Composition for composite antibacterial gel, composite antibacterial gel prepared from composition and application of composite antibacterial gel

Abstract

The invention discloses a composition for composite antibacterial gel, the composite antibacterial gel prepared from the composition and application of the composite antibacterial gel. The composition for the composite antibacterial gel comprises carboxymethyl chitosan, natural polyphenol, metal chloride, sodium bicarbonate / ammonia water / formaldehyde and water. Wherein the mass ratio of the carboxymethyl chitosan to the natural polyphenols to the metal chloride to the sodium bicarbonate is (0.3-1.5): 1: (10-30): (8-40); the metal chloride is selected from at least one of CuCl2, MnCl2 or CeCl3. According to the method, carboxymethyl chitosan and tannic acid are combined through the hydrogen bond combination effect, meanwhile, metal ions and tannic acid are subjected to coordination combination, after a reducing agent is added, the metal hydroxide particle-loaded antibacterial repairing composite antibacterial gel is formed, and the preparation process is simple, convenient, green and easy to implement.

Description

Technical Field

[0001] The invention relates to the technical field of medical materials, and in particular to a composite antibacterial gel composition and the composite antibacterial gel prepared therefrom and its application. Background Art

[0002] Skin tissue is the largest organ of the human body and the first line of defense of the human body. Its structure and function can protect the human body from injury and invasion of microorganisms. However, in a complex external environment, the skin is prone to a series of injuries, such as surgical wounds, burns, abrasions, etc. During the injury process, the skin will produce open wounds, and its epidermis, dermis and subcutaneous tissue will be damaged to varying degrees. Wound repair involves many complex factors such as inflammation, epithelial cell proliferation, and vascular remodeling. Most wounds can heal slowly under the action of immunity and proliferation of skin cells. However, due to infection with drug-resistant Staphylococcus aureus or Escherichia coli, the self-repair ability of the wound is greatly damaged, which may cause serious bacterial infectious diseases, especially chronic wounds or bacteremia. Therefore, it is particularly important to develop gel dressings with high safety, excellent antibacterial properties, and wound healing.

[0003] Hydrogels are considered to be the most ideal wound healing dressings because they have a highly humid microenvironment and extracellular matrix-like properties that can protect wounds from bacteria and microorganisms. Due to their non-toxicity, high bioactivity and degradability, natural polymer materials are increasingly attracting attention for wound gel dressings. Chitin is a polysaccharide extracted from the shells of marine crustaceans and is a natural antibacterial agent, but chitin is difficult to dissolve in water. Carboxymethyl chitosan is a derivative of chitin after acetylation and methylation. It contains rich carboxyl and amino groups, which greatly improves its solubility while retaining its degradability and antibacterial properties. Therefore, carboxymethyl chitosan is widely used in the fields of biomedicine and biological tissue engineering. In addition, natural polyphenols have anti-cancer, antioxidant, antibacterial and antiviral effects. In addition to having multiple biological functions, natural polyphenols also have unique physical and chemical properties and can bind to a variety of substances through hydrogen bonds, hydrophobic interactions, π-π interactions and cationic coordination. Based on these characteristics, natural polyphenols are widely used in the manufacture of biomaterials, such as nanocapsules and nanogels for the delivery of various cargo molecules. The preparation of chitosan derivatives and natural polyphenols has important application prospects in the field of wound antibacterial and repair.

[0004] In addition, copper ions react chemically under aerobic conditions to produce hydroxyl radicals and active oxygen ions, which have strong redox effects and can destroy the reproduction ability of microbial cells, thereby inhibiting and killing microorganisms. It is reported that copper antibacterial liquid can kill 650 types of bacteria within 5 minutes, with a bactericidal rate of 99.99%, including Staphylococcus aureus, Escherichia coli, intestinal pathogens, pyogenic cocci, pathogenic yeasts, common bacteria in hospital infections, Candida albicans, etc. From a biological point of view, copper, as an essential trace element for the human body, not only has a good antibacterial effect, but copper ions in the body can combine with proteases to play an anti-inflammatory and angiogenic role in wound repair. Therefore, how to combine with hydrogel to achieve the loading and slow release of copper ions is another problem that needs to be solved. Summary of the invention

