasis, and promotion of wound healing.

[0013] In some embodiments, the modified carboxymethyl chitosan is used in an amount of 100-400 parts;

[0014] In some embodiments, the carboxymethyl chitosan is used in an amount of 1000-3000 parts;

[0015] In some embodiments, the amount of sodium alginate is 200-1000 parts

[0016] In some embodiments, the amount of fucoidan is 1-5 parts;

[0017] In some embodiments, the amount of chitosan quaternary ammonium salt is 500-2000 parts;

[0018] In some embodiments, the hyaluronic acid is 500-2000 parts.

[0019] Preferably, the polysaccharide composition is composed of the following raw materials in parts by weight: 1000-3000 parts of carboxymethyl chitosan, 200-1000 parts of sodium alginate, and 1-5 parts of fucoidan.

[0020] More preferably, the polysaccharide composition is composed of the following raw materials in parts by weight: 2000 parts of carboxymethyl chitosan, 1000 parts of sodium alginate, and 1 part of fucoidan.

[0021] In some embodiments, the modified carboxymethyl chitosan is a modified carboxymethyl chitosan modified with a catechol group.

[0022] In some embodiments, the preparation method of the modified carboxymethyl chitosan modified with catechol groups includes: fully dissolving the carboxymethyl chitosan, adding an N,N-dimethylformamide solution of 3,4-dihydroxybenzaldehyde under the protection of an inert atmosphere to react, adding a NaBH3CN solution to continue the reaction after the reaction is completed, adding an ethanol solution to the reaction solution after the reaction is completed to obtain a flocculent precipitate, re-dissolving the precipitate with water after centrifugation, dialyzing, and freeze-drying to obtain a modified carboxymethyl chitosan modified with catechol groups.

[0023] In some embodiments, the molecular weights of sodium alginate and fucoidan are both 20 kDa-300 kDa.

[0024] In some embodiments, the cross-linking agent consists of oxidized starch and tannic acid, and the mass ratio of the two is 1-2:2-4.

[0025] In some embodiments, the aldehyde content of the oxidized starch is not less than 60%.

[0026] The second aspect of the present invention also provides a method for preparing a marine polymer polysaccharide hydrogel wound dressing, comprising:

[0027] uniformly mixing at least three of modified carboxymethyl chitosan, carboxymethyl chitosan, sodium alginate, fucoidan, chitosan quaternary ammonium salt, and hyaluronic acid to obtain a polysaccharide composition;

[0028] The oxidized starch and tannic acid are uniformly mixed to obtain a cross-linking agent;

[0029] The polysaccharide composition is reacted with a cross-linking agent to obtain a marine polymer polysaccharide hydrogel wound dressing.

[0030] In some embodiments, the gel time is 10-20 s, or 15 s.

[0031] More specifically, it includes:

[0032] S1. Extracting seaweed polysaccharides from brown algae, including sodium alginate, fucoidan, etc.;

[0033] S2, using carboxymethyl chitosan as a raw material to prepare modified carboxymethyl chitosan modified with catechol groups;

[0034] S3, mixing three or more of modified carboxymethyl chitosan, carboxymethyl chitosan, chitosan quaternary ammonium salt, sodium alginate, hyaluronic acid and fucoidan in a certain proportion to obtain a polysaccharide hemostatic composition, preferably a polysaccharide composition with good coagulation activity;

[0035] S4. Weigh oxidized starch and tannic acid in a mass percentage ratio of 1:2, mix them thoroughly, and prepare a cross-linking agent, wherein the aldehyde content of the oxidized starch is not less than 60%.

[0036] S5. Based on the polysaccharide composition, a cross-linking agent is added, and the mass percentage of the cross-linking agent and the cross-linking agent is 1-3:1-2 to prepare a hydrogel wound dressing;

[0037] The hydrogel dressing prepared by the present invention has a short gelation time of about 15 seconds; the pore distribution is uniform and moderate; it has self-healing property, and can be placed at room temperature to recombine to form a complete hydrogel without being stimulated by the outside world, and will not separate under the action of external force; it has adhesion, and can adhere to plastic, rubber, metal, glass, skin, and paper; it has high swelling property, and the swelling rate can reach about 2000% in 20 minutes; it has an inhibitory effect on Escherichia coli and Staphylococcus aureus, and the inhibition rate is higher than 90%; it has no skin irritation and skin allergy, and has high biological safety; it can quickly stop bleeding within 10 seconds, and the amount of bleeding is significantly reduced, and the effect is significantly better than the commercially available hydrogel dressing (Germany Buchmann Hydrosorb Gel); the wound contraction, scab formation, and wound healing promotion effects are all significantly better than the commercially available hydrogel dressing.

