Natural polymer-based multifunctional hydrogel wound dressing as well as preparation method and application thereof

Abstract

The invention provides a natural polymer-based multifunctional hydrogel wound dressing as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, oxidizing sodium alginate with sodium periodate to generate oxidized sodium alginate with aldehyde groups; meanwhile, dopamine is subjected to self-polymerization to form a polydopamine network, then the cuttlefish element nanoparticles are doped into the polydopamine network, and polydopamine wrapping the cuttlefish element nanoparticles is obtained; then connecting the oxidized sodium alginate with the polydopamine wrapping the cuttlefish element nano particles through EDC (Ethylene Dichloride) and NHS (N-Hydroxysuccinimide); finally, carboxymethyl chitosan is connected with oxidized sodium alginate connected with dopamine to form the natural polymer-based multifunctional hydrogel wound dressing. The raw materials used in the invention are natural polymer-based materials, and the obtained dressing is green, environment-friendly, excellent in biocompatibility and more suitable for human skin; the anti-oxidation antibacterial coating has the advantages of excellent anti-oxidation and antibacterial properties, good adhesion and excellent photo-thermal performance, and can effectively inhibit the growth of bacteria without hurting skin tissues.

Description

Technical Field

[0001] This invention belongs to the field of multifunctional wound dressings, specifically relating to a natural polymer-based multifunctional hydrogel wound dressing, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Diabetes is a chronic disease with a rising incidence rate worldwide. The global prevalence of diabetes is 88%, with 20% of cases involving diabetic wounds. Diabetic wounds are a typical complication of diabetes, often accompanied by cellular dysfunction, prolonged inflammation, and insufficient angiogenesis. These factors increase medical costs, complicate treatment, and reduce patients' quality of life. Once a wound develops, chronic inflammation maintains its inflammatory state, hindering healing. Typically, chronic inflammation is primarily caused by significant levels of reactive oxygen species, pro-inflammatory factors, and cellular pathogens, and is a major cause of persistent wounds. Wound healing in diabetic patients is often challenging because their skin no longer possesses typical regenerative capabilities. Therefore, actively seeking and identifying new and effective methods for treating diabetic wounds is crucial.

[0004] Hydrogels are considered ideal dressings due to their 3D structure, good permeability, excellent biocompatibility, and ability to provide a moist environment for wound healing, overcoming the shortcomings of traditional dressings. Sodium alginate and carboxymethyl chitosan are the most commonly used monomers for hydrogel wound dressings. Sodium alginate can activate macrophages and stimulate monocytes to produce interleukins and tumor necrosis factor, accelerating the healing of chronic wounds. Carboxymethyl chitosan has broad-spectrum antibacterial activity, inhibiting bacterial growth at the wound site. Furthermore, sodium alginate and carboxymethyl chitosan, as natural polymer-based hydrogels, exhibit excellent biocompatibility and degradation ability. However, hydrogel wound dressings synthesized solely from sodium alginate and carboxymethyl chitosan do not perform exceptionally well, particularly in antibacterial, antioxidant, and adhesive properties, and lack photothermal properties. Dopamine, as a mussel material, can improve the adhesion and antioxidant capacity of hydrogels. Chinese patent document CN106075553A discloses a biomedical adhesive capable of cross-linking with wound skin tissue and its preparation method. The adhesive is prepared as follows: First, sodium alginate is oxidized with sodium periodate to obtain dialdehyde sodium alginate. After dialyzing and drying, the product is dissolved in MES buffer solution, and then carboxymethyl chitosan is added. After stirring for 6-12 hours, a certain amount of N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and dopamine are added sequentially. The reaction is continued under nitrogen protection for 8-24 hours, followed by dialyzing, freeze-drying, and storage in a cool, dry place. However, this adhesive does not address antioxidant properties, photothermal properties, etc., and both of these properties are also suboptimal. Chinese patent document CN106581734A discloses a preparation method for a highly antibacterial alginate dressing. This invention uses sodium periodate to oxidize sodium alginate and mixes silver nitrate, zinc nitrate, and trisodium citrate solution to prepare a silver-zinc antibacterial agent. Dopamine hydrochloride is then combined with the oxidized sodium alginate, adhering to the surface of the oxidized sodium alginate through a dopamine polymerization reaction. The silver and zinc ions are uniformly loaded onto the surface of the oxidized sodium alginate using the reduction and chelation effect of polydopamine, forming a spinning solution. After vacuum degassing, the solution is spun, coagulated, stretched, washed, wound, and dried, then needle-punched to form a dressing, resulting in a highly antibacterial alginate dressing. However, this invention's dressing does not address antioxidant or photothermal properties, and both of these properties are suboptimal. Furthermore, the invention uses metallic silver and zinc as antibacterial agents, which are toxic and could harm the human body, and pose a risk of environmental pollution. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a natural polymer-based multifunctional hydrogel wound dressing, its preparation method, and its applications. The invention first oxidizes sodium alginate with sodium periodate to generate sodium alginate oxide with aldehyde groups; simultaneously, dopamine self-polymerizes to form a polydopamine network, then incorporates cucurbitacin nanoparticles into the polydopamine network to obtain polydopamine encapsulating the cucurbitacin nanoparticles; next, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are used to link the sodium alginate oxide to the polydopamine encapsulating the cucurbitacin nanoparticles; finally, carboxymethyl chitosan is linked to the dopamine-linked sodium alginate oxide to form the natural polymer-based multifunctional hydrogel wound dressing. The raw materials used in this invention are all natural polymer-based materials, resulting in a green, environmentally friendly dressing with excellent biocompatibility, making it more suitable for human skin; it possesses excellent antioxidant and antibacterial properties, good adhesion, and excellent photothermal properties, effectively inhibiting bacterial growth without harming skin tissue.