[0005] In view of the above-mentioned problems, the present invention aims to expand the application of carboxymethyl chitosan and metal-based materials in the field of wound repair technology, and provides a composition for composite antibacterial gel, and a composite antibacterial gel prepared by the same and its application for inhibiting bacteria in infected wounds and promoting wound healing. The antibacterial gel selects carboxymethyl chitosan, natural polyphenols and metal ions as antibacterial agents, and forms a carboxymethyl chitosan-tannic acid-metal hydroxide particle composite antibacterial gel in situ by introducing a weak reducing agent (such as sodium bicarbonate), which plays a role in highly efficient bacterial killing and promoting rapid wound healing.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A composite antibacterial gel composition comprising carboxymethyl chitosan, natural polyphenols, metal chloride, a reducing agent and water;

[0008] Wherein, the mass ratio of carboxymethyl chitosan, natural polyphenols, metal chloride and reducing agent is (0.3-1.5):1:(10-30):(8-40);

[0009] The metal chloride is selected from at least one of CuCl2, MnCl2 or CeCl3.

[0010] According to an embodiment of the present invention, the concentration of the metal chloride in water is 50-200 μg / mL.

[0011] According to an embodiment of the present invention, the concentration of the carboxymethyl chitosan in water is 1-5% w / v, for example, 1% w / v, 2% w / v, 3% w / v, 4% w / v or 5% w / v.

[0012] According to an embodiment of the present invention, the concentration of the natural polyphenols in water is 1-5% w / v, for example, 1% w / v, 2% w / v, 3% w / v, 4% w / v or 5% w / v.

[0013] According to an embodiment of the present invention, the reducing agent is selected from at least one of sodium bicarbonate, ammonia water and formaldehyde, preferably sodium bicarbonate.

[0014] According to an embodiment of the present invention, the concentration of the reducing agent in water is 100-300 μg / mL.

[0015] According to an embodiment of the present invention, the natural polyphenol is at least one of tannic acid, tea polyphenol and grape polyphenol.

[0016] The present invention also provides a composite antibacterial gel, which is obtained by gelling the composite antibacterial gel composition.

[0017] According to an embodiment of the present invention, the composite antibacterial gel comprises carboxymethyl chitosan, natural polyphenols and metal hydroxide particles;

[0018] The metal hydroxide particles are Cu(OH)2, Mn(OH)2 or Ce(OH)3.

[0019] In the present invention, carboxymethyl chitosan forms hydrogen bonds with natural polyphenols (such as tannic acid), and metal ions (i.e., Cu 2+ , Mn 2+ or Ce 3+ ) coordinates with the phenolic hydroxyl groups of natural polyphenols (such as tannic acid) and forms metal hydroxide particles in situ in the gel in the presence of a reducing agent.

[0020] According to an embodiment of the present invention, the metal hydroxide particles are generated by reacting metal ions with a reducing agent.

[0021] According to an embodiment of the present invention, the size of the metal hydroxide particles is 100 nm to 1000 nm.

[0022] According to the embodiment of the present invention, the composite antibacterial gel can inhibit drug-resistant S. aureus and / or E. coli wound infection.

[0023] The present invention also provides a method for preparing the composite antibacterial gel, which comprises:

[0024] The composite antibacterial gel is prepared by mixing carboxymethyl chitosan, natural polyphenol, metal chloride, a reducing agent and water.

[0025] According to an embodiment of the present invention, the method is: carboxymethyl chitosan, natural polyphenols, metal chloride and water are mixed, and then a reducing agent is added and mixed to obtain a composite antibacterial gel.

[0026] According to an embodiment of the present invention, the concentration of the metal chloride in water is 50-200 μg / mL.

[0027] According to an embodiment of the present invention, the concentration of the carboxymethyl chitosan in water is 1-5% w / v, for example, 1% w / v, 2% w / v, 3% w / v, 4% w / v or 5% w / v.

[0028] According to an embodiment of the present invention, the concentration of the natural polyphenols in water is 1-5% w / v, for example, 1% w / v, 2% w / v, 3% w / v, 4% w / v or 5% w / v.

[0029] According to an embodiment of the present invention, the concentration of the reducing agent in water is 100-300 μg / mL.

[0030] According to an embodiment of the present invention, the carboxymethyl chitosan can be prepared by deacetylation and carboxymethylation of chitin.

[0031] Preferably, deacetylation may be performed first, and then carboxymethylation may be performed; deacetylation and carboxymethylation may also be performed simultaneously.

[0032] According to an embodiment of the present invention, the mixing temperature is 30-60° C., such as 45° C.; and the mixing time is 1-60 min.

[0033] According to an embodiment of the present invention, the carboxymethyl chitosan, natural polyphenols, metal chloride and water are mixed for 1-60 minutes; and the mixing time after adding the reducing agent is 10 seconds to 30 seconds.