[0038] The third aspect of the present invention also provides the use of the above-mentioned marine polymer polysaccharide hydrogel wound dressing in the preparation of medical materials for hemostasis or promoting wound healing.

[0039] The fourth aspect of the present invention also provides the use of the above-mentioned marine polymer polysaccharide hydrogel wound dressing in the preparation of medical devices.

[0040] The beneficial effects of the present invention are:

[0041] The hydrogel dressing prepared in this project:

[0042] (1) Rapid gelation, forming a hydrogel in about 15 seconds;

[0043] (2) It has strong self-healing properties, which is conducive to filling irregularly shaped wounds and has a high degree of fit with the wound;

[0044] (3) High adhesion, can adhere to plastic, rubber, metal, glass, skin, and paper;

[0045] (4) It has high swelling capacity, strong ability to absorb wound fluid, and is conducive to maintaining a moist environment at the wound site;

[0046] (5) Strong antibacterial properties, with an inhibition rate of over 90% against Escherichia coli and Staphylococcus aureus;

[0047] (6) No skin irritation or skin allergy, high biological safety;

[0048] (7) Rapid hemostasis within 10 seconds, significantly reducing the amount of bleeding, and the effect was significantly better than the commercially available hydrogel dressing (Hydrosorb Gel, Germany) (P < 0.05);

[0049] (8) It can cause the wound surface to shrink and form a scab quickly, which can effectively protect the wound and reduce the risk of infection. Commercially available hydrogel dressings do not have this effect. It also has the effect of promoting wound healing, and the effects are significantly better than commercially available hydrogel dressings (P<0.05).

[0050] (9) Compared with the hydrogel wound dressings in the current literature and patents, the hydrogel of the present application has a simple formula, does not use toxic and harmful reagents, has no skin irritation and allergy, has high biosafety, can quickly stop bleeding and promote wound healing, and its effects are better than those of commercially available hydrogel dressings (Hydrosorb Gel from Germany) (P<0.05). It has a very high prospect for industrialization and is expected to be developed into new wound dressings and medical devices, breaking the dilemma of domestic hydrogel dressings relying mostly on imports. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0052] Figure 1 This is a test chart of the coagulation time of the polysaccharide composition of Example 1 of the present invention;

[0053] Figure 2 This is a comparison diagram of the coagulation effect of the polysaccharide composition of Example 1 of the present invention;

[0054] Figure 3 This is a schematic diagram of the hydrogel preparation and product planning of Example 1 of the present invention;

[0055] Figure 4 This is a scanning electron microscope image of the microstructure of the hydrogel of Example 1 of the present invention;

[0056] Figure 5 This is a test diagram of the hydrogel self-healing performance of Example 1 of the present invention;

[0057] Figure 6 This is a test diagram of the hydrogel adhesion of Example 1 of the present invention;

[0058] Figure 7 This is a test diagram of the swelling performance of the hydrogel in Example 1 of the present invention;

[0059] Figure 8 This is a test diagram of the antibacterial performance of the hydrogel in Example 1 of the present invention;

[0060] Fig. 9The hydrogel skin irritation test and HE staining diagram of Example 1 of the present invention;

[0061] Fig.10 This is a test diagram of the skin allergy of the hydrogel in Example 1 of the present invention;

[0062] Fig.11 The hemostatic performance test diagram of the hydrogel in Example 1 of the present invention; wherein a is a hemostatic performance test diagram, and b is a local enlarged diagram after applying the hydrogel;

[0063] Fig.12 This is a test chart of the hemostatic performance and bleeding volume of the hydrogel in Example 1 of the present invention;

[0064] Fig.13 This is a test diagram of the healing performance of the hydrogel in Example 1 of the present invention. DETAILED DESCRIPTION

[0065] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0066] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0067] In view of the problems of traditional hydrogels having complex formulas, insufficient performance, insufficient and poor safety evaluation, and low industrialization prospects, the present invention provides a safe and effective hydrogel wound dressing based on marine polymer polysaccharides with simple formula, high performance, and strong industrialization prospects.

[0068] A typical embodiment of the present invention provides a marine polymer polysaccharide hydrogel wound dressing, wherein the hydrogel dressing is formed by cross-linking a polysaccharide composition and a cross-linking agent, and the coagulation ability of the polysaccharide composition is better than that of Yunnan Baiyao and commercial chitosan hemostatic powder; the gelation time is short, about 15 seconds; the pore distribution is uniform and moderate; it has self-healing properties, and can be placed at room temperature to form a complete hydrogel again without external stimulation, and will not separate under the action of external force; it has adhesion and can adhere to plastic, rubber, metal, glass, skin, and paper; it has high swelling properties, and the swelling rate can reach about 2000% in 20 minutes; it has an inhibitory effect on Escherichia coli and Staphylococcus aureus, and the inhibition rate is higher than 90%; it has no skin irritation and skin allergy, and has high biological safety; it can quickly stop bleeding within 10 seconds, and the amount of bleeding is significantly reduced; it can cause the wound surface to shrink and scab quickly, and it has the effect of promoting wound healing, and the hemostasis and wound healing effects are significantly better than those of commercially available hydrogel dressings (Germany Buchmann Hydrosorb Gel)(P<0.05) is a high-performance wound dressing with the characteristics of short gelation time, strong adhesion, high fit with the wound, high biosafety, rapid hemostasis, infection prevention, and promotion of wound healing.

[0069] On the other hand, in the preparation method of the above hydrogel wound dressing, the hydrogel includes a polysaccharide composition and a cross-linking agent, and the mass percentage of the two is 1-3:1-2.

[0070] S1. The polysaccharide composition is a mixture of three or more of modified carboxymethyl chitosan, carboxymethyl chitosan, chitosan quaternary ammonium salt, sodium alginate, hyaluronic acid and fucoidan in a certain proportion to optimize the polysaccharide composition with good coagulation activity;

[0071] In some embodiments, the components of the polysaccharide hemostatic composition are mixed in proportion and stirred for 1-2 hours to achieve sufficient mixing between the components of the polysaccharide composition. The polysaccharide composition is obtained after freeze-drying. The coagulation activity of the polysaccharide composition is better than that of commercially available chitosan hemostatic powder and Yunnan Baiyao.

[0072] S2, the preparation method of the modified carboxymethyl chitosan is to fully dissolve the carboxymethyl chitosan, slowly drop 3,4-dihydroxybenzaldehyde N, N-dimethylformamide solution under nitrogen protection, stir at room temperature for 7 hours, add NaBH3CN solution, and continue to react for 7 hours. After the reaction is completed, 4 times ethanol solution is added to the reaction solution to obtain a large amount of flocculent precipitate, and the precipitate is redissolved with deionized water after centrifugation, dialyzed at room temperature for 3d and freeze-dried to obtain modified carboxymethyl chitosan modified with catechol groups.

[0073] S3. The sodium alginate and fucoidan are both extracted from brown algae, and have a molecular weight of 20 kDa-300 kDa.

[0074] S4. The brown algae include kelp, ascophylla, undaria pinnatifida, etc.

[0075] S5. The sodium alginate is extracted by placing brown algae powder in water and heating it for extraction. In one or more embodiments, the heating temperature is 60-70° C., the extraction time is 2-3 hours, and the extraction is repeated at least 2 times, and the supernatant is collected for acid precipitation and alkali dissolution.

[0076] In some examples of this implementation mode, the process of acid precipitation and alkali dissolution is: adding acid to the material solution to adjust the pH to acidic, generating precipitation, collecting the precipitation and then dissolving it with alkaline solution.

[0077] In some embodiments, the pH is adjusted to 0.8-1.2. Under this condition, alginate can be better extracted. The alkaline solution is a sodium carbonate solution, which has a weak alkalinity and can prevent the structure of sodium alginate from being damaged.

[0078] S6. The fucoidan is extracted by placing brown algae powder in a CaCl2 solution and heating for extraction. In one or more embodiments, the heating temperature is 70-80°C, the extraction time is 2-4 hours, and the process is repeated at least 2 times, and the supernatant is collected.

[0079] In some embodiments, the mass percentage of the CaCl2 solution is 1.5%-2%.

[0080] In some embodiments, after the supernatant is filtered, the liquid volume is concentrated to 1 / 10 to 1 / 6 of the original volume, and then alcohol precipitation is performed.

[0081] Furthermore, the solution used for alcohol precipitation is 95% ethanol.

[0082] S7. Weigh oxidized starch and tannic acid in a mass percentage ratio of 1:2, mix them thoroughly, and obtain a cross-linking agent, wherein the aldehyde content of the oxidized starch is not less than 60%.

