A nanocellulose-based antibacterial and antioxidant wound indicating hydrogel and a method of preparing the same

Abstract

The application belongs to the technical field of hydrogel, and particularly relates to a nanocellulose-based antibacterial and antioxidant wound indicating hydrogel and a preparation method thereof. The hydrogel is prepared by physical cross-linking and chemical cross-linking of water, aldehyde-modified nanocellulose, water-soluble polysaccharide derivatives and anthocyanin. The aldehyde-modified nanocellulose is prepared from nanocellulose extracted from cotton and hemp plants through aldehyde modification treatment of sodium periodate. The water-soluble polysaccharide derivatives are one or more of carboxymethyl chitosan or quaternary ammonium salt chitosan. The anthocyanin is blueberry anthocyanin. Hydrogen bonds are formed between the aldehyde-modified nanocellulose, the water-soluble polysaccharide derivatives and the blueberry anthocyanin, and imine bonds are formed between the aldehyde-modified nanocellulose and the water-soluble polysaccharide derivatives. The hydrogel is used as a wound monitoring dressing.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel technology, and particularly relates to a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel and its preparation method. Background Technology

[0002] Wound healing is a complex physiological process that is susceptible to interference from factors such as infection, oxidative stress, and abnormal wound microenvironment. Traditional wound dressings can only provide basic physical protection and moisture retention, which is insufficient to meet the comprehensive clinical needs for dynamic wound monitoring, infection control, and antioxidant intervention, and thus have significant technical shortcomings.

[0003] In terms of wound infection control, existing dressings mostly rely on single antibacterial components (such as silver ions and antibiotics). Among them, silver ion dressings pose a risk of heavy metal accumulation, and long-term use may cause skin irritation or cytotoxicity. Antibiotic dressings are prone to bacterial resistance, especially against common wound infection strains such as Staphylococcus aureus and Escherichia coli, where the stability of antibacterial efficacy and safety are difficult to balance. At the same time, most antibacterial dressings lack the ability to provide real-time feedback on wound infection. Clinicians need to rely on visual observation of exudate color and odor or sampling tests to determine the infection status, which is not only delayed but also prone to misdiagnosis, thus delaying treatment.

[0004] In wound oxidative stress management, a large amount of reactive oxygen species (ROS) are generated during wound healing. Excessive ROS can damage cell structure, inhibit fibroblast proliferation and collagen synthesis, and delay the healing process. Existing antioxidant dressings often use small-molecule antioxidants such as vitamin C and glutathione. These ingredients are prone to degradation and inactivation during storage or use, and have weak binding force with the dressing matrix, making them easy to be lost with exudate. This results in a short duration of antioxidant effect and fails to provide long-term protection for the wound.

[0005] In terms of wound monitoring, wound pH is a key indicator reflecting the healing status. Healthy skin has a pH of approximately 5.5 (slightly acidic), while chronic or infected wounds, due to the accumulation of bacterial metabolites, can have a pH above 7.0 (neutral or slightly alkaline). Currently, most wound dressings with pH-responsive functions use synthetic pigment indicators (such as bromocresol green and phenol red). These indicators suffer from poor biocompatibility, low color change sensitivity, and susceptibility to interference from wound exudate components. Furthermore, they are difficult to integrate synergistically with the antibacterial and antioxidant functions of dressings, resulting in products with limited functionality that cannot meet the multi-dimensional care needs of complex wounds.

[0006] Furthermore, regarding the selection of dressing matrix materials, traditional hydrogel matrices (such as polyacrylamide and polyvinyl alcohol), while possessing good moisturizing properties, suffer from poor biodegradability, insufficient mechanical strength, and poor adhesion to skin tissue. This makes them prone to displacement due to movement, affecting the stability of the wound healing environment. Natural polymer matrices (such as unmodified cellulose and ordinary chitosan), while exhibiting excellent biocompatibility, lack functional modification design, making it difficult to simultaneously achieve the integration of antibacterial, antioxidant, and pH-responsive indication functions, thus limiting their application in the field of smart wound dressings. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned technical problems by providing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel and its preparation method.

[0008] In view of this, the present invention provides a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel and its preparation method, wherein the hydrogel is prepared by physical crosslinking and chemical crosslinking of water, aldehyde-modified nanocellulose, water-soluble polysaccharide derivatives and anthocyanins;

[0009] The aldehyde-modified nanocellulose is prepared by aldehyde treatment with sodium periodate using nanocellulose extracted from cotton and linen plants as raw material.

[0010] The water-soluble polysaccharide derivative is one or more of carboxymethyl chitosan or quaternary ammonium salt chitosan;

[0011] The anthocyanin is blueberry anthocyanin;

[0012] Hydrogen bonds are formed between the aldehyde-modified nanocellulose, the water-soluble polysaccharide derivative, and the blueberry anthocyanin; and imine bonds are formed between the aldehyde-modified nanocellulose and the water-soluble polysaccharide derivative.

[0013] The hydrogel is used as a wound monitoring dressing.

[0014] Preferably, the nanocellulose extracted from cotton and linen plants is one or both of nanocellulose crystals and nanocellulose fibers.

[0015] Preferably, the water-soluble polysaccharide derivative is carboxymethyl chitosan.

[0016] Preferably, when preparing the aldehyde-modified nanocellulose, the weight ratio of sodium periodate to nanocellulose extracted from cotton and linen plants is 1:1-3.98.

[0017] A method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel includes the following steps:

[0018] Step 1: Mix the nanocellulose solution with the sodium periodate solution and stir the mixture at 30℃-60℃ and 100-200r / min for 3h-24h to obtain the first solution; wherein the weight ratio of sodium periodate to nanocellulose is 1:1-3.98.

[0019] Step 2: Add ethylene glycol to the first solution, wherein the molar ratio of ethylene glycol to sodium periodate in Step 1 is 1:1. Then, adjust the pH of the mixture to 4.5 using PBS solution, and stir the mixture at 20℃-40℃ and 100-200r / min for 1h-2h to obtain the second solution.

[0020] Step 3: The second solution is placed into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3-5 days. After dialysis, the solution is freeze-dried for 3 days. Deionized water is added to the dried product and ultrasonically dispersed for 24 hours to obtain a third solution with a mass fraction of 1.0 wt%.

[0021] Step 4: Add anthocyanin to the third solution and ultrasonically disperse for 1-2 hours to obtain the fourth solution;

[0022] Step 5: Add the water-soluble polysaccharide derivative to deionized water, wherein the ratio of the water-soluble polysaccharide derivative to deionized water is 1g:24ml, and stir for 3h-6h at 50℃-60℃ and 100-200r / min to obtain the fifth solution;

[0023] Step Six: Mix the fourth solution and the fifth solution in equal volumes, and stir for 20-30 minutes at 20℃-35℃ and 100-150r / min to complete physical and chemical crosslinking. Then centrifuge at 5000r / min for 10-20 minutes to remove air bubbles generated during the crosslinking process and ensure sufficient crosslinking to obtain the nanocellulose-based antibacterial and antioxidant wound indicator hydrogel.

[0024] Preferably, in step one, the nanocellulose is nanocellulose crystals, the reaction temperature is 45℃, and the stirring reaction time is 4h.

[0025] Preferably, in step three, the deionized water dialysis time is 4 days.

[0026] Preferably, in step four, the anthocyanin is blueberry anthocyanin, and the ultrasonic dispersion time is 1 hour.

[0027] Preferably, in step five, the water-soluble polysaccharide derivative is carboxymethyl chitosan, the stirring temperature is 60°C, and the stirring time is 3 hours.

[0028] Preferably, in step six, the stirring temperature is 20°C, the stirring time is 30 min, and the centrifugation time is 10 min.

[0029] The beneficial effects of this invention are:

[0030] It has a highly effective and safe antibacterial effect, significantly reducing the risk of wound infection;

[0031] Broad-spectrum antibacterial with excellent antibacterial rate: The core antibacterial component in the hydrogel, carboxymethyl chitosan (99% purity), works through a dual mechanism of disrupting bacterial cell membrane structure and inhibiting bacterial enzyme activity. It achieves an antibacterial rate of over 94% against common wound infection strains such as Staphylococcus aureus and Escherichia coli (far exceeding the conventional level of 80%-90% of traditional single antibacterial dressings), effectively inhibiting bacterial reproduction and blocking the spread of infection.

[0032] Safe and without side effects: Compared with traditional antibacterial dressings containing silver ions and antibiotics, this invention uses carboxymethyl chitosan, a natural polymer-derived antibacterial agent, to avoid skin irritation and cytotoxicity caused by heavy metal accumulation, as well as bacterial resistance caused by antibiotics. Biocompatibility tests have verified that it has no adverse effects on human cells and is suitable for long-term wound care.

[0033] Long-lasting and stable antioxidant properties accelerate the wound healing process;

[0034] High antioxidant efficiency and long duration: The blueberry anthocyanins (25% purity) in the hydrogel remove excess reactive oxygen species (ROS) from the wound through hydrogen ion transfer mechanism. Even when the anthocyanin content is only 0.1%, the hydrogel can still remove DPPH free radicals with an efficiency of 64% within 30 minutes. In addition, the anthocyanins are tightly bound to nanocellulose and carboxymethyl chitosan through hydrogen bonds, avoiding the defects of traditional small molecule antioxidants (such as vitamin C) that are easy to degrade and easily lost with exudate. It can provide long-lasting antioxidant protection for the wound, reduce the damage of ROS to fibroblast proliferation and collagen synthesis, and significantly shorten the healing period.

[0035] Antioxidant properties are adjustable: By adjusting the amount of blueberry anthocyanins added (such as the gradient change from 10mg to 35mg in Examples 1-5), it can be flexibly adapted to wounds with different degrees of oxidative stress (such as mild abrasions and deep burns), achieving precise matching of antioxidant effects and broadening the application scenarios of the product.

[0036] Precise pH-responsive color change indication enables real-time monitoring of wound condition;

[0037] High sensitivity to distinguish wound condition: The blueberry anthocyanins in the hydrogel also have pH-responsive colorimetric function. In an environment simulating the pH of healthy skin (5.5), it can show a significant color difference from an environment simulating the pH of chronic / infected wounds (7.2). Moreover, as the anthocyanin content increases, the color depth gradient changes clearly and can be directly observed with the naked eye without relying on professional testing equipment. This solves the problem of the lag in traditional dressings that require exudate sampling and laboratory testing to determine infection, and achieves real-time monitoring that is "instantaneous".

[0038] High indicator stability: Anthocyanins are bound to the hydrogel matrix through chemical bonds, making them less susceptible to interference from components such as proteins and electrolytes in wound exudate. The color change effect is stable and reproducible. At the same time, the hydrogel can maintain its structural integrity after swelling under different pH conditions, avoiding indicator failure due to matrix rupture and ensuring the reliability of monitoring results.

[0039] Excellent biocompatibility and mechanical stability ensure the safety and comfort of wound care;

[0040] Outstanding biocompatibility: After incubation with the hydrogel extract in human foreskin fibroblast culture test, the cell viability was no lower than that of the blank control group. Moreover, as the anthocyanin content increased, the cell density and morphology slightly improved, proving that the material has no inhibitory effect on cell proliferation, no sensitization or cytotoxicity, can adhere well to wound tissue, reduce foreign body stimulation, and reduce the risk of inflammatory response.

[0041] Stable structure and good fit: Using aldehyde-modified nanocellulose (extracted from cotton and linen plants) as the matrix, a three-dimensional network structure is formed through double cross-linking of hydrogen bonds (nanocellulose-anthocyanin, nanocellulose-carboxymethyl chitosan) and imine bonds (nanocellulose-carboxymethyl chitosan). The mechanical strength is significantly better than that of traditional unmodified cellulose hydrogels. At the same time, the hydrogel has moderate elasticity and swelling properties, which can closely adhere to the skin surface. Even in dynamic scenarios such as joint movement, it is not easy to shift or break. It can continuously maintain a moist environment on the wound and avoid secondary damage caused by dressing loosening.

[0042] Fifth, the preparation process is highly controllable, which is conducive to industrial production;

[0043] The preparation method of this invention has clear steps (aldehyde modification - dialysis dispersion - component mixing - crosslinking centrifugation), and key process parameters (such as the ratio of sodium periodate to nanocellulose 1:1-3.98, reaction temperature 30℃-60℃, centrifugation speed 5000r / min) are clearly defined and can be quantitatively controlled, without the need for special high-end equipment; at the same time, the raw materials (nanocellulose, carboxymethyl chitosan, blueberry anthocyanins) are widely available and cost-controllable, and the preparation process leaves no toxic solvent residues, which is in line with the concept of green production, facilitates large-scale mass production, and provides technical support for the industrialization and promotion of the product. Attached Figure Description

[0044] Figure 1 The image shows bacterial colonies on agar plates after the antibacterial and antioxidant wound indicator hydrogel prepared in Case 1-5 was co-cultured with Staphylococcus aureus and Escherichia coli.

[0045] Figure 2 This is a graph showing the Staphylococcus aureus inhibition rate of the antibacterial and antioxidant wound indicator hydrogels prepared in Case 1-5;

[0046] Figure 3 This is a graph showing the Escherichia coli inhibition rate of the antibacterial and antioxidant wound indicator hydrogels prepared in Case 1-5.

[0047] Figure 4 This is a staining image of live and dead cells 24 hours after preparation of antibacterial and antioxidant wound indicator hydrogels in Case 1-5;

[0048] Figure 5 This is a cell viability graph of the antibacterial and antioxidant wound indicator hydrogel prepared in Case 1-5 after 24 hours;

[0049] Figure 6 The following are pH color change graphs for implementation cases 1-5: (a) Actual images of cases 1-5 after different times at different pH levels; (b) Changes in a value and ΔE of implementation case 3 at different times at pH 5.5; (c) Changes in a value and ΔE of implementation case 3 at different times at pH 7.2; (d) Actual images of implementation case 3 on a human forearm after 20 minutes at different pH levels.

[0050] Figure 7 This is an antioxidant data graph of the antibacterial and antioxidant wound indicator hydrogels prepared in Case 1-5.

[0051] Figure 8 This is an electron microscope image of nanocellulose.

[0052] Figure 9 XRD patterns of nanocellulose and aldehyde-modified nanocellulose.

[0053] Figure 10 This is a Fourier transform infrared spectrum.

[0054] Figure 11 This is a molecular structure diagram of anthocyanins. Detailed Implementation

[0055] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0056] It should be noted that all directional and positional terms used in this invention, such as "up," "down," "left," "right," "front," "back," "vertical," "horizontal," "inner," "outer," "top," "lower," "lateral," "longitudinal," and "center," are only used to explain the relative positional relationships and connections between components in a specific state (as shown in the accompanying drawings). They are merely for the convenience of describing the invention and do not require the invention to be constructed and operated in a specific orientation; therefore, they should not be construed as limitations on the invention. Furthermore, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0057] In the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0058] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0059] Control represents the blank control group;

[0060] DACSA-0 represents the hydrogel prepared in Example 1;

[0061] DACSA-1 represents the hydrogel prepared in Example 2;

[0062] DACSA-2 represents the hydrogel prepared in Implementation Case 3;

[0063] DACSA-3 represents the hydrogel prepared in Implementation Case 4;

[0064] DACSA-4 represents the hydrogel prepared in Implementation Case 5;

[0065] aureus represents Staphylococcus aureus;

[0066] E. coli represents Escherichia coli;

[0067] A nanocellulose-based antibacterial and antioxidant wound indicator hydrogel, wherein the materials are arranged in mass percentages and the components include water, antibacterial agent, nanocellulose, and pH-responsive colorimetric indicator.

[0068] A nanocellulose-based antibacterial and antioxidant wound indicator hydrogel is composed of water, nanocellulose, water-soluble polysaccharide derivatives, and anthocyanins.

[0069] The nanocellulose is extracted from cotton and linen plants and modified by aldehyde alkylation; the water-soluble polysaccharide derivative is one or more of carboxymethyl chitosan or quaternary ammonium salt chitosan; the anthocyanin is blueberry anthocyanin.

[0070] The nanocellulose, carboxymethyl chitosan, and blueberry anthocyanin form hydrogen bonds, and the nanocellulose and carboxymethyl chitosan form imine bonds. Through physical and chemical cross-linking, a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel is prepared, which is suitable for wound monitoring dressings.

[0071] The carboxymethyl chitosan has a purity of 99%, and the anthocyanin has a purity of 25%.

[0072] The present invention discloses a method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel, comprising mixing a nanocellulose solution with a sodium periodate solution, heating, stirring, and performing aldehyde modification to obtain a first solution;

[0073] Ethylene glycol was mixed with the first solution, the pH was adjusted with PBS solution, and then the mixture was heated and stirred to obtain the second solution.

[0074] The second solution from step (2) was dialyzed with deionized water and then freeze-dried. Deionized water was added and the solution was ultrasonically dispersed to obtain a third solution with a mass fraction of 1.0 wt%.

[0075] The pH-responsive colorimetric indicator was mixed with the third solution from step (3) and ultrasonically dispersed to obtain the fourth solution.

[0076] Carboxymethyl chitosan was added to deionized water, heated, stirred, and mixed to obtain the fifth solution;

[0077] The fourth solution from step (4) and the fifth solution from step (5) were mixed in equal volumes, stirred, crosslinked, and centrifuged to obtain a nanocellulose-based hydrogel.

[0078] In step (1), nanocellulose includes one or more of nanocellulose crystals and nanocellulose fibers, the weight ratio of sodium periodate to nanocellulose is 1:1-3.98, the temperature is 30℃-60℃, the stirring speed is 100-200r / min, and the reaction time is 3h-24h.

[0079] In step (2), the molar ratio of ethylene glycol to sodium periodate in step (1) is 1:1, pH is 4.5, reaction temperature is 20℃-40℃, stirring speed is 100-200r / min, and reaction time is 1h-2h.

[0080] In step (3), the molecular weight cutoff of the dialysis bag is 3500 Da, the dialysis time is 3-5 days, the freeze-drying is 3 days, and the ultrasonic dispersion is 24 hours.

[0081] In step (4), the pH indicator includes one or more of blueberry anthocyanins and proanthocyanidins, and the ultrasonic dispersion time is 1-2 hours.

[0082] In step (5), the ratio of carboxymethyl chitosan to deionized water is 1g:24ml, the temperature is 50℃-60℃, the stirring speed is 100-200r / min, and the stirring time is 3h-6h.

[0083] In step (6), the reaction temperature is 20℃-35℃, the stirring time is 20min-30min, the stirring speed is 100-150r / min, the centrifugation speed is 5000r / min, and the centrifugation time is 10min-20min.

[0084] A nanocellulose-based antibacterial and antioxidant wound indicator hydrogel prepared by the method described above.

[0085] Implementation Case 1;

[0086] A method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel includes the following steps: dissolving sodium periodate in deionized water to prepare a 10% sodium periodate solution;

[0087] Sodium periodate solution was added to 1% nanocellulose solution, with a molar ratio of sodium periodate to nanocellulose of 3.98:1. PBS solution with pH 4 was added to adjust the pH of the mixed solution to 4.5, and then the mixture was magnetically stirred at 45°C for 4 hours in the dark.

[0088] Add ethylene glycol in an amount equal to sodium periodate to the solution in step (2), then stir magnetically for 1 hour, and pour the resulting solution into a dialysis bag and dialyze with deionized water for 4 days.

[0089] The solution after dialysis in step (3) was freeze-dried for 3 days, then removed and ultrasonically dispersed in deionized water to prepare a 1.0% aldehyde-modified nanocellulose solution.

[0090] Add 10 mg of anthocyanin to the solution obtained in step (4) and disperse it by ultrasonication for 1 h to obtain a cellulose-anthocyanin mixed solution.

[0091] Carboxymethyl chitosan was dissolved in deionized water under magnetic stirring at 60 degrees Celsius to prepare a 4.0% carboxymethyl chitosan solution.

[0092] Take 10 ml of each of the solutions obtained in steps (5) and (6), mix them, stir for 30 min at 20°C, and obtain a hydrogel through physical and chemical cross-linking.

[0093] The hydrogel obtained in step (7) was centrifuged at 5000 r / min for 10 min to eliminate air bubbles generated during cross-linking and to ensure sufficient cross-linking, thus producing an antibacterial and antioxidant wound indicator hydrogel.

[0094] Implementation Case 2;

[0095] Dissolve sodium periodate in deionized water to prepare a 10% sodium periodate solution.

[0096] Sodium periodate solution was added to 1% nanocellulose solution, with a molar ratio of sodium periodate to nanocellulose of 3.98:1. PBS solution with pH 4 was added to adjust the pH of the mixed solution to 4.5, and then the mixture was magnetically stirred at 45°C for 4 hours in the dark.

[0097] Add ethylene glycol in an amount equal to sodium periodate to the solution in step (2), then stir magnetically for 1 hour, and pour the resulting solution into a dialysis bag and dialyze with deionized water for 4 days.

[0098] The solution after dialysis in step (3) was freeze-dried for 3 days, then removed and ultrasonically dispersed in deionized water to prepare a 1.0% aldehyde-modified nanocellulose solution.

[0099] Add 20 mg of anthocyanin to the solution obtained in step (4) and disperse it by ultrasonication for 1 h to obtain a cellulose-anthocyanin mixed solution.

[0100] Carboxymethyl chitosan was dissolved in deionized water under magnetic stirring at 60 degrees Celsius to prepare a 4.0% carboxymethyl chitosan solution.

[0101] Take 10 ml of each of the solutions obtained in step (5) and step (6) and mix them in equal volumes. Stir for 30 min at 20°C and then crosslink the mixture with physical and chemical agents to obtain a hydrogel.

[0102] The hydrogel obtained in step (7) was centrifuged at 5000 r / min for 10 min to eliminate air bubbles generated during cross-linking and to ensure sufficient cross-linking, thus producing an antibacterial and antioxidant wound indicator hydrogel.

[0103] Implementation Case 3;

[0104] Dissolve sodium periodate in deionized water to prepare a 10% sodium periodate solution.

[0105] Sodium periodate solution was added to 1% nanocellulose solution, with a molar ratio of sodium periodate to nanocellulose of 3.98:1. PBS solution with pH 4 was added to adjust the pH of the mixed solution to 4.5, and then the mixture was magnetically stirred at 45°C for 4 hours in the dark.

[0106] Add ethylene glycol in an amount equal to sodium periodate to the solution in step (2), then stir magnetically for 1 hour, and pour the resulting solution into a dialysis bag and dialyze with deionized water for 4 days.

[0107] The solution after dialysis in step (3) was freeze-dried for 3 days, then removed and ultrasonically dispersed in deionized water to prepare a 1.0% aldehyde-modified nanocellulose solution.

[0108] Add 25 mg of anthocyanin to the solution obtained in step (4) and disperse it by ultrasonication for 1 h to obtain a cellulose-anthocyanin mixed solution.

[0109] Carboxymethyl chitosan was dissolved in deionized water under magnetic stirring at 60 degrees Celsius to prepare a 4.0% carboxymethyl chitosan solution.

[0110] Take 10 ml of each of the solutions obtained in step (5) and step (6) and mix them in equal volumes. Stir for 30 min at 20°C and then crosslink the mixture with physical and chemical agents to obtain a hydrogel.

[0111] The hydrogel obtained in step (7) was centrifuged at 5000 r / min for 10 min to eliminate air bubbles generated during cross-linking and to ensure sufficient cross-linking, thus producing an antibacterial and antioxidant wound indicator hydrogel.

[0112] Implementation Case 4;

[0113] Dissolve sodium periodate in deionized water to prepare a 10% sodium periodate solution.

[0114] Sodium periodate solution was added to 1% nanocellulose solution, with a molar ratio of sodium periodate to nanocellulose of 3.98:1. PBS solution with pH 4 was added to adjust the pH of the mixed solution to 4.5, and then the mixture was magnetically stirred at 45°C for 4 hours in the dark.

[0115] Add ethylene glycol in an amount equal to sodium periodate to the solution in step (2), then stir magnetically for 1 hour, and pour the resulting solution into a dialysis bag and dialyze with deionized water for 4 days.

[0116] The solution after dialysis in step (3) was freeze-dried for 3 days, then removed and ultrasonically dispersed in deionized water to prepare a 1.0% aldehyde-modified nanocellulose solution.

[0117] Add 30 mg of anthocyanin to the solution obtained in step (4) and disperse it by ultrasonication for 1 h to obtain a cellulose-anthocyanin mixed solution.

[0118] Carboxymethyl chitosan was dissolved in deionized water under magnetic stirring at 60 degrees Celsius to prepare a 4.0% carboxymethyl chitosan solution.

[0119] Take 10 ml of each of the solutions obtained in step (5) and step (6) and mix them in equal volumes. Stir for 30 min at 20°C and then crosslink the mixture with physical and chemical agents to obtain a hydrogel.

[0120] The hydrogel obtained in step (7) was centrifuged at 5000 r / min for 10 min to eliminate air bubbles generated during cross-linking and to ensure sufficient cross-linking, thus producing an antibacterial and antioxidant wound indicator hydrogel.

[0121] Implementation Case 5;

[0122] Dissolve sodium periodate in deionized water to prepare a 10% sodium periodate solution.

[0123] Sodium periodate solution was added to 1% nanocellulose solution, with a molar ratio of sodium periodate to nanocellulose of 3.98:1. PBS solution with pH 4 was added to adjust the pH of the mixed solution to 4.5, and then the mixture was magnetically stirred at 45°C for 4 hours in the dark.

[0124] Add ethylene glycol in an amount equal to sodium periodate to the solution in step (2), then stir magnetically for 1 hour, and pour the resulting solution into a dialysis bag and dialyze with deionized water for 4 days.

[0125] The solution after dialysis in step (3) was freeze-dried for 3 days, then removed and ultrasonically dispersed in deionized water to prepare a 1.0% aldehyde-modified nanocellulose solution.

[0126] Add 35 mg of anthocyanin to the solution obtained in step (4) and disperse it by ultrasonication for 1 h to obtain a cellulose-anthocyanin mixed solution.

[0127] Carboxymethyl chitosan was dissolved in deionized water under magnetic stirring at 60 degrees Celsius to prepare a 4.0% carboxymethyl chitosan solution.

[0128] Take 10 ml of each of the solutions obtained in step (5) and step (6) and mix them in equal volumes. Stir for 30 min at 20°C and then crosslink the mixture with physical and chemical agents to obtain a hydrogel.

[0129] The hydrogel obtained in step (7) was centrifuged at 5000 r / min for 10 min to eliminate air bubbles generated during cross-linking and to ensure sufficient cross-linking, thus producing an antibacterial and antioxidant wound indicator hydrogel.

[0130] Analysis of the antibacterial effect of antibacterial and antioxidant wound indicator hydrogels;

[0131] The prepared hydrogel was sterilized under ultraviolet light and then mixed with Staphylococcus aureus and Escherichia coli bacterial suspensions for 12 hours. The bacterial suspensions were then diluted with PBS and transferred to agar plates. After 18 hours of incubation, the colony counts were photographed and determined. Figures 1-3 As shown in the implementation cases 1-5, the antibacterial agent carboxymethyl chitosan effectively inhibited bacterial reproduction by disrupting bacterial cell membrane structure and inhibiting bacterial enzyme activity, compared with the blank control group. The inhibition rate against Staphylococcus aureus and Escherichia coli both reached over 97%.

[0132] Biocompatibility analysis of antibacterial and antioxidant wound indicator hydrogels;

[0133] After sterilizing the prepared hydrogel with UV light, it was incubated in a humid environment containing 5% CO2 for 3 hours. Human foreskin fibroblasts were incubated in a culture dish in a humid environment containing 5% CO2 for 3 hours. Then, hydrogel extraction solution was added, and incubation was carried out for 24 hours. The culture medium was then replaced with CCK-8 containing virgin medium, and incubation was continued for 4 hours. The absorbance was measured at 450 nm, and live and dead cells were stained and photographed. Figure 4 As can be seen from the stained and photographed images of live and dead cells, with the increase of anthocyanin content in Cases 1-5, cell density and morphology were slightly improved compared to the control group. Figure 5 The cell viability data also showed that the cell viability of the samples in Case 1-5 was no less than that of the blank control group, confirming the excellent biocompatibility of the hydrogel.