[0034] According to an embodiment of the present invention, the stirring rate during mixing is 100-1000 rpm / min, for example 400 rpm / min.

[0035] According to an embodiment of the present invention, the composite antibacterial gel is stored at 0 to -10°C, for example -4°C.

[0036] According to an embodiment of the present invention, the method further comprises a post-treatment step: washing the prepared product by centrifugation at a washing rate of 2000-4000 rpm / min, for example 3000 rpm / min.

[0037] As an exemplary embodiment of the present invention, the preparation method of the composite antibacterial gel specifically comprises:

[0038] A 50-200 μg / mL CuCl2 aqueous solution, a 1-5% w / v carboxymethyl chitosan aqueous solution, a 1-5% w / v tannic acid aqueous solution, and a 100-300 μg / mL NaHCO3 aqueous solution are prepared;

[0039] 0.5 mL, 1-5% w / v carboxymethyl chitosan aqueous solution, 0.5 mL, 1-5% w / v tannic acid aqueous solution, and 0.1 mL, 50-200 μg / mL CuCl2 aqueous solution are continuously and vigorously stirred at a speed of 300-600 r / min in an oil bath and mixed evenly; after half an hour, 0.1 mL, 100-300 μg / mL NaHCO3 aqueous solution is added and stirred for 10-30 seconds to obtain the composite antibacterial gel.

[0040] The present invention also provides application of the composite antibacterial gel in preparing antibacterial products for skin wounds.

[0041] Beneficial effects of the present invention:

[0042] (1) The method of the present invention uses carboxymethyl chitosan and tannic acid to combine through hydrogen bonding, and at the same time, metal ions are coordinated with tannic acid. After adding a reducing agent (such as sodium bicarbonate), a composite antibacterial gel loaded with metal hydroxide particles for antibacterial repair is formed. The preparation process is simple, green and easy.

[0043] (2) The composite antibacterial gel of the present invention has a triple antibacterial effect of metal ions, carboxymethyl chitosan and tannic acid, and has a killing effect on Gram-negative, Gram-positive and drug-resistant bacteria, and has a broad-spectrum and efficient bactericidal performance.

[0044] (3) The metal ion loading in the composite antibacterial gel of the present invention can achieve keratinocyte migration and angiogenesis in wounds infected with drug-resistant strains.

[0045] (4) The present invention uses carboxymethyl chitosan and copper ions or manganese ions or cerium ions, and does not contain any drug components or any toxic side effects, thereby greatly improving the safety of the organism.

[0046] (5) The hydrogel drug delivery method is easier to apply / spray on the wound, which improves drug delivery efficiency and reduces the number of dressing changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 , wherein A is a transmission electron micrograph of carboxymethyl chitosan-tannic acid (CMC / TA) prepared in Example 1 of the present invention at different reaction times;

[0048] B is the element distribution diagram of CMC / TA / Cu prepared in Example 1 of the present invention;

[0049] C is a transmission electron micrograph of methyl chitosan-tannic acid-copper hydroxide (CMC / TA / Cu) prepared in Example 1 at different reaction times;

[0050] D is the strain amplitude scanning curve of CMC / TA / Cu prepared in Example 1;

[0051] E is the expansion ratio of CMC / TA / Cu and CMC / TA prepared in Example 1 of the present invention;

[0052] F is the release curve of copper ions and tannic acid when the CMC / TA / Cu prepared in Example 1 of the present invention is immersed in a weak acid solution.

[0053] Figure 2 In the figure, A and D are the fluorescence photos and fluorescence quantitative analysis diagrams of the effects of CMC / TA and CMC / TA / Cu on keratinocyte proliferation in Example 2 of the present invention;

[0054] B is a graph showing the results of toxicological experiments of CMC / TA / Cu at different mass concentrations on keratinocytes and endothelial cells in Example 2 of the present invention;

[0055] C and E are the changes of CMC / TA / Cu and CMC / TA on keratinocyte scratches in Example 2 of the present invention and the quantitative analysis of the scratch size;

[0056] F and G are the quantitative analysis diagrams of the in vitro angiogenesis experiment of endothelial cells and tubule connection of CMC / TA / Cu and CMC / TA in Example 2 of the present invention.

[0057] Figure 3 In the figure, A is a scanning electron microscope image of MRSA after MRSA was incubated with CMC / TA / Cu or PBS for 4 hours in Example 3 of the present invention;

[0058] B is a graph showing the inverted plate results of wound colonies after applying CMC / TA / Cu and CMC / TA to the wound surface infected with MRSA in Example 4 of the present invention, 1 day, 8 days, and 12 days;

[0059] C is a graph showing the pathological sections of the heart, liver, spleen, lung and kidney of mice 12 days after smearing CMC / TA / Cu and CMC / TA on the wound surface infected with MRSA in Example 4 of the present invention, and the normal group of mice was used as a control.

[0060] Figure 4 AB is the regional trace diagram of wound healing and wound closure when CMC / TA / Cu gel or PBS is applied on the surface of a living MRSA-infected wound in Example 5 of the present invention;

[0061] C is a diagram showing the wound healing efficiency of applying CMC / TA / Cu gel or PBS on the surface of a living MRSA-infected wound in Example 5 of the present invention;

[0062] D is the weight change of mice in different groups in Example 5 of the present invention;

[0063] EI is H&E staining-cross section, H&E staining-longitudinal section (number of new hair follicles), CD86+ immunostaining, Mason staining and Gissam staining of the mouse wound after 12 days of CMC / TA / Cu gel or PBS treatment in Example 5 of the present invention.

[0064] Among them, "CMC" is carboxymethyl chitosan; "TA" is tannic acid; "Cu" is Cu(OH)2 particles; "CMC / TA / Cu" is carboxymethyl chitosan-tannic acid-copper hydroxide gel; "CMC / TA" is carboxymethyl chitosan-tannic acid gel.

[0065] Figure 5 The present invention is a reaction flow chart of the composite antibacterial gel. DETAILED DESCRIPTION

[0066] Carboxymethyl chitosan can be prepared by sequential deacetylation, carboxymethylation or simultaneous deacetylation and carboxymethylation of chitin.

[0067] The preparation method of the above-mentioned carboxymethyl chitosan aqueous solution is as follows:

[0068] First, chitosan powder (4% w / v) is added to a solution of KOH (15% w / v), and the mixture is frozen at -30°C for 6 hours. After the sample is thawed, it is filtered and centrifuged to remove the undissolved chitosan, thereby obtaining a chitosan solution dissolved in a strong base. Then the chitosan-strong base solution is placed in an ice bath at 0°C, and KOH powder is continued to be added to the reaction system so that the KOH concentration in the reaction system reaches more than 41% w / v. Then, the reaction is kept in an oil bath at a temperature of 100°C for 2 to 6 hours for subsequent deacetylation. Then, chloroacetic acid (20% v / v) is added to the reaction solution, and then kept in a microwave (600 to 800W) for 4 to 8 hours for subsequent carboxymethylation. Or after the deacetylation reaction is completed, centrifugal washing and drying are performed, and the sample is redispersed in 41% w / v of KOH, and a subsequent carboxymethylation reaction is performed by 20% v / v chloroacetic acid.

[0069] Preferably, in the preparation of the chitosan derivative in step 1, deacetylation and carboxymethylation can be performed without the need for centrifugation, washing, drying and other steps after deacetylation.

[0070] Preferably, the deacetylation reaction in step 1 is maintained in an oil bath at 100° C. for 4 hours.

[0071] Preferably, the retention time of the carboxymethylation reaction in step 1 in a microwave apparatus is 6 hours.

[0072] Preferably, in step 1, the power of the microwave instrument should be 700W.

[0073] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0074] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0075] Example 1

[0076] A method for preparing a composite antibacterial gel, the method specifically comprising:

[0077] (1) Preparation of carboxymethyl chitosan: Chitosan powder (4% w / v) was added to a solution of KOH (15% w / v), and the mixture was frozen at -30°C for 6 hours. After the sample was thawed, it was filtered and centrifuged to remove the undissolved chitosan, thereby obtaining a chitosan solution dissolved in a strong base. The chitosan-strong base solution was then placed in an ice bath at 0°C, and KOH powder was continued to be added to the reaction system so that the KOH concentration in the reaction system reached 41% w / v or more. Then, it was kept in an oil bath at a temperature of 100°C for 4 hours for subsequent deacetylation reaction. Chloroacetic acid (20% v / v) was added to the reaction solution, and then it was kept in a microwave (700W) for 6 hours for subsequent carboxymethylation reaction. Finally, ultrapure water with a volume twice that of the total liquid was added to the reaction system, and after adjusting the pH to 7.0 with glacial acetic acid, it was filtered with a Buchner funnel and then centrifuged and washed three times with ultrapure water (10,000 rpm / min). Finally, the sample was washed with anhydrous ethanol to replace the water molecules in the carboxymethyl chitosan, and the final sample was dried in an oven at 65°C overnight to obtain carboxymethyl chitosan, which was used for the subsequent preparation of gel.

[0078] (2) Preparation of composite antibacterial gel (i.e., carboxymethyl chitosan-tannic acid-copper hydroxide gel, CMC / TA / Cu): 0.1 mL of 3% w / v tannic acid was added with 0.25 mL of 5% w / v carboxymethyl chitosan at room temperature. Then, 0.05 mL of 100 μg / mL CuCl2 aqueous solution was added to the mixture, and after stirring (400 rpm / min) on a 45°C oil bath for half an hour, 0.05 mL of 100 μg / mL NaHCO3 aqueous solution was added and reacted for 15 seconds to obtain composite antibacterial gel CMC / TA / Cu.

[0079] Preparation of antibacterial gel CMC / TA (i.e., carboxymethyl chitosan-tannic acid gel): 0.1 mL of 3% w / v tannic acid was added with 0.25 mL of 3% w / v carboxymethyl chitosan at room temperature, and the mixture was stirred (400 rpm / min) in a 45°C oil bath for half an hour to obtain the antibacterial gel CMC / TA.

[0080] Characterization of CMC / TA / Cu composite antibacterial gel and antibacterial gel CMC / TA:

[0081] Characterization of dynamic rheological properties: The dynamic rheological properties were characterized by a rotational rheometer (Malvern Kinexus Pro + , UK), the specific process is as follows: the gel is prepared into a cube with a size of 20mm×20mm×20mm. In order to carry out viscoelastic area measurement, the strain amplitude sweep is set from 0.01% to 500% at 1Hz.

[0082] Regarding the determination of the swelling coefficient, the mass of the gel (W0) was first measured, and then it was placed in a small glass bottle containing 5 mL of PBS and the mass of the gel (W t ), so the swelling coefficient can be calculated according to: Swelling coefficient = (W0-W t ) / W0. At the same time, the gel was placed in a small glass bottle containing 5 mL of PBS buffer, pH = 5.5 (main components of Na2HPO4, KH2PO4, NaCl and KCl), and the copper ions and tannic acid released in the solution were measured at different times at room temperature.

[0083] Result analysis:

[0084] Figure 1 A and C are transmission electron micrographs of carboxymethyl chitosan-tannic acid (CMC / TA) and methyl chitosan-tannic acid-copper hydroxide (CMC / TA / Cu) prepared in Example 1 at different reaction times, wherein the reaction time of Ai and Ci is 10 s, and the reaction time of Aii and Aii is 30 s; Figure 1 It can be seen from A and C that compared with CMC / TA, the presence of Cu(OH)2 can be clearly found in CMC / TA / Cu, and the size of Cu(OH)2 is 100nm~1000nm.

[0085] Figure 1 B is the element distribution diagram of CMC / TA / Cu prepared in Example 1, wherein i refers to the high-resolution transmission-scanning electron microscopy diagram of the gel, ii refers to the Cu element distribution diagram, iii is the O element distribution diagram, and iv refers to the superposition diagram of the Cu and O element distributions; Figure 1B It can be seen that the main elements observed in the CMC / TA / Cu composite antibacterial gel are Cu and O, which proves the in-situ generation of Cu(OH)2.

[0086] Figure 1 D is the strain amplitude scanning curve of CMC / TA / Cu; Figure 1 E is the expansion ratio of CMC / TA / Cu and CMC / TA prepared in Example 1; Figure 1 F is the release curve of copper ions and tannic acid when the CMC / TA / Cu prepared in Example 1 is immersed in a weak acid solution, indicating that the gel can achieve efficient wound repair and sterilization at the wound.

[0087] from Figure 1 It can be seen from D and E that when the elastic modulus and storage modulus are the same, the strain coefficient of CMC / TA / Cu is 221%, which is close to the reported CMC / TA; at the same time, CMC / TA and CMC / TA / Cu have similar swelling coefficients (i.e., expansion rates), proving that the in-situ generation of Cu(OH)2 will not affect the mechanical properties of the gel.

[0088] Example 2

[0089] Effects of composite antibacterial gel on cell migration and angiogenesis

[0090] Composite antibacterial gels (CMC / TA / Cu) of different masses were prepared, and their concentrations in cell culture medium were 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, respectively.

[0091] Cytotoxicity: 1×10 4 Keratinocytes and endothelial cells were seeded in 96-well plates. After 12 hours, different mass concentrations of CMC / TA / Cu were added. After incubation for 12 hours, the cytotoxicity was determined by CCK-8 kit.