[0083] S8. Based on the polysaccharide composition, a cross-linking agent is added and mixed to form a hydrogel dressing, and the mass percentage of the two is 1-3:1-2.

[0084] In some embodiments, the polysaccharide composition is weighed according to mass percentage and then water is added to fully dissolve it.

[0085] In some embodiments, the cross-linking agent is weighed by mass percentage and then added with water to fully dissolve it.

[0086] In some embodiments, the polysaccharide composition and the cross-linking agent solution are placed separately and mixed before use to form a hydrogel dressing.

[0087] The present invention also provides applications of the hydrogel in antibacterial, rapid hemostasis, wound healing promoting wound dressings and medical devices.

[0088] Example 1

[0089] A marine polymer polysaccharide hydrogel wound dressing comprises the following steps:

[0090] S1. Preparation of polysaccharide composition

[0091] Preparation of modified carboxymethyl chitosan: In a complex and humid marine environment, mussels still maintain strong adhesion ability, which is mainly attributed to the rich catechol groups in mussel byssus protein. Catechol groups are considered to be the main reason for the adhesion function of mussel byssus protein. At present, many research groups at home and abroad are conducting bionic research on mussel mucin, modifying natural polysaccharides such as chitosan to increase its adhesion.

[0092] The carboxymethyl chitosan was fully dissolved and placed in a round-bottom flask. Under nitrogen protection, 60 mg / mL of 3,4-dihydroxybenzaldehyde in N,N-dimethylformamide solution was slowly added dropwise, stirred at room temperature for 7 hours, and 300 mg / mL of NaBH3CN solution was added, and the reaction continued for 7 hours. After the reaction was completed, 4 times ethanol solution was added to the reaction solution to obtain a large amount of flocculent precipitate. After centrifugation, the precipitate was redissolved with deionized water, dialyzed at room temperature for 3 days, and freeze-dried to obtain modified carboxymethyl chitosan modified with catechol groups, which was stored at -20°C.

[0093] Preparation of sodium alginate: Dry and crush the brown algae to obtain brown algae powder. Add it to water at a solid-liquid ratio of 1:20, stir and extract at 60-70°C for 3 hours, repeat twice, and collect the supernatant.

[0094] The pH of the supernatant was adjusted to 1.0, allowed to stand overnight, centrifuged, and the precipitate was collected.

[0095] The precipitate was redissolved with 2% sodium carbonate solution, the pH was adjusted to 1.0 again, centrifuged, washed with distilled water for several times, dehydrated with anhydrous ethanol and then dried.

[0096] The precipitate was redissolved with 2% sodium carbonate solution, dialyzed (molecular weight cut-off: 3.5 kDa), and the remaining portion was concentrated and freeze-dried to obtain crude sodium alginate.

[0097] The crude sodium alginate is dissolved in water, the pH is adjusted to neutral, ultrafiltration is performed (molecular weight cut-off is 10 kDa), the retained fraction is collected, concentrated and freeze-dried to obtain sodium alginate.

[0098] Preparation of fucoidan: Dry and crush brown algae to obtain brown algae powder, add it to 1.56% CaCl2 solution at a solid-liquid ratio of 1:30, extract at 80°C for 2h, repeat twice, centrifuge, and collect the supernatant;

[0099] The supernatant was concentrated to 1 / 6 to 1 / 10 of the original volume, 4 times the volume of 95% ethanol was added, precipitated, left to stand overnight at 4°C, centrifuged, the precipitate was collected, and dried at 40°C. The sample was redissolved, ultrafiltered (molecular weight cutoff 30000 kDa), concentrated, and spray-dried to obtain fucoidan.

[0100] Preparation of polysaccharide composition: Select modified carboxymethyl chitosan (modified carboxymethyl chitosan modified with catechol groups), carboxymethyl chitosan, sodium alginate, fucoidan, chitosan quaternary ammonium salt, and hyaluronic acid as influencing factors, determine the dosage level, determine the proportion of each influencing factor of the polysaccharide composition by means of orthogonal experiment, and apply the orthogonal experimental design table L 32 (4 9 ), design a six-factor four-level experiment.

[0101] Table 1 Factors and dosage ranges

[0102]

[0103]

[0104] According to the requirements of Table 2, six polysaccharide solutions were mixed and fully stirred, and then freeze-dried to obtain polysaccharide compositions, totaling 32 types.

[0105] Table 2 Orthogonal experimental design

[0106]

[0107]

[0108] Coagulation time determination: Take 100 μL of anticoagulated rabbit blood, add 100 μL (20 mg / mL) of polysaccharide composition solution and 100 μL of calcium chloride solution (preheated at 37°C), start coagulation and start timing, take the centrifuge tube out of the water bath every 10 seconds, and continuously tilt the centrifuge tube in a bright environment to observe the blood flow state. Once the blood coagulation is observed, stop the clock immediately and record the time. Repeat 3 times for each group. Take another 100 μL of anticoagulated rabbit blood, add 100 μL of water and 100 μL of calcium chloride solution (preheated at 37°C), and record the coagulation time as a blank control group. After the coagulation time exceeds 600s, it is no longer recorded and is counted as 600s. Polysaccharide compositions with short coagulation times are preferred, and are compared with Yunnan Baiyao and commercially available chitosan hemostatic powder.

[0109] S2. Preparation of cross-linking agent:

[0110] A certain amount of oxidized starch and tannic acid are weighed, the mass percentage of the two is 1:2, wherein the aldehyde content of the oxidized starch is not less than 60%.

[0111] S3. Preparation of hydrogel dressing:

[0112] The prepared cross-linking agent solution is added to the polysaccharide composition solution in a mass percentage of 1-3:1-2. After being fully mixed, a hydrogel dressing is formed, and the gelation time is recorded. The hydrogel with the best gel state and the shortest gelation time is preferred.

[0113] The following are the test results of the physicochemical properties, biosafety, and bioactivity of hydrogel wound dressings. The physicochemical properties include the gelation time, internal morphology, self-healing properties, adhesion properties, and swelling properties of the hydrogel; the biosafety includes skin irritation and skin allergy tests; and the bioactivity tests include antibacterial, hemostatic, and wound healing activity tests.

[0114] The coagulation time of 32 polysaccharide compositions Figure 1 As shown, polysaccharide composition 10 and polysaccharide composition 14 have the shortest coagulation time, so these two combinations are preferred for subsequent experiments.

[0115] The coagulation effect of the polysaccharide composition is as follows Figure 2 As shown, after being evenly mixed with the blood, the blood in the two polysaccharide composition groups could coagulate and stop flowing in a short time. At the same time, the blood in the Yunnan Baiyao and commercially available chitosan hemostatic powder groups could not coagulate. Therefore, the coagulation activity of the polysaccharide composition was better than that of Yunnan Baiyao and commercially available chitosan hemostatic powder.

[0116] The hydrogels based on polysaccharide composition 10 and polysaccharide composition 14 were named H1 and H2, respectively. The gelation time is shown in Table 3. H1 does not form a gel or the gelation time is generally long, and H2 has a short gelation time. H2-2 with a short gelation time and good gelation state is preferred for subsequent experiments.

[0117] Schematic diagram of hydrogel preparation Figure 3 As shown, the polysaccharide composition and the cross-linking agent can be quickly gelled after being fully mixed. The hydrogel product of the present application can be designed in a double tube form, and the polysaccharide composition and the cross-linking agent in the tube are fully mixed through the transfer interface and then applied to the wound.

[0118] Table 3 Statistics of gelation time of a series of hydrogels

[0119]

[0120] The internal morphology of the hydrogel is Figure 4 As shown, usually there are certain pores inside the hydrogel. For biomedical materials used for wound healing, larger pores can ensure sufficient oxygen to enter, which is beneficial to wound healing, but too large pores often lead to loose hydrogel structure. The pores of the hydrogel dressing obtained in the present application are evenly distributed and of moderate size, which can ensure sufficient oxygen to enter, which is beneficial to wound healing without causing the hydrogel structure to loosen.

[0121] The self-healing properties of hydrogels Figure 5 As shown, heart-shaped hydrogels of different colors were spliced ​​together and placed at room temperature for 1 hour. It was found that the two could be recombined to form a complete hydrogel without being stimulated by external factors. By observing the gel interface, it can be seen that the hydrogel has been re-cross-linked into a whole and does not separate under the action of external force, indicating that the hydrogel has good self-healing properties. The self-healing property can enable the hydrogel to effectively fill irregular shaped wounds.

[0122] Adhesion is one of the important indicators of hydrogel wound dressings. The hydrogel obtained in this application can adhere to plastic, rubber, metal, glass, skin and paper with good adhesion ( Figure 6 ).

[0123] When the hydrogel is immersed in a solution, it will continue to swell over time until the swelling reaches equilibrium, which is an important feature of the gel. The hydrogel dressing obtained in this application has good water absorption capacity, with a swelling rate of about 2000% in 20 minutes and a swelling equilibrium in about 1 hour. The swelling performance reflects the water content and water retention capacity of the hydrogel. The hydrogel with a high swelling degree can absorb the sore fluid, and is also conducive to maintaining a moist environment at the wound site and promoting wound healing ( Figure 7 ).

[0124] Infection is a common complication of war trauma, which can cause a large number of casualties to become ill and die. Escherichia coli and Staphylococcus aureus are two common pathogens that can cause wound infection in clinical practice. The hydrogel dressing obtained in this application has a high resistance to Escherichia coli (1×10 6 CFU / mL) and Staphylococcus aureus (1×10 6 CFU / mL) had an inhibitory effect, with inhibition rates of 92.37% and 95.01% respectively, and the antibacterial effect was strong ( Figure 8 ).

[0125] Biosafety is an important indicator for evaluating hydrogel dressings and is also a basic indicator for the future industrialization of hydrogel dressings. At present, there are many patents and literature reports related to hydrogels, but some of them have problems such as high toxicity of the reagents used and insufficient safety evaluation. This application conducted skin irritation and skin allergy tests to evaluate the biosafety of hydrogels.

[0126] Skin irritation evaluation:

[0127] Twenty healthy male Kunming (KM) mice aged 4-5 weeks were fed adaptively for 7 days and randomly divided into two groups, a hydrogel group and a commercially available hydrogel group (Hydrosorb Gel, Germany), with 10 mice in each group. 24 hours before the experiment, the hair on both sides of the spine of the back of the mice was shaved and depilated with 6% sodium sulfide, 3*3cm on each side. The integrity of the depilated skin was checked before administration, and those with damage were not included.

[0128] Administration: Before administration, the subjects were anesthetized and the hydrogel was directly applied to the hairless skin on the left side using the self-comparison method. The right side was applied with warm water as a blank control. After 6 hours of application, the test substance was removed and the administration site was cleaned with warm water. The administration was repeated once a day for 14 consecutive days.

[0129] Observation index: Skin irritation was evaluated according to the skin irritation reaction scoring standards in the "Technical Guidelines for Drug Irritation, Allergy and Hemolysis Research" (Table 4-Table 5). Observe the application site for erythema and edema with the naked eye 1 hour after each drug removal, 1, 24, 48, and 72 hours after the last drug administration, and observe whether there is pigmentation, bleeding spots, rough skin or thin skin at the application site, as well as the time of occurrence and disappearance, and score erythema and edema. According to the scoring results, calculate the mean irritation score for each mouse every day during the observation period.

[0130] The skin irritation reaction and irritation intensity were scored according to the table. The average skin irritation reaction value = (total score of erythema formation + total score of edema formation) / total number of animals.

[0131] After observing the reaction for 72 hours, the mice were killed, the skin and subcutaneous tissue at the administration site were cut off, fixed, dehydrated, transparent and wax-impregnated, and routine paraffin-embedded sections were performed. The tissue pathological changes were observed after HE staining.

[0132] Table 4 Skin irritation response scoring criteria

[0133]

[0134]

[0135] Table 5 Evaluation criteria for skin irritation intensity

[0136]

[0137] The results showed that during the administration period and the recovery observation period, no irritation symptoms such as erythema and edema occurred on the administration side and the control side of the hydrogel group in KM mice. The skin irritation score was 0, the skin irritation intensity was non-irritating, and there was no pathological changes such as epidermal necrosis and inflammatory infiltration ( Fig. 9 ), while the commercially available hydrogel group showed mild irritation in the early stage of administration (Tables 6-7).

[0138] Table 6 Skin irritation scores of mice after administration ( n=10)

[0139]

[0140]

[0141] Table 7 Skin irritation intensity of mice after multiple administration

[0142]

[0143] Skin allergy evaluation:

[0144] Forty male Kunming (KM) mice aged 4-5 weeks were randomly divided into 4 groups after 7 days of adaptive feeding, namely, blank group (warm water), positive group (2-4 dinitrochlorobenzene, sensitization 1.0% 0.2ml / mouse, stimulation 0.1% 0.2ml / mouse), commercial hydrogel group (Hydrosorb Gel, Germany) and hydrogel group, with 10 mice in each group. 24 hours before sensitization, the hair on both sides of the back of the mice was removed, and 6% sodium sulfide was used for hair removal, with an area of ​​about 3cm×3cm. Hydrogel, commercial hydrogel, warm water, and 1.0% 2-4 dinitrochlorobenzene were applied to the hairless skin on the left side, maintained for 6 hours, and then washed with warm water; the right side was depilated 24 hours before stimulation.

[0145] Sensitization administration: The corresponding drugs were applied to the shaved area on the left side of the mice on days 0, 7, and 14, respectively. Six hours after administration, the test substances were removed and the administration site was cleaned with warm water.

[0146] Provocative administration: On the 14th day after the last sensitization, apply the corresponding drug to the shaved area on the right side for provocative contact, maintain for 6 hours and then wash with warm water, and observe the mouse skin erythema, edema and other allergic reactions at 1, 24, 48 and 72 hours. The skin allergy evaluation was carried out according to the skin allergy reaction scoring standard and evaluation standard in the "Technical Guidelines for Drug Irritation, Allergy and Hemolysis Research" (Table 8).

[0147] Calculate the average value and sensitization rate of skin allergic reaction. Average value of skin allergic reaction = (total score of erythema formation + total score of edema formation) / total number of animals, sensitization rate = number of animals with positive skin allergic reaction (regardless of the severity) / total number of tested animals, sensitization rate: 0-10%, no sensitization; 11%-30%, mild sensitization; 31%-60%, moderate sensitization; 61%-80%, high sensitization; 81%-100%, extreme sensitization.

[0148] Table 8 Scoring criteria for skin allergic reactions

[0149]

[0150] The results of skin allergic reactions at 1, 24, 48, and 72 hours after stimulation are shown in Table 9. No erythema or edema was observed on the skin of mice at the administration site in the control group and the administration groups (hydrogel group and commercially available hydrogel group). Fig. 9 ), the score was 0, and no systemic allergic reactions such as asthma and unstable standing were observed. The sensitization rate was 0, and the sensitization rate of the positive control group was 100%.

[0151] Table 9 Mouse skin allergic reaction scores (n=10)

[0152]

[0153]

[0154] In the skin irritation test, the average skin irritation reaction on the administration side of the hydrogel group was 0, and there were no abnormal manifestations such as erythema and edema on the administration side, nor were there pathological changes such as epidermal necrosis and inflammatory infiltration. The commercially available hydrogel group showed mild skin irritation in the early stage of administration. Therefore, it is believed that the hydrogel has no irritating reaction to the mouse skin and is superior to the commercially available hydrogel group.

[0155] In the skin allergy test, after three sensitizations, no abnormal manifestations such as erythema and edema were observed in the hydrogel group, the commercial hydrogel group and the control group. During the stimulation phase, no abnormal manifestations such as erythema and edema, as well as severe systemic allergic reactions such as asthma, unsteady standing and shock were observed at the administration site of the hydrogel group, the commercial hydrogel group and the control group. The sensitization rate was 0, and the average value of skin allergic reaction was 0. Therefore, it is believed that the hydrogel dressing and the commercial hydrogel dressing have no allergic reaction to the mouse skin.

[0156] In summary, the hydrogel obtained in the present application has no skin irritation and no allergy, while commercially available hydrogels have mild skin irritation and no allergy in the early stage of administration. Therefore, the hydrogel obtained in the present application is safer than commercially available hydrogels.

[0157] The first hour of rescue after trauma is the "golden hour" that matters life and death. During this hour, rapid hemostasis of the wound is the key to saving the wounded's life. Rapid and effective hemostasis can improve the survival rate and reduce complications of the wounded. This application uses a mouse liver puncture model to evaluate the hemostatic properties of a hydrogel dressing. The results showed that the hydrogel wound dressing can quickly stop bleeding within 10 seconds, significantly reduce the amount of bleeding, and is more effective than commercially available hydrogel dressings ( Figure 11-Figure 12 The rapid hemostatic performance of the hydrogel is not only due to the good coagulation ability of the polysaccharide composition, but also related to its excellent adhesion. Fig.11 As can be seen in b, after the hydrogel is applied, it can adhere to the mouse liver, while the commercially available hydrogel will slide off the surface of the liver, resulting in reduced hemostatic effect.

[0158] The skin is the largest, most exposed, and most fragile organ in the human body. Once damaged, it often needs to undergo a long self-repair process. Severe skin trauma, especially full-thickness skin defect injuries, will form chronic wounds if not treated and are difficult to heal. This application uses a full-thickness skin defect model to evaluate the effect of a series of hydrogels on promoting wound healing. Wound repair and healing usually require two main processes: rapid coagulation and scab formation to seal the wound, followed by tissue regeneration to heal the wound. The hydrogel obtained in this application can cause the wound surface to shrink rapidly and form a scab (the scab is hard to the touch and has no sense of fluctuation), which can quickly and effectively protect the wound and reduce the risk of infection ( Fig.13 ), while the commercial hydrogel group had no such effect; it has excellent wound healing effect. The wound healing rate in the early stage of the wound (1 day) was 68%, which was much higher than the 29% of the commercial hydrogel group. After 7 days, the wound healing rate could reach 98%, which was much higher than the 76% of the commercial hydrogel group. The wound healing effect was significantly better than that of the commercial hydrogel group (P<0.05)( Fig.13 ).

[0159] Example 2

[0160] The difference from Example 1 is that the polysaccharide composition is composed of the following raw materials in parts by weight: 1000 parts of carboxymethyl chitosan, 200 parts of sodium alginate, and 1 part of fucoidan.

[0161] Example 3

[0162] The difference from Example 1 is that the polysaccharide composition is composed of the following raw materials in parts by weight: 3000 parts of carboxymethyl chitosan, 1000 parts of sodium alginate, and 5 parts of fucoidan.

[0163] The performance of the medical dressings prepared in Examples 2 and 3 was tested by the same method as in Example 1. The results showed that the medical dressings prepared in Examples 2 and 3 also have the advantages of strong self-healing ability, high adhesion, high swelling ability, strong antibacterial performance, no skin irritation and allergy, high biosafety, rapid hemostasis, rapid contraction and scab formation on the wound surface, and promoting wound healing.

[0164] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A marine polymer polysaccharide hydrogel wound dressing, characterized in that: It is composed of a polysaccharide composition and a cross-linking agent, and the mass ratio of the two is 1-3:1-2: The polysaccharide composition is selected from at least three of modified carboxymethyl chitosan, carboxymethyl chitosan, sodium alginate, fucoidan, chitosan quaternary ammonium salt and hyaluronic acid.

2. The marine polymer polysaccharide hydrogel wound dressing according to claim 1, characterized in that: The modified carboxymethyl chitosan is used in an amount of 100-400 parts; Or, the amount of the carboxymethyl chitosan is 1000-3000 parts; Or, the amount of sodium alginate is 200-1000 parts Or, the amount of fucoidan is 1-5 parts; Or, the dosage of chitosan quaternary ammonium salt is 500-2000 parts; Or, 500-2000 parts of hyaluronic acid.

3. The marine polymer polysaccharide hydrogel wound dressing according to claim 1, characterized in that: The polysaccharide composition is composed of the following raw materials in parts by weight: 1000-3000 parts of carboxymethyl chitosan, 200-1000 parts of sodium alginate, and 1-5 parts of fucoidan.

4. The marine polymer polysaccharide hydrogel wound dressing according to claim 1, characterized in that: The polysaccharide composition is composed of the following raw materials in parts by weight: 2000 parts of carboxymethyl chitosan, 1000 parts of sodium alginate and 1 part of fucoidan.

5. The marine polymer polysaccharide hydrogel wound dressing according to claim 1, characterized in that: The modified carboxymethyl chitosan is a modified carboxymethyl chitosan modified with a catechol group; Alternatively, the molecular weights of sodium alginate and fucoidan are both 20 kDa-300 kDa.

6. The marine polymer polysaccharide hydrogel wound dressing according to claim 1, characterized in that: The cross-linking agent consists of oxidized starch and tannic acid, and the mass ratio of the two is 1-2:2-4.

7. The marine polymer polysaccharide hydrogel wound dressing according to claim 1, characterized in that: The aldehyde content of the oxidized starch is not less than 60%.

8. A method for preparing a marine polymer polysaccharide hydrogel wound dressing, characterized in that: include: uniformly mixing at least three of modified carboxymethyl chitosan, carboxymethyl chitosan, sodium alginate, fucoidan, chitosan quaternary ammonium salt, and hyaluronic acid to obtain a polysaccharide composition; The oxidized starch and tannic acid are uniformly mixed to obtain a cross-linking agent; The polysaccharide composition is reacted with a cross-linking agent to obtain a marine polymer polysaccharide hydrogel wound dressing.

9. Use of the marine polymer polysaccharide hydrogel wound dressing according to any one of claims 1 to 7 in the preparation of medical materials for hemostasis or promoting wound healing.

10. Use of the marine polymer polysaccharide hydrogel wound dressing according to any one of claims 1 to 7 in the preparation of medical devices.

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