[0134] Analysis of pH-responsive colorimetric properties of antibacterial and antioxidant wound indicator hydrogels;

[0135] Samples prepared in Examples 1-5 were swollen for 20 min in PBS solution simulating healthy skin (pH 5.5) and for 20 min in PBS solution simulating chronic wound (pH 7.2), respectively. They were then removed, photographed, and measured using a Datacolor 850 colorimeter to record the hydrogel's red-green (a), yellow-blue (b), and total color difference (ΔE). Figure 6 As shown in (a), after 20 min, the hydrogel turned red and purple at pH 5.5 and pH 7.2, respectively, demonstrating a significant pH color change indication effect. Figure 6(b) At pH 5.5, the a value in Example 3 changed from 1.1 to 4.34, and the red saturation of the hydrogel increased. With increasing time, the a value increased, and the hydrogel color gradually turned redder. At 20 min, the ΔE value increased to 8.02, indicating that the color change was observable to the naked eye. This is because some anthocyanins bind protons, and their molecular structure changes to anthocyanin cations (flavylium cations), exhibiting a red color. Similarly, Figure 6 In (c), at pH 7.2, the b-value of the hydrogel increased from -6.36 to -1.7 after 20 minutes, and the blue saturation of the hydrogel decreased. With increasing time, the change in b-value was not significant. This is because the pH during hydrogel preparation was also 7.2, so the increase in swelling led to an increase in b-value, resulting in a lighter color. However, even when ΔE increased to 6.28, this color change was still noticeable to the naked eye. Figure 6 d).

[0136] Analysis of the antioxidant properties of antibacterial and antioxidant wound indicator hydrogels;

[0137] The in vitro antioxidant properties of hydrogels prepared in Case Studies 1-5 were studied using the DPPH free radical scavenging method. Figure 7 As the anthocyanin content increases, the phenolic hydroxyl content of the hydrogel also increases. Through the hydrogen ion transfer mechanism, free radicals are reduced, thus increasing the antioxidant properties of the hydrogel. The hydrogel with an anthocyanin content of only 0.1% achieved a free radical scavenging efficiency of 66.9% within 30 minutes, demonstrating the excellent antioxidant properties of the hydrogel.

[0138] Nanocellulose, 1 wt%, produced by Shandong Yuyue Home Textiles Co., Ltd., from textile waste cotton using the Tempo oxidation method. The nanocellulose fibers have a diameter of 10-14 nm, an average aspect ratio of 61.3, and an absolute value of 38.6 mV for the zeta potential. Figure 8 Electron micrograph of nanocellulose (provided by Joy Company);

[0139] Aldehyde-modified nanocellulose was prepared by sodium periodate oxidation, with an aldehyde content of 9.89 mmol / g.

[0140] The aldehyde content was tested using a semi-micro hydroxylamine hydrochloride determination method, with the following procedure: 1 g of hydroxylamine hydrochloride was weighed into an Erlenmeyer flask, 50 ml of methanol was added, and the solution was sonicated to obtain a hydroxylamine hydrochloride methanol solution. 0.5 ml of thymol blue was added as an indicator, and the solution turned red. 0.03 mol / L sodium hydroxide-methanol solution (concentration denoted as C) was added to the mixed solution until the pH reached 4.5, at which point the solution turned yellow. The volume of sodium hydroxide-methanol solution added at this point was recorded as V1. Then, 0.1 g (dry weight denoted as m) of wet aldehyde cellulose solution (pH 4) was added to the Erlenmeyer flask, and the solution turned red again. After reacting at room temperature for 24 hours, it was titrated with 0.03 mol / L sodium hydroxide-methanol solution until the pH reached 4.5, at which point the solution turned yellow and remained so for 10-15 seconds. The volume of sodium hydroxide-methanol solution consumed at this point was recorded as V2.

[0141] ;

[0142] The crystal structures of CNF, DACNF, CMCS, and freeze-dried hydrogel samples were analyzed using a Bruker D8 X-ray diffractometer. The scan rate was 1° / min, the scan range (2θ) was 5°–60°, the voltage was 20 kV, and the current was 20 mA. XRD patterns of nanocellulose and aldehyde-modified nanocellulose are shown below. Figure 9 As shown, by Figure 9 It can be seen that CNF has diffraction characteristic peaks at 2θ=16.5° and 22.7°, corresponding to the (110) and (200) crystal planes of cellulose, respectively. After oxidation with NaIO4, the diffraction characteristic peaks of CNF are consistent with those of CNF, indicating that NaIO4 oxidation did not change the crystal structure of cellulose. The formula for calculating the crystallinity of nanocellulose, CrI, is as follows, where I... 200 I represents the intensity of the diffraction peak in the crystalline region, specifically the maximum intensity at a diffraction angle of approximately 2θ = 22.6 °. am The intensity of the diffraction peak in the amorphous region is approximately the intensity of the diffraction peak at 2θ = 18.7 °. The crystallinity of the nanocellulose fiber is 77.1%, while that of the aldehyde-modified nanocellulose is 56.9%. This indicates that NaIO4 oxidation destroys part of the crystalline structure of CNF. On the other hand, the breakage and recombination of the pyranose ring during oxidation reduces the crystallinity of DACNF.

[0143] ;

[0144] The chemical composition of CNF, DACNF, CMCS, and freeze-dried hydrogel samples was determined and analyzed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), with a scanning range of 500-4000 cm⁻¹. -1 Compared to CNF, DACNF is at 1731 cm.-1 A significant peak appeared at the (C=O stretching) position, indicating successful oxidation of CNF; this is the aldehyde absorption peak. When DACNF is mixed with CMCS, the aldehyde groups on the DACNF molecular chain can undergo a Schiff base reaction with the amino groups in CMCS to form covalent crosslinks. The FTIR of the hydrogel DACSA-0 prepared in Example 1... Figure 10 It can be found that 1731 cm -1 The disappearance of the aldehyde absorption peak indicates that the aldehyde group on DACNF reacted successfully with the amino Schiff base of CMCS.

[0145] Carboxymethyl chitosan, degree of substitution ≥90, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0146] Anthocyanins, 25%, derived from blueberry anthocyanins, Shanghai Aladdin Biochemical Technology Co., Ltd. Figure 11 Provide molecular structure diagrams for Aladdin Company.

[0147] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A nanocellulose-based antibacterial and antioxidant wound indicator hydrogel, characterized in that: The hydrogel was prepared by physical and chemical crosslinking of water, aldehyde-modified nanocellulose, water-soluble polysaccharide derivatives and anthocyanins. The mixture was stirred for 20-30 minutes at 20℃-35℃ and 100-150r / min to complete the physical and chemical cross-linking, thereby obtaining the nanocellulose-based antibacterial and antioxidant wound indicator hydrogel. The aldehyde-modified nanocellulose is prepared by aldehyde treatment with sodium periodate using nanocellulose extracted from cotton and linen plants as raw material. The water-soluble polysaccharide derivative is one or more of carboxymethyl chitosan or quaternary ammonium salt chitosan; The anthocyanin is blueberry anthocyanin; Hydrogen bonds are formed between the aldehyde-modified nanocellulose, the water-soluble polysaccharide derivative, and the blueberry anthocyanin; and imine bonds are formed between the aldehyde-modified nanocellulose and the water-soluble polysaccharide derivative. The hydrogel is used as a wound monitoring dressing. Anthocyanins bind to the hydrogel matrix via chemical bonds.

2. The nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to claim 1, characterized in that: The nanocellulose extracted from cotton and linen plants is one or both of nanocellulose crystals and nanocellulose fibers.

3. The nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to claim 2, characterized in that: When preparing the aldehyde-modified nanocellulose, the weight ratio of sodium periodate to nanocellulose extracted from cotton and hemp plants is 1:1-3.

98.

4. A method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel, based on the nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to any one of claims 1-3, characterized in that: Includes the following steps: Step 1: Mix the nanocellulose solution with the sodium periodate solution and stir the mixture at 30℃-60℃ and 100-200r / min for 3h-24h to obtain the first solution; wherein the weight ratio of sodium periodate to nanocellulose is 1:1-3.

98. Step 2: Add ethylene glycol to the first solution, wherein the molar ratio of ethylene glycol to sodium periodate in Step 1 is 1:

1. Then, adjust the pH of the mixture to 4.5 using PBS solution, and stir the mixture at 20℃-40℃ and 100-200r / min for 1h-2h to obtain the second solution. Step 3: The second solution is placed into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3-5 days. After dialysis, the solution is freeze-dried for 3 days. Deionized water is added to the dried product and ultrasonically dispersed for 24 hours to obtain a third solution with a mass fraction of 1.0 wt%. Step 4: Add anthocyanin to the third solution and ultrasonically disperse for 1-2 hours to obtain the fourth solution; Step 5: Add the water-soluble polysaccharide derivative to deionized water, wherein the ratio of the water-soluble polysaccharide derivative to deionized water is 1g:24ml, and stir for 3h-6h at 50℃-60℃ and 100-200r / min to obtain the fifth solution; Step Six: Mix the fourth solution and the fifth solution in equal volumes, and stir for 20-30 minutes at 20℃-35℃ and 100-150r / min to complete physical and chemical crosslinking. Then centrifuge at 5000r / min for 10-20 minutes to remove air bubbles generated during the crosslinking process and ensure sufficient crosslinking to obtain the nanocellulose-based antibacterial and antioxidant wound indicator hydrogel.

5. The method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to claim 4, characterized in that: In step one, the nanocellulose is nanocellulose crystals, the reaction temperature is 45℃, and the stirring reaction time is 4h.

6. The method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to claim 5, characterized in that: In step three, the deionized water dialysis time is 4 days.

7. The method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to claim 6, characterized in that: In step four, the anthocyanin is blueberry anthocyanin, and the ultrasonic dispersion time is 1 hour.

8. The method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to claim 7, characterized in that: In step five, the water-soluble polysaccharide derivative is carboxymethyl chitosan, the stirring temperature is 60°C, and the stirring time is 3 hours.

9. The method for preparing a nanocellulose-based antibacterial and antioxidant wound indicator hydrogel according to claim 5, characterized in that: In step six, the stirring temperature is 20℃, the stirring time is 30 min, and the centrifugation time is 10 min.

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