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

[0007] The first aspect of this invention provides a method for preparing a natural polymer-based multifunctional hydrogel wound dressing, comprising the steps of:

[0008] (1) Sodium alginate (SA) was dissolved in water, sodium periodate was added, and an oxidation reaction was carried out; then ethylene glycol was added, and after reaction, dialysis and freeze drying, oxidized sodium alginate (OSA) was obtained.

[0009] (2) Dopamine (DA) was dissolved in NaOH aqueous solution and polymerized to obtain polydopamine (PDA); cuttlefish olein nanoparticles were added and reacted to obtain polydopamine (cuttlefish olein@PDA) encapsulated with cuttlefish olein nanoparticles.

[0010] (3) Dissolve sodium oxidized alginate (OSA) in water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate it; then add polydopamine (cucurbitacin@PDA) encapsulating cucurbitacin nanoparticles, and obtain OSA-cucurbitacin@PDA through reaction, dialysis and freeze drying;

[0011] (4) Mix OSA-cuttlefish extract@PDA solution with carboxymethyl chitosan solution evenly, and then react to form a gel to obtain a natural polymer-based multifunctional hydrogel wound dressing.

[0012] According to a preferred embodiment of the present invention, in step (1), the mass ratio of sodium alginate to water is 0.001-0.1 g / mL.

[0013] According to a preferred embodiment of the present invention, in step (1), the mass ratio of sodium alginate (SA) to sodium periodate is 1:1-1.5.

[0014] According to a preferred embodiment of the present invention, in step (1), the oxidation reaction temperature is room temperature, the oxidation reaction time is 3-8 hours, and the oxidation reaction is carried out under light-proof and stirring conditions.

[0015] According to a preferred embodiment of the present invention, in step (1), the mass ratio of sodium alginate (SA) to ethylene glycol is 0.1-1 g / mL.

[0016] According to a preferred embodiment of the present invention, in step (1), the reaction temperature after adding ethylene glycol is room temperature, the reaction time is 0.5-2h, and the reaction is carried out under stirring conditions.

[0017] According to a preferred embodiment of the present invention, in step (1), dialysis is performed using a dialysis bag with a molecular weight cutoff Mw of 8000 Da, and the dialysis time is 2-4 days, with the water being changed every 5-7 hours during the dialysis process.

[0018] According to a preferred embodiment of the present invention, in step (2), the pH of the NaOH aqueous solution is 10-12; and the mass ratio of dopamine (PDA) to the volume ratio of the NaOH aqueous solution is 0.001-0.01 g / mL.

[0019] According to a preferred embodiment of the present invention, in step (2), the polymerization reaction temperature is room temperature, the polymerization reaction time is 10-30 min, and the polymerization reaction is carried out under stirring conditions.

[0020] According to a preferred embodiment of the present invention, in step (2), the cuttlefish oleoresin nanoparticles have a particle size of 100-200 nm. The cuttlefish oleoresin nanoparticles are prepared by existing methods.

[0021] According to a preferred embodiment of the present invention, in step (2), the mass ratio of dopamine (DA) to cucurbitacin nanoparticles is 1:1-3.

[0022] According to a preferred embodiment of the present invention, in step (2), the reaction temperature after adding cuttlefish oleoresin nanoparticles is room temperature, the reaction time is 20-40 min, and the reaction is carried out under stirring conditions.

[0023] According to a preferred embodiment of the present invention, in step (3), the mass ratio of oxidized sodium alginate (OSA) to water is 0.01-0.1 g / mL.

[0024] According to a preferred embodiment of the present invention, in step (3), the mass ratio of sodium oxidized alginate (OSA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is 1:0.5-1.5:0.5-0.6.

[0025] According to a preferred embodiment of the present invention, in step (3), the activation temperature is 30-40°C, the activation time is 20-40 min, and the activation is carried out under inert gas protection and stirring conditions. Preferably, the inert gas is nitrogen or argon.

[0026] According to a preferred embodiment of the present invention, in step (3), the mass ratio of oxidized sodium alginate (OSA) to dopamine in step (2) is 1:0.2-0.4.

[0027] According to a preferred embodiment of the present invention, in step (3), the reaction temperature after adding polydopamine (cucurbitacin@PDA) encapsulating cucurbitacin nanoparticles is 30-40°C, the reaction time is 10-14 h, and the reaction is carried out under inert gas protection and stirring conditions. Preferably, the inert gas is nitrogen or argon.

[0028] According to a preferred embodiment of the present invention, in step (3), dialysis is performed using a dialysis bag with a molecular weight cutoff of Mw = 8000 Da, the dialysis time is 2-4 days, and the water is changed every 5-7 hours during the dialysis process.

[0029] According to a preferred embodiment of the present invention, in step (4), the OSA-cucurbitacin@PDA solution is a PBS solution of OSA-cucurbitacin@PDA with a mass concentration of 2-6%; the carboxymethyl chitosan solution is a PBS solution of carboxymethyl chitosan with a mass concentration of 2-6%; and the pH of the PBS buffer used as the solvent is 7.4.

[0030] According to a preferred embodiment of the present invention, in step (4), the mass ratio of OSA-cucurbitacin@PDA in the OSA-cucