A multifunctional antibacterial hydrogel dressing and its preparation method and application

Abstract

The present invention discloses a layered multifunctional antibacterial hydrogel dressing, a preparation method, and an application thereof. In situ oxidation is used to form CuO2 on the surface of needle-shaped nano-Cu(OH)2 to obtain copper-based nanoneedles; titanium nitride nanopowder is evenly dispersed in an acetic acid solution of chitosan, which is then soaked in an ethanol suspension of the copper-based nanoneedles and vacuum dried to obtain a chitosan layer; an aldehyde-modified polyaspartic acid aqueous solution and an acylhydrazide hyaluronic acid aqueous solution are evenly mixed and allowed to stand to form a hyaluronic acid hydrogel layer; a chitosan layer is placed on the hyaluronic acid hydrogel layer, and freeze-dried to obtain a layered multifunctional hydrogel dressing. The layered hydrogel dressing of the present invention organically integrates the functions of a one-way pump for removing exudate, supplying oxygen, antibacterial, hemostatic, and promoting angiogenesis, providing an innovative design solution for a multifunctional antibacterial dressing and having good application prospects in wound hemostasis and the treatment of infectious wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a layered multifunctional antibacterial hydrogel dressing, a preparation method and an application thereof. Background Art

[0002] Exudate and bleeding are common phenomena in skin wounds, especially large open wounds and burn wounds, but they are extremely prone to infection. Currently, exudate absorption and hemostasis materials are mainly traditional dressings such as gauze and bandages. They have the advantages of low price, wide availability, and easy production. They play an important role in protecting wounds and draining exudate. However, traditional dressings can neither promote wound healing nor treat wound infections. At the same time, it is difficult to maintain a moist environment in the wound, resulting in adhesion between the dressing and the wound, causing secondary damage to the wound and causing pain to the patient.

[0003] Hydrogel dressings are polymeric hydrogel dressings that can hold tens of times their own weight. They possess unique properties such as extracellular matrix-like properties and structure, exudate absorption, and the ability to maintain a moist wound microenvironment. They not only serve as a barrier to protect the wound surface and resist external infection, but also facilitate oxygen penetration, promote cell migration and tissue regeneration, and accelerate wound healing. Furthermore, hydrogels can carry antibacterial and pro-healing drugs. Currently, hydrogel dressings have become the most attractive type of wound dressing.

[0004] Wound exudate not only affects the speed of wound healing, but also easily irritates the skin around the wound, causing eczema and even bacterial infection. Bacterial infection, in particular, can seriously affect the speed of wound healing. In order to give hydrogel dressings antibacterial properties, antibiotics and other antibacterial drugs are usually added to the hydrogel, but there are still problems such as antibacterial resistance or side effects caused by long-term use. On the other hand, maintaining a certain level of oxygen concentration in the wound plays an important role in ensuring normal cell growth and promoting tissue regeneration. Therefore, the development of hydrogels with multiple functions such as rapid hemostasis, rapid absorption of wound exudate, antibacterial, oxygen supply, and promotion of healing is expected to solve the problem of difficult healing of complex infected wounds. Summary of the Invention

[0005] To overcome the problems of antimicrobial resistance, alleviate wound hypoxia, and promote the healing of chronic wounds, the present invention aims to provide a layered multifunctional antimicrobial hydrogel dressing, preparation method, and application thereof. This gel dressing has excellent biocompatibility, antimicrobial properties, one-way water pumping performance, hemostasis, and other advantages, and can significantly promote the healing of infected wounds. The chitosan layer rapidly transfers wound exudate to the hyaluronic acid hydrogel layer via capillary action, which helps to increase the concentration of coagulation factors in the wound and quickly stop bleeding. The chitosan, titanium nitride photothermal effect, and copper ions and their Fenton-like catalytic properties exhibit excellent antimicrobial properties. The copper-based nanoneedles can continuously release oxygen and copper ions, which can resolve wound hypoxia, promote cell migration and angiogenesis, and thus accelerate wound healing.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a layered multifunctional antibacterial hydrogel dressing comprises the following steps:

[0008] The in-situ oxidation method was used to form CuO2 on the surface of needle-shaped nano-Cu(OH)2 to obtain copper-based nanoneedles;

[0009] Titanium nitride nanopowder is uniformly dispersed in an acetic acid solution of chitosan, freeze-dried, and then soaked in an ethanol suspension of copper-based nanoneedles and vacuum-dried to obtain a chitosan layer;

[0010] The aldehyde-modified polyaspartic acid aqueous solution and the hydrazide-modified hyaluronic acid aqueous solution are mixed evenly and allowed to stand to form a hyaluronic acid hydrogel layer. A chitosan layer is placed on the hyaluronic acid hydrogel layer and freeze-dried to obtain a layered multifunctional hydrogel dressing.

[0011] Furthermore, the length of the copper-based nanoneedles is 50 to 600 nm, and the diameter is 5 to 10 nm.

[0012] Furthermore, the preparation steps of copper-based nanoneedles are as follows:

[0013] The copper salt aqueous solution is mixed with the dispersant aqueous solution, and then NaOH solution is added and stirred to form needle-shaped nano Cu(OH)2, and then H2O2 solution is added to react to obtain copper-based nanoneedles.