[0092] Figure 2 B is the result of the toxicological experiment of CMC / TA / Cu with different mass concentrations on keratinocytes and endothelial cells in Example 2. Figure 2 As can be seen from B, CMC / TA / Cu has less toxicity to keratinocytes and endothelial cells. When the concentration reaches 500 μg / mL, the cell activity can still reach more than 80%.

[0093] Cell proliferation: 2×10 4Keratinocytes were seeded in a 96-well plate. After 6 hours, the old culture medium (the old culture medium refers to the culture medium after 6 hours of culture with the new culture medium) was replaced with a fresh culture medium (pH = 5.5-6.5) mixed with 300 μg / mL CMC / TA or CMC / TA / Cu, and the cells were stained with Annexin V-FITC / PI after continued culture for 0, 2, 4 and 6 hours, and then photographed with a fluorescence microscope (Ex 488nm, Em 550nm) and analyzed by fluorescence quantification.

[0094] Figure 2 A and D are respectively the fluorescence photographs and fluorescence quantitative analysis diagrams of the effects of 300 μg / mL CMC / TA and CMC / TA / Cu (or CMC / Cu / TA) on keratinocyte proliferation at different times (0, 2h, 4h and 6h, respectively) in Example 2. Figure 2 As shown in A and D, CMC / TA / Cu can significantly enhance the proliferation of keratinocytes due to the slow release of copper ions.

[0095] Cell scratching: 1×10 5 Keratinocytes were seeded in 12-well plates and monolayered after 12 hours. Then, the cells were starved with PBS for 6 hours, and scratches of the same size were made with the cells. The cells were divided into 2 groups, CMC / TA group and CMC / TA / Cu group. The old culture medium was replaced with fresh culture medium (pH=5.5-6.5) containing 300 μg / mL gel (i.e., CMC / TA or CMC / TA / Cu), and the changes in the size of cell scratches were photographed after 0, 2, 4 and 6 hours of incubation, and statistical analysis was performed.

[0096] Figure 2 C and E are respectively the changes of the scratches on keratinocytes by CMC / TA / Cu and CMC / TA at different time (0, 2h, 4h and 6h, respectively) and the quantitative analysis of the scratch size in Example 2; Figure 2 As shown in Figures C and E, the healing efficiency of CMC / TA / Cu is significantly higher than that of CMC / TA at each time point. Especially at 6 hours, CMC / TA / Cu can lead to 94.2% healing of the scratch, while CMC / TA / Cu can only lead to 64% healing.

[0097] Angiogenesis: 5×10 4Endothelial cells were seeded in a 24-well plate. After 12 hours, the cells were divided into two groups, CMC / TA and CMC / TA / Cu. The old culture medium was replaced with fresh culture medium (pH = 5.5-6.5) containing 300 μg / mL gel. After incubation for 24 hours, the endothelial cells of each group were collected. Then, 50 μL of BD matrix gel diluted with 2 times endothelial cell culture medium was added to the 96-well plate. After incubation in a 37°C incubator for half an hour, the collected endothelial cells (1×10 4 ) were added to the above 96-well plate and cultured for 0, 2, 4 and 6 hours. The complete microscopic closed network formed by endothelial cells was photographed under a microscope, and the tubule connections were quantitatively analyzed using Image J.

[0098] Figure 2 F and G are the quantitative analysis diagrams of the in vitro angiogenesis experiment of endothelial cells and tubule connection of CMC / TA / Cu and CMC / TA in Example 2; Figure 2 As shown in F and G, the angiogenesis experiment of endothelial cells was consistent with the results of keratinocyte proliferation and scratch experiments. Compared with CMC / TA, CMC / TA / Cu greatly promoted angiogenesis, especially at 6 hours.

[0099] Example 3

[0100] In vitro antimicrobial performance evaluation

[0101] The in vitro antibacterial steps are as follows: Disperse the MRSA frozen solution stored in a -80°C refrigerator in 10% glycerol. Take 10 μL of MRSA bacterial solution and add it to LB liquid culture medium (tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L). After 24 hours, take 100 μL of the above-prepared bacterial suspension and add it to 5mL of fresh LB liquid culture medium (pH=5.5-6.5). The bacterial solution is divided into 2 groups, each with three parts, and the same volume of PBS and CMC / TA / Cu gel (wherein, CMC / TA / Cu gel is sterilized at 121°C for half an hour) are added respectively, and then placed in a 37°C shaker (180rpm / min) for 2 hours.