[0014] Furthermore, the copper salt is CuCl2 or Cu(NO3)2, and the dispersant is polyvinyl pyrrolidone, hexadecyltrimethylammonium bromide or polyvinyl alcohol.

[0015] Furthermore, the concentration of the copper salt aqueous solution is 10 to 100 mmol / L, the mass concentration of the dispersant aqueous solution is 0.1% to 10%, the volume ratio of the copper salt aqueous solution to the dispersant aqueous solution is 1:1 to 10:1, the molar ratio of the copper salt to NaOH is 1:50 to 1:4, the concentration of the H2O2 solution is 0.1 to 1 mol / L, and the molar ratio of the copper salt to the H2O2 solution is 1:50 to 1:2.

[0016] Further, the chitosan layer is prepared by the following steps:

[0017] Chitosan was dissolved in acetic acid solution, and then titanium nitride powder was added. After uniform dispersion by ultrasonication and freeze-drying, the mixture was immersed in copper-based nanoneedle ethanol suspension and vacuum dried to obtain a chitosan layer.

[0018] Furthermore, the particle size of the titanium nitride powder is 10 to 300 nm, and the performance parameters of chitosan are: the deacetylation degree of the chitosan is ≥95%, and the viscosity is 100 to 200 mPa·s; the mass ratio of chitosan to acetic acid is 2:1 to 50:1, the mass ratio of chitosan to titanium nitride powder is 5:1 to 400:1, and the mass fraction of the ethanol suspension of copper-based nanoneedles is 0.01% to 10%.

[0019] Furthermore, the mass ratio of the aldehyded polyaspartic acid and the hydrazide hyaluronic acid is 1:10 to 1:2, and the thickness ratio of the hyaluronic acid hydrogel layer to the chitosan layer is 3:1 to 1:1.

[0020] A layered multifunctional antibacterial hydrogel dressing.

[0021] A layered multifunctional antibacterial hydrogel dressing is used in the treatment of infected skin wounds and local drug delivery of tumors, as well as in the field of tissue engineering.

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

[0023] 1. This invention uses an in-situ oxidation method to form CuO2 on the surface of needle-shaped nano-Cu(OH)2 to produce copper-based nanoneedles. Copper-based nanoneedles can regulate wound pH, oxygen levels, and angiogenesis, thereby promoting wound healing. Copper-based nanoneedles are needle-shaped structures composed of copper hydroxide inside and copper peroxide on the surface. In response to wound exudate, they decompose and release copper ions, oxygen, hydroxide, and other substances. Copper ions promote angiogenesis in the wound, oxygen alleviates wound hypoxia and promotes cell growth and proliferation in the wound, and hydroxide regulates the wound pH, thereby promoting wound healing.

[0024] 2. In the preparation of the chitosan layer, copper-based nanoneedles and titanium nitride nanopowder were added, which effectively improved the strength and modulus of the chitosan layer. The chitosan layer in the present invention integrates the antibacterial properties of chitosan, copper-based nanoneedles and titanium nitride, namely the cationic antibacterial properties of chitosan, the antibacterial properties of copper ions and their Fenton-like reactions to generate hydroxyl radicals, and the photothermal antibacterial properties of titanium nitride, thereby achieving an organic fusion of ionic antibacterial, chemical antibacterial and physical antibacterial properties, and having excellent comprehensive antibacterial properties. Compared with currently commonly used inorganic antibacterial agents such as nanogold, the antibacterial agent of the present invention has the advantages of low cost, simple and mild process, and scalable preparation.

[0025] 3. The chitosan layer in contact with the wound has low hydrophilicity and water absorption without deformation. The hyaluronic acid hydrogel layer has high water absorption and water retention capacity, and the layer interface has a porous structure, which ensures that the wound exudate can move quickly from the chitosan surface to the hyaluronic acid hydrogel layer through capillary action, thereby clearing the wound exudate and coagulation factors such as platelets / proteins, preventing infection and promoting hemostasis.

[0026] 4. In the preparation of the hyaluronic acid hydrogel layer, aldehyde-modified polyaspartic acid was used as a cross-linking agent, which significantly improved the strength and dimensional stability of the hyaluronic acid hydrogel.

[0027] 5. The layered multifunctional hydrogel dressing of the present invention has outstanding one-way water pumping properties and improves the interfacial bonding strength and structural stability of the layered hydrogel. At the interface between the two polysaccharide hydrogels, the copper-based nanoneedles in the chitosan layer can coordinate with the hydrazide hyaluronic acid in the hyaluronic acid hydrogel layer, while the aldehyde-modified polyaspartic acid in the hyaluronic acid hydrogel layer can react with the chitosan in the chitosan layer to produce a Schiff base reaction. The two work together to significantly improve the interfacial bonding strength and structural stability. The layered hydrogel dressing of the present invention organically integrates the functions of one-way pump exudate removal, oxygen supply, antibacterial, hemostasis, and angiogenesis promotion, providing an innovative design solution for multifunctional antibacterial dressings, and has good application prospects in wound hemostasis and infectious wound treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a photo of freeze-dried sample C prepared in Example 1; the white upper layer is the hyaluronic acid hydrogel layer, and the black lower layer is the chitosan layer;

[0029] Figure 2 is a transmission electron microscope photograph of the copper-based nanoneedles prepared in Example 1;

[0030] Figure 3 is the X-ray diffraction pattern of the copper-based nanoneedles prepared in Example 1;

[0031] Figure 4 1 is a scanning electron microscope photograph of sample C prepared in Example 1; wherein, (a) is the hyaluronic acid hydrogel layer, (b) is the interface connection of the layered multifunctional gel dressing, and (c) is the chitosan layer.

[0032] Figure 5 These are the frequency sweep curves of Comparative Examples 1, 2, and Example 1;

[0033] Figure 6 1 is the strain sweep curve of Comparative Examples 1, 2 and Example 1;

[0034] Figure 7 is the shear thinning curve and injectable photograph of Comparative Example 1;

[0035] Figure 8 This is a photo of the injectable sample of Comparative Example 1;

[0036] Figure 9 is the alternating strain curve of comparative example 1;

[0037] Figure 10 are the swelling curves of Comparative Example 1 and Example 1;

[0038] Figure 11 This is a physical picture of the water absorption and swelling of Example 1;

[0039] Figure 12 This is the temperature rise curve of Example 1 under laser irradiation of different powers;

[0040] Figure 13 This is the photothermal cycle stability of Example 1 at 1W power.

[0041] Figure 14 These are the antibacterial results of Examples 1 to 3;

[0042] Figure 15 The biocompatibility results of Comparative Examples 1 and 2 and Examples 1 to 3 are as follows;

[0043] Figure 16 This is a diagram showing the effect of oxygen release at different pH values ​​of the copper-based nanoneedles prepared in Example 1 measured by a dissolved oxygen meter;

[0044] Figure 17 This is a graph showing the effect of copper-based nanoneedles prepared in Example 1 releasing hydroxyl radicals at different addition amounts using TMB as an indicator, with the absorbance at 650 nm being used;

[0045] Figure 18 This is a diagram showing the effect of promoting cell migration in Example 1;

[0046] Figure 19 3 is a graph showing the hemostatic effect of Example 1 tested by constructing a SD mouse liver bleeding model, wherein (a) is the control group and (b) is sample C. DETAILED DESCRIPTION

[0047] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in a variety of different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0048] Chitosan is a cationic polysaccharide with excellent biocompatibility and biodegradability as well as good antibacterial, hemostatic and healing promoting effects, and is a commonly used wound dressing raw material. However, chitosan is relatively low in hydrophilicity under neutral or weakly alkaline conditions, and does not deform after absorbing water, and its ability to absorb exudate and antibacterial is limited, and its advantages can not be fully exerted in the case of a large amount of exudate from wounds. Hyaluronic acid is a kind of anionic polysaccharide, and is also one of the main components of the extracellular matrix, with excellent biocompatibility, biodegradability and water retention, and is widely used in hydrogel preparation. However, the strength of pure hyaluronic acid hydrogel is relatively poor, and the deformation amount is large and shape is unstable after absorbing water, and hyaluronic acid itself lacks functions such as antibacterial. So the present invention utilizes chitosan and hyaluronic acid to prepare antibacterial hydrogel dressing.

[0049] A layered multifunctional antibacterial hydrogel dressing of the present invention comprises a lower layer and an upper layer, wherein the lower layer is an antibacterial chitosan containing copper-based nanoneedles and titanium nitride nanopowder, and the upper layer is a hyaluronic acid cross-linked with aldehyde-modified polyaspartic acid with a water storage function. This structure can unidirectionally transfer wound exudate from the chitosan layer to the hyaluronic acid hydrogel layer, thereby significantly increasing the concentration of coagulation factors at the wound and accelerating hemostasis. The cationic properties of chitosan, the copper ions released by the copper-based nanoneedles and the hydroxyl radicals generated by hydrogen peroxide at the wound, and the photothermal properties of titanium nitride give the chitosan lower layer excellent antibacterial properties. At the same time, the copper-based nanoneedles release oxygen, which can solve the problem of wound hypoxia, accelerate cell migration and wound healing. The hyaluronic acid hydrogel layer has a high water absorption capacity and can pump wound exudate out through the chitosan layer, maintaining a moist environment and preventing bacterial infection. The hydrogel dressing has the advantages of good biocompatibility, strong water absorption capacity, and excellent antibacterial properties, and has broad application prospects in wound hemostasis, anti-infection, and wound healing.

[0050] The method for preparing a layered multifunctional antibacterial hydrogel dressing of the present invention comprises the following steps:

[0051] The in-situ oxidation method was used to form CuO2 on the surface of needle-shaped nano-Cu(OH)2 to obtain copper-based nanoneedles;

[0052] Titanium nitride nanopowder is uniformly dispersed in an acetic acid solution of chitosan under ultrasonication, then injected into a mold and freeze-dried. After alternately washing with alkali and alcohol, it is vacuum-dried. It is then soaked in an ethanol suspension of copper-based nanoneedles and vacuum-dried to obtain a non-swelling chitosan layer.

[0053] An aqueous solution of aldehyde-modified polyaspartic acid and an aqueous solution of hydrazide-modified hyaluronic acid are mixed evenly, placed in a mold, and allowed to stand to form a swelling layer. A chitosan layer is then added, and freeze-dried to produce a layered multifunctional hydrogel dressing. The swelling layer and chitosan layer are linked by Schiff base bonds and copper-hydrazide coordination, exhibiting unidirectional transport of aqueous liquid from the chitosan layer to the hyaluronic acid hydrogel layer. The chitosan layer possesses cationic, copper ion, and photothermal antibacterial properties, as well as hemostatic and hypoxia-relieving properties. The hyaluronic acid hydrogel layer has high water absorption and water retention capabilities, serving as a water reservoir.

[0054] Specifically, the preparation steps of copper-based nanoneedles are:

[0055] A 10-100 mmol / L aqueous copper salt (CuCl2 or Cu(NO3)2) solution and a 0.1%-10% aqueous polyvinyl pyrrolidone solution (polyvinyl pyrrolidone is used as a dispersant, and the dispersant can also be CTAB or polyvinyl alcohol) are mixed in a volume ratio of 1:1-10:1. A 0.1-1 mol / L NaOH solution is then added dropwise. After the addition is complete, the mixture is stirred at 10,000-13,000 rpm for 5-30 minutes to form needle-shaped nano-Cu(OH)2. A 0.1-1 mol / L H2O2 solution is then added. After reacting for 0.1-6 hours, the copper-based nanoneedles are collected by centrifugation. The length of the copper-based nanoneedles is 50-600 nm and the diameter is 5-10 nm. The molar ratio of the copper-based nanoneedles to the NaOH solution is 1:50-1:4, and the molar ratio of the copper-based nanoneedles to the H2O2 solution is 1:50-1:2.

[0056] Specifically, the chitosan layer is prepared by the following steps:

[0057] Chitosan is dissolved in a 0.2% to 5% acetic acid (hydrochloric acid, formic acid, or oxalic acid) solution to obtain a chitosan solution with a mass fraction of 0.1% to 10%. Titanium nitride powder with a particle size of 10 to 300 nm is then added at a chitosan to titanium nitride powder mass ratio of 5:1 to 400:1. The mixture is ultrasonically dispersed and then injected into a mold. After freeze-drying for 1 to 3 days, the mixture is washed alternately with 0.1 to 1 mol / L NaOH solution and ethanol for 3 to 6 times before vacuum drying. Finally, the dried sample is immersed in a 0.01% to 10% copper-based nanoneedle ethanol suspension for 2 to 24 hours and vacuum dried to obtain a chitosan layer. The chitosan has a degree of deacetylation of ≥95% and a viscosity of 100 to 200 mPa·s.

[0058] Specifically, the layered hydrogel dressing is prepared by the following process:

[0059] A solution of aldehyde-modified polyaspartic acid (cross-linker) with a mass fraction of 0.025% to 30%, a degree of aldehyde modification of 10% to 60%, and a molecular weight of 5kDa to 1000kDa is dropwise added to an aqueous solution of hydrazide-modified hyaluronic acid with a mass fraction of 0.1% to 10%, a degree of hydrazide modification of 10% to 40%, and a molecular weight of 10kDa to 3000kDa. The mass ratio of aldehyde-modified polyaspartic acid to hydrazide-modified hyaluronic acid is 1:10 to 1:2. After mixing for 1 to 5 minutes, the mixture is injected into a mold, and a chitosan layer is subsequently placed. The mixture is allowed to stand for 5 to 30 minutes and then freeze-dried to form a layered hydrogel dressing. The layered hydrogel dressing includes a hyaluronic acid hydrogel layer and a chitosan layer with a thickness ratio of 1:1 to 3:1.

[0060] The layered multifunctional antibacterial hydrogel dressing prepared by the present invention can be used in the fields of treating infected skin wounds, local administration of tumors, and tissue engineering, thereby reducing the risk of wound infection.

[0061] The following are specific examples.

[0062] Example 1

[0063] Preparation of copper-based nanoneedles: A 40 mmol / L aqueous solution of CuCl2 and a 1% aqueous solution of polyvinyl pyrrolidone were mixed in a 1:1 ratio to prepare a 10 mL solution. A 0.4 mol / L NaOH solution was then added dropwise. After the addition was complete, stirring was performed at 10,000 rpm for 10 minutes to form needle-shaped nano-Cu(OH)2. Subsequently, 2 mL of a 0.5 mol / L H2O2 solution was added. After reacting for 0.5 hours, the copper-based nanoneedles were collected by centrifugation. The molar ratio of CuCl2 to NaOH was 1:4.

[0064] The chitosan layer was prepared by the following steps:

[0065] 1g of chitosan was dissolved in a 1% acetic acid solution to obtain a 2% chitosan solution; then 5mg of titanium nitride powder with a particle size of 100nm was added, uniformly dispersed by ultrasound, and injected into a mold; after freeze-drying for 1 day, the sample was alternately washed with a 1mol / L NaOH solution and ethanol for 5 times and then vacuum-dried; finally, the dried sample was immersed in a 5% copper-based nanoneedle ethanol suspension for 2 hours and vacuum-dried to obtain a chitosan layer.

[0066] Prepare the layered hydrogel dressing:

[0067] 0.25 mL of a 5% by mass, 40% degree of aldehyde-modified polyaspartic acid solution with a molecular weight of 1000 kDa was dropwise added to 1 mL of a 3% by mass, 40% degree of hydrazide hyaluronic acid aqueous solution with a molecular weight of 3000 kDa. After mixing for 3 minutes, the mixture was injected into a mold to form a hyaluronic acid hydrogel layer. A chitosan layer was then placed on top. After standing for 10 minutes, the dressing was freeze-dried to obtain a layered hydrogel dressing, designated as Sample C. The thickness ratio of the hyaluronic acid hydrogel layer to the chitosan layer was 1:1.

[0068] See also Figure 2 , the average length of the copper-based nanoneedles is about 400 nanometers and the diameter is about 10 nanometers;

[0069] See also Figure 3 The X-ray diffraction pattern of copper-based nanoneedles shows that the main body of the nanoneedles is CuO2 and Cu(OH)2.

[0070] See also Figure 4 The scanning electron microscope image of sample C shows that the layered interface is clear and tightly connected, the pore structure of each layer is complete, and the pore size distribution is uniform.

[0071] See also Figure 11 After sample C absorbs water and swells, the chitosan layer can maintain a stable morphology, and the hyaluronic acid hydrogel layer can store a large amount of water.

[0072] See also Figure 12 , sample C has excellent photothermal performance

[0073] See also Figure 13 , sample C has excellent photothermal cycling stability.

[0074] See also Figure 16 The copper-based nanoneedles can slowly release oxygen at different pH levels, with the release rate and concentration being highest under acidic conditions, indicating their potential to alleviate wound hypoxia.

[0075] See also Figure 17 The higher the peak value, the more hydroxyl radicals are released. As the amount of copper-based nanoneedles added increases, the amount of hydroxyl radicals generated increases, indicating that it has the ability to release hydroxyl radicals to kill bacteria.

[0076] See also Figure 18 Compared with the control group, sample C could significantly promote cell migration.

[0077] See also Figure 19 In (a) and (b), sample C has excellent hemostatic function. Compared with the control group in Figure (a), the amount of bleeding in sample C in Figure (b) is significantly reduced.

[0078] Example 2

[0079] Preparation of copper-based nanoneedles: A 40 mmol / L aqueous solution of CuCl2 and a 1% aqueous solution of polyvinyl pyrrolidone were mixed in a 1:1 ratio to prepare a 10 mL solution. 2 mL of a 0.4 mol / L NaOH solution was then added dropwise. After the addition was complete, stirring was performed at 10,000 rpm for 10 minutes to form needle-shaped nano-Cu(OH)2. Then, 2 mL of a 0.5 mol / L H2O2 solution was added. After reacting for 0.5 hours, the copper-based nanoneedles were collected by centrifugation. The molar ratio of CuCl2 to NaOH was 1:4.

[0080] The chitosan layer was prepared by the following steps:

[0081] 1g of chitosan was dissolved in a 1% acetic acid solution to obtain a 2% chitosan solution; then 5mg of titanium nitride powder with a particle size of 100nm was added, dispersed evenly by ultrasound, and injected into a mold; after freeze-drying for 1 day, the sample was alternately washed with a 1mol / L NaOH solution and ethanol for 5 times and then vacuum-dried; finally, the dried sample was immersed in a 1% copper-based nanoneedle ethanol suspension for 2 hours and vacuum-dried to obtain a chitosan layer.

[0082] Prepare the layered hydrogel dressing:

[0083] 0.25 mL of a 5% by mass, 40% degree of aldehyde-modified polyaspartic acid solution with a molecular weight of 1000 kDa was dropwise added to 1 mL of a 3% by mass, 40% degree of hydrazide hyaluronic acid aqueous solution with a molecular weight of 3000 kDa. After mixing for 3 minutes, the mixture was injected into a mold, followed by a chitosan layer. After standing for 10 minutes, the mixture was freeze-dried to obtain a layered hydrogel dressing, designated as Sample D. The thickness ratio of the hyaluronic acid hydrogel layer to the chitosan layer was 1:1.

[0084] Example 3

[0085] Preparation of copper-based nanoneedles: A 40 mmol / L aqueous solution of CuCl2 and a 1% aqueous solution of polyvinyl pyrrolidone were mixed in a 1:1 ratio to prepare a 10 mL solution. 2 mL of a 0.4 mol / L NaOH solution was then added dropwise. After the addition was complete, stirring was performed at 10,000 rpm for 10 minutes to form needle-shaped nano-Cu(OH)2. Then, 2 mL of a 0.5 mol / L H2O2 solution was added. After reacting for 0.5 hours, the copper-based nanoneedles were collected by centrifugation. The molar ratio of CuCl2 to NaOH was 1:4.

[0086] The chitosan layer was prepared by the following steps:

[0087] 1g of chitosan was dissolved in a 1% acetic acid solution to obtain a 2% chitosan solution; then 5mg of titanium nitride powder with a particle size of 100nm was added, uniformly dispersed by ultrasound, and injected into a mold; after freeze-drying for 1 day, the sample was alternately washed with a 1mol / L NaOH solution and ethanol for 5 times and then vacuum-dried; finally, the dried sample was immersed in a 5% copper-based nanoneedle ethanol suspension for 2 hours and vacuum-dried to obtain a chitosan layer.

[0088] Prepare the layered hydrogel dressing:

[0089] 0.25 mL of a 5% by mass, 40% degree of aldehyde-modified polyaspartic acid solution with a molecular weight of 1000 kDa was dropwise added to 1 mL of a 3% by mass, 40% degree of hydrazide hyaluronic acid aqueous solution with a molecular weight of 3000 kDa. After mixing for 3 minutes, the mixture was injected into a mold, followed by a chitosan layer. After standing for 10 minutes, the mixture was freeze-dried to obtain a layered hydrogel dressing, designated as Sample E. The thickness ratio of the hyaluronic acid hydrogel layer to the chitosan layer was 3:1.

[0090] See also Figure 5 ,Due to the high strength of titanium nitride and copper-based nanoneedle modified chitosan, the strength of sample B is higher than that of sample A and C. The strength of sample C is similar to that of sample A, indicating that after the double layer composite, the strength of the sample is close to that of the single layer with lower strength.

[0091] See also Figure 6 Because the modified chitosan is not easily deformed, Sample B fails at a relatively low strain. Samples A and C, on the other hand, exhibit good toughness and can withstand larger deformations, indicating that the double-layer composite samples have significantly improved toughness compared to the modified chitosan layer. Because the modified chitosan is not easily deformed, Sample B fails at a relatively low strain. Samples A and C, on the other hand, exhibit good toughness and can withstand larger deformations, indicating that the double-layer composite samples have significantly improved toughness compared to the modified chitosan layer.

[0092] See also Figure 10 It can be seen that the swelling rate and equilibrium swelling ratio of sample C are significantly higher than those of sample B, indicating that sample C has excellent ability to absorb wound exudate. It can be seen that the water absorption rate and equilibrium swelling ratio of the double-layer composite gel are significantly greater than those of a single material.

[0093] See also Figure 14 It can be seen that the inhibition rate of different materials against Staphylococcus aureus and Escherichia coli was characterized by the colony counting method. Sample C has excellent antibacterial properties, and the bactericidal rate can reach more than 99%. The chitosan layer in the layered gel dressing provides antibacterial properties.

[0094] See also Figure 15By adding CCK-8 working solution and measuring the absorbance with a microplate reader to calculate the cell survival rate, the extract of sample C has good cell compatibility, and the cells proliferate normally as the culture time increases.

[0095] Example 4

[0096] Preparation of copper-based nanoneedles: A 10 mmol / L copper salt (CuCl2) aqueous solution and a 10% polyvinyl pyrrolidone aqueous solution were mixed in a volume ratio of 1:1 to prepare a 10 mL solution, and then a 0.6 mol / L NaOH solution was added dropwise. After the addition was completed, the solution was stirred at 10,000 rap / min for 30 minutes to form needle-shaped nano-Cu(OH)2. Then, a 0.1 mol / L H2O2 solution was added dropwise. After reacting for 0.1 hour, the copper-based nanoneedles were collected by centrifugation. The molar ratio of copper salt to NaOH was 1:4, and the molar ratio of copper salt to H2O2 solution was 1:30.

[0097] Preparation of chitosan layer:

[0098] Chitosan was dissolved in a 3% hydrochloric acid solution to obtain a 5% chitosan solution. Titanium nitride powder with a particle size of 10 to 300 nm was then added at a chitosan to titanium nitride ratio of 5:1. The mixture was ultrasonically dispersed and then injected into a mold. After freeze-drying for two days, the sample was washed three times with a 0.5 mol / L NaOH solution and ethanol, followed by vacuum drying. Finally, the dried sample was immersed in a 0.01% copper-based nanoneedle suspension in ethanol for 10 hours and vacuum dried to obtain a chitosan layer. The chitosan had a degree of deacetylation of ≥95% and a viscosity of 100 to 200 mPa·s.

[0099] Preparation of the layered hydrogel dressing: A 0.025% (mass fraction), 30% (formaldehyde) polyaspartic acid (crosslinker) solution with a molecular weight of 5 kDa was added dropwise to a 6% (mass fraction), 250% (hydrazide) degree, 1000 kDa (molecular weight) hydrazide hyaluronic acid aqueous solution. The mass ratio of the aldehyde-modified polyaspartic acid to the hydrazide hyaluronic acid was 1:10. After mixing for 1 minute, the mixture was injected into a mold, followed by a chitosan layer. The mixture was allowed to stand for 20 minutes and then freeze-dried to form a layered hydrogel dressing. The layered hydrogel dressing consisted of a hyaluronic acid hydrogel layer and a chitosan layer with a thickness ratio of 2:1.

[0100] Example 5

[0101] Preparation of copper-based nanoneedles: A 100 mmol / L copper salt (Cu(NO3)2) aqueous solution and a 4% polyvinyl alcohol aqueous solution were mixed in a volume ratio of 10:1 to prepare a 10 mL solution, and then a 0.1 mol / L NaOH solution was added dropwise. After the addition was complete, the solution was stirred at 13000 rap / min for 5 min to form needle-shaped nano-Cu(OH)2. Then, a 1 mol / L H2O2 solution was added. After the reaction for 6 hours, the copper-based nanoneedles were collected by centrifugation. The molar ratio of copper salt to NaOH was 1:20, and the molar ratio of copper salt to H2O2 solution was 1:2.

[0102] Preparation of chitosan layer:

[0103] Chitosan was dissolved in a 5% hydrochloric acid solution to obtain a 0.1% chitosan solution. Titanium nitride powder with a particle size of 10 to 300 nm was then added at a chitosan to titanium nitride ratio of 400:1. The mixture was ultrasonically dispersed and then injected into a mold. After freeze-drying for three days, the sample was washed six times alternately with a 1 mol / L NaOH solution and ethanol, and then vacuum-dried. Finally, the dried sample was immersed in a 10% ethanol suspension of copper-based nanoneedles for 24 hours and vacuum-dried to obtain a chitosan layer. The chitosan had a degree of deacetylation of ≥95% and a viscosity of 100 to 200 mPa·s.

[0104] Preparation of the layered hydrogel dressing: A 30% by mass, 10% degree of aldehyde-modified polyaspartic acid (crosslinker) solution with a molecular weight of 1000 kDa was added dropwise to a 0.1% by mass, 40% degree of hydrazide, and 3000 kDa aqueous solution of hydrazide-modified hyaluronic acid. The mass ratio of aldehyde-modified polyaspartic acid to hydrazide-modified hyaluronic acid was 1:2. After mixing for 5 minutes, the mixture was injected into a mold, and a chitosan layer was then placed. After standing for 30 minutes, the mixture was freeze-dried to form a layered hydrogel dressing. The layered hydrogel dressing includes a hyaluronic acid hydrogel layer and a chitosan layer with a thickness ratio of 3:1.

[0105] Example 6

[0106] Preparation of copper-based nanoneedles: A 50 mmol / L copper salt (CuCl2) aqueous solution and a 0.1% CTAB (hexadecyltrimethylammonium bromide) aqueous solution were mixed in a volume ratio of 5:1 to prepare a 10 mL solution, and then a 1 mol / L NaOH solution was added dropwise. After the addition was complete, the mixture was stirred at 12000 rap / min for 20 min to form needle-shaped nano Cu(OH)2. Then, a 0.5 mol / L H2O2 solution was added. After reacting for 2 hours, the copper-based nanoneedles were collected by centrifugation. The molar ratio of copper salt to NaOH was 1:50, and the molar ratio of copper salt to H2O2 solution was 1:50.

[0107] Preparation of chitosan layer:

[0108] Chitosan was dissolved in a 0.2% hydrochloric acid solution to obtain a 10% chitosan solution. Titanium nitride powder with a particle size of 10 to 300 nm was then added at a chitosan to titanium nitride ratio of 100:1. The mixture was ultrasonically dispersed and then injected into a mold. After freeze-drying for one day, the sample was washed five times alternately with a 0.1 mol / L NaOH solution and ethanol, and then vacuum-dried. Finally, the dried sample was immersed in a 4% copper-based nanoneedle suspension in ethanol for two hours and vacuum-dried to obtain a chitosan layer. The chitosan had a degree of deacetylation of ≥95% and a viscosity of 100 to 200 mPa·s.

[0109] Preparation of the layered hydrogel dressing: A 10% by mass solution of aldehyde-modified polyaspartic acid (crosslinker) with a 60% degree of aldehyde modification and a molecular weight of 400 kDa is added dropwise to a 10% by mass solution of hydrazide-modified hyaluronic acid with a 10% degree of hydrazide modification and a molecular weight of 10 kDa. The mass ratio of aldehyde-modified polyaspartic acid to hydrazide-modified hyaluronic acid is 1:6. After mixing for 3 minutes, the mixture is injected into a mold, and a chitosan layer is then placed. The mixture is allowed to stand for 5 minutes and then freeze-dried to form a layered hydrogel dressing. The layered hydrogel dressing includes a hyaluronic acid hydrogel layer and a chitosan layer with a thickness ratio of 1:1.

[0110] Comparative Example 1

[0111] A 5% aldehyde-modified polyaspartic acid solution was added dropwise to a 3% hydrazide-modified hyaluronic acid solution and mixed for 3 minutes to form a homogeneous solution. The polyaspartic acid had a 40% aldehyde modification degree and a molecular weight of 1000 kDa, while the hyaluronic acid had a 40% hydrazide modification degree and a molecular weight of 3000 kDa. This solution was injected into a mold and allowed to solidify for 5-30 minutes, forming a hyaluronic acid reservoir gel (Sample A).

[0112] See also Figure 7 and Figure 8 , Sample A has good shear thinning properties, indicating that Sample A has injectable properties, can be smoothly extruded from a syringe, and can fill irregularly shaped wounds by injection.

[0113] See also Figure 8 Specimen A has excellent self-healing properties and can quickly recover to its original state under alternating strain, indicating that specimen A can adapt to wounds of different shapes and sizes, making it convenient for practical application. The surface specimen can quickly recover to its initial state after being damaged by external force, showing self-healing ability and can adapt to wounds of different shapes and sizes.

[0114] Comparative Example 2

[0115] Preparation of copper-based nanoneedles: A 40 mmol / L aqueous solution of CuCl2 and a 1% aqueous solution of polyvinyl pyrrolidone were mixed in a 1:1 ratio. A 0.4 mol / L NaOH solution and a H2O2 solution were then added dropwise. The mixture reacted for 0.5 hours and then centrifuged to collect the copper-based nanoneedles. The molar ratio of CuCl2 to NaOH was 1:4.

[0116] The chitosan layer was prepared by the following steps:

[0117] Chitosan was dissolved in a 1% acetic acid solution to obtain a chitosan solution with a mass fraction of 2%. Titanium nitride powder with a particle size of 100 nm was then added, dispersed evenly by ultrasound, and injected into a mold. After freeze-drying for one day, the sample was alternately washed five times with a 1 mol / L NaOH solution and ethanol and then vacuum-dried. Finally, the dried sample was immersed in a 5% copper-based nanoneedle ethanol suspension for two hours and vacuum-dried to obtain sample B.

[0118] Comparative Example 1 is a hyaluronic acid hydrogel layer (single layer), and Comparative Example 2 is a chitosan layer (single layer). The mechanical properties are compared with the layered gel dressing of the present invention to illustrate the role of each layer.

[0119] See also Figure 5 ,It can be seen that the layered gel dressing has higher strength and cross-linking degree than the single hyaluronic acid hydrogel layer because the chitosan layer provides support and interlayer cross-linking.

[0120] See also Figure 6 It can be seen that the layered gel dressing has much higher toughness than the single chitosan layer because the hyaluronic acid hydrogel layer bears the deformation and interlayer cross-linking effect, and can withstand larger external deformation.

[0121] The layered gel dressing prepared by the present invention combines the mechanical performance advantages of two single layers (the hyaluronic acid hydrogel layer and the chitosan layer) and has better strength and toughness.

[0122] The part of the layered gel dressing prepared by the present invention that provides injectability and self-healing properties is the hyaluronic acid hydrogel layer.

[0123] The above description is merely a description of the preferred embodiment of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiment, and variations in the specific structure are permitted. Any variations made within the scope of the independent claims of the present invention are also within the scope of protection of the present invention.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

Claims

1. A method for preparing a layered multifunctional antibacterial hydrogel dressing, characterized in that: The following steps are involved: The in-situ oxidation method was used to form CuO2 on the surface of needle-shaped nano-Cu(OH)2 to obtain copper-based nanoneedles; Titanium nitride nanopowder is uniformly dispersed in an acetic acid solution of chitosan, freeze-dried, and then soaked in an ethanol suspension of copper-based nanoneedles and vacuum-dried to obtain a chitosan layer; The aldehyde-modified polyaspartic acid aqueous solution and the hydrazide-modified hyaluronic acid aqueous solution are mixed evenly and allowed to stand to form a hyaluronic acid hydrogel layer. A chitosan layer is placed on the hyaluronic acid hydrogel layer and freeze-dried to obtain a layered multifunctional hydrogel dressing.

2. The method for preparing the layered multifunctional antibacterial hydrogel dressing according to claim 1, characterized in that: The length of the copper-based nanoneedles is 50 to 600 nm and the diameter is 5 to 10 nm.

3. The method for preparing the layered multifunctional antibacterial hydrogel dressing according to claim 1, characterized in that: The preparation steps of copper-based nanoneedles are: The copper salt aqueous solution is mixed with the dispersant aqueous solution, and then NaOH solution is added and stirred to form needle-shaped nano Cu(OH)2, and then H2O2 solution is added to react to obtain copper-based nanoneedles.

4. The method for preparing the layered multifunctional antibacterial hydrogel dressing according to claim 3, characterized in that: The copper salt is CuCl2 or Cu(NO3)2, and the dispersant is polyvinyl pyrrolidone, hexadecyltrimethylammonium bromide or polyvinyl alcohol.

5. The method for preparing the layered multifunctional antibacterial hydrogel dressing according to claim 3, characterized in that: The concentration of the copper salt aqueous solution is 10-100 mmol / L, the mass concentration of the dispersant aqueous solution is 0.1%-10%, the volume ratio of the copper salt aqueous solution to the dispersant aqueous solution is 1:1-10:1, the molar ratio of the copper salt to NaOH is 1:50-1:4, the concentration of the H2O2 solution is 0.1-1 mol / L, and the molar ratio of the copper salt to the H2O2 solution is 1:50-1:

2.

6. The method for preparing the layered multifunctional antibacterial hydrogel dressing according to claim 1, characterized in that: The chitosan layer was prepared by the following steps: Chitosan was dissolved in acetic acid solution, and then titanium nitride powder was added. After uniform dispersion by ultrasonication and freeze-drying, the mixture was immersed in copper-based nanoneedle ethanol suspension and vacuum dried to obtain a chitosan layer.

7. The method for preparing the layered multifunctional antibacterial hydrogel dressing according to claim 1, characterized in that: The particle size of the titanium nitride powder is 10 to 300 nm, and the performance parameters of chitosan are as follows: the deacetylation degree of chitosan is ≥95%, and the viscosity is 100 to 200 mPa·s; the mass ratio of chitosan to acetic acid is 2:1 to 50:1, the mass ratio of chitosan to titanium nitride powder is 5:1 to 400:1, and the mass fraction of the ethanol suspension of copper-based nanoneedles is 0.01% to 10%.

8. The method for preparing the layered multifunctional antibacterial hydrogel dressing according to claim 6, characterized in that: The mass ratio of aldehyde-modified polyaspartic acid to hydrazide-modified hyaluronic acid is 1:10 to 1:2, and the thickness ratio of the hyaluronic acid hydrogel layer to the chitosan layer is 3:1 to 1:

1.

9. A layered multifunctional antibacterial hydrogel dressing prepared according to the method according to any one of claims 1 to 8.

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