[0102] Add 1 mL of the treated bacterial suspension to a 5 mL centrifuge tube, centrifuge at 3000-5000 rpm for 5 min, obtain MRSA precipitate, then add 5 mL of saline solution to resuspend for later use. Centrifuge the MRSA sample dispersed in saline solution, obtain precipitate, add 5 mL of 2.5% paraformaldehyde to evenly precipitate, and place the bacterial sample in a 4°C refrigerator for half an hour.

[0103] The MRSA sample prepared above was centrifuged at 3000-5000 rpm for 5 min, and the centrifugal product finally obtained was dispersed by blowing with 30% ethanol / water solution, incubated for 5 min at room temperature, and centrifuged at 3000-5000 rpm for 5 min to obtain a preliminary dehydrated MRSA sample. The MRSA sample was then dehydrated step by step with 40%, 50%, 60%, 70%, 80%, 90% and 100% ethanol / water solution.

[0104] Finally, the MRSA samples dispersed in anhydrous ethanol were dropped onto a SiO2 wafer, and the morphologies of MRSA in different groups were observed under a scanning electron microscope (LEO1530VP, Germany) at 80 KV.

[0105] Figure 3 A is a scanning electron micrograph of MRSA after incubation with CMC / TA / Cu or PBS for 4 hours in Example 3; Figure 3 As shown in A, the spherical morphology of MRSA was maintained in the PBS group, while almost all MRSA in the CMC / TA / Cu gel group was deformed and destroyed, demonstrating its efficient antibacterial effect.

[0106] Example 4

[0107] In vivo antimicrobial and systemic toxicity assessment

[0108] The steps for in vivo antibacterial effect are as follows: First, construct a MRSA infection wound model: clean the hair on the back of the right hind leg of a female Balb / C mouse (19-22g), punch the mouse skin with a skin sampler with a diameter of 2-8mm, and remove the skin tissue to obtain a circular wound. Use a syringe to inject 100μL of MRSA (10 4 ~10 9 CFU / mL), and apply the bacterial suspension to the entire wound.

[0109] The model mice were divided into two groups (5 in each group). 24 hours after the bacterial solution was injected, 10-100 μL of CMC / TA / Cu and CMC / TA gel were applied to the wounds respectively. MRSA from the wounds of the two groups of mice was scraped with a cotton swab after 1, 8 and 12 days, and placed in 5 mL of LB liquid culture medium, and the culture medium was placed in a 37°C shaker for shaking (180 rpm / min) overnight. 100 μL of the above bacterial suspension was placed on an LB solid agar plate (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L and agar powder 15 g / L), and the bacterial solution was evenly applied using a glass scraper. The above LB plate was placed in a 37°C incubator for 24 hours and photographed.

[0110] Figure 3B is the inverted plate result of wound colonies after applying CMC / TA / Cu and CMC / TA to the wound surface infected with MRSA in Example 4, 1 day, 8 days, and 12 days; wherein, Figure 3 In B, the upper layer is coated with CMC / TA, and the lower layer is coated with CMC / TA / Cu.

[0111] like Figure 3 As shown in B, the number of bacteria in both CMC / TA / Cu and CMC / TA groups decreased with time. Compared with CMC / TA, there was almost no MRSA in the wounds treated with CMC / TA / Cu after 12 days, proving that it has a more effective antibacterial effect.

[0112] Systemic toxicity assessment: On the 12th day, the mice in the CMC / TA / Cu group were killed, and the wound tissues and important organ tissues (heart, liver, spleen, lung, and kidney) were removed and placed in 4% paraformaldehyde for subsequent H&E staining. At the same time, mice in the healthy group were set up as controls.

[0113] Figure 3 C is the pathological section results of the heart, liver, spleen, lung and kidney of mice smeared with CMC / TA / Cu on the wound surface infected with MRSA in Example 4 12 days later. The normal group of mice was used as the control (i.e. Figure 3 C in normal); result analysis: Figure 3 As shown in C, compared with the pathological staining of organs of mice in the healthy group, no abnormalities or lesions were found in the organs of mice in the CMC / TA / Cu group, proving that the gel has good biosafety.

[0114] Example 5

[0115] Evaluation of wound healing in living MRSA infected wounds

[0116] Reference Figure 4 A, B, the CMC / TA / Cu gel and PBS in Example 1 were applied to the MRSA infected wound in Example 3, respectively, to study the therapeutic efficiency of CMC / TA / Cu and PBS on MRSA infected wounds. The specific process is as follows:

[0117] The mice with MRSA-infected wounds were divided into two groups (5 mice in each group). After 24 hours of bacterial injection, CMC / TA / Cu and PBS were applied to the wounds. The wound healing was recorded by taking photos, and the weight of the mice was measured. The wound healing rate and the schematic diagram of the wound healing process were analyzed by Image J.

[0118] On day 12, the mice were sacrificed, and the wound tissues were removed, and then the wound tissues were subjected to H&E staining, Mason staining, Gissam staining, and CD86+ cell immunostaining.

[0119] Result analysis:

[0120] Figure 4 AB is the regional trace diagram of wound healing and wound closure when CMC / TA / Cu gel or PBS is applied on the surface of a living MRSA-infected wound in Example 5; Figure 4 C is a diagram showing the wound healing efficiency of applying CMC / TA / Cu gel or PBS on the surface of a living MRSA-infected wound in Example 5; Figure 4 It can be seen from AC that the antibacterial and wound healing efficiency of the two gels is: CMC / TA / Cu>PBS.

[0121] The healing efficiency was calculated by Image J and the area of ​​the healed wound was divided by the initial wound area.

[0122] Figure 4 D is the weight changes of mice in different groups in Example 5; Figure 4 In D, no significant changes were found in the body weight of the two groups of mice during the treatment, which once again proved the biocompatibility of the gel.

[0123] Figure 4 EI is the H&E staining-cross section, H&E staining-longitudinal section (number of new hair follicles), CD86+ immunostaining, Mason staining and Guisam staining of the mouse wound after 12 days of CMC / TA / Cu gel or PBS treatment in Example 5. Figure 4 EI pathological staining showed that the CMC / TA / Cu gel-treated group had more complete wound tissue, a higher amount of new hair follicles, larger collagen deposition, lower bacterial residues, and lower CD86+ distribution, proving that CMC / TA / Cu gel has the best performance in antibacterial and wound healing.

[0124] The above is an exemplary description of the embodiments of the present invention. However, the protection scope of the present invention is not limited to the above embodiments. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composite antibacterial gel composition, characterized in that: It includes carboxymethyl chitosan, natural polyphenols, metal chloride, reducing agent and water; Wherein, the mass ratio of carboxymethyl chitosan, natural polyphenols, metal chloride and sodium bicarbonate is (0.3-1.5):1:(10-30):(8-40); The metal chloride is selected from at least one of CuCl2, MnCl2 or CeCl3.

2. The composite antibacterial gel composition according to claim 1, characterized in that: The concentration of the metal chloride in water is 50-200 μg / mL. Preferably, the concentration of the carboxymethyl chitosan in water is 1-5% w / v.

3. The composite antibacterial gel composition according to claim 1, characterized in that: The concentration of the natural polyphenol in water is 1-5% w / v. Preferably, the reducing agent is selected from at least one of sodium bicarbonate, aqueous ammonia and formaldehyde. Preferably, the concentration of the reducing agent in water is 100-300 μg / mL. Preferably, the natural polyphenol is at least one of tannic acid, tea polyphenol and grape polyphenol.

4. A composite antibacterial gel, characterized in that: The composite antibacterial gel is obtained by gelling the composite antibacterial gel composition according to any one of claims 1 to 3.

5. The composite antibacterial gel according to claim 4, characterized in that: The composite antibacterial gel comprises carboxymethyl chitosan, natural polyphenols and metal hydroxide particles; The metal hydroxide particles are Cu(OH)2, Mn(OH)2 or Ce(OH)3.

6. The composite antibacterial gel according to claim 4 or 5, characterized in that: The size of the metal hydroxide particles is 100nm to 1000nm. Preferably, the composite antibacterial gel can inhibit drug-resistant S. aureus and / or E. coli wound infections.

7. The method for preparing the composite antibacterial gel according to any one of claims 4 to 6, characterized in that: The method is: The composite antibacterial gel is prepared by mixing carboxymethyl chitosan, natural polyphenol, metal chloride, a reducing agent and water.

8. The method according to claim 7, characterized in that The concentration of the metal chloride in water is 50-200 μg / mL.

9. The method according to claim 7 or 8, characterized in that: The concentration of the carboxymethyl chitosan in water is 1-5% w / v. Preferably, the concentration of the natural polyphenols in water is 1-5% w / v. Preferably, the concentration of the reducing agent in water is 100-300 μg / mL.

10. Use of the composite antibacterial gel according to any one of claims 1 to 3 in the preparation of antibacterial products for skin wounds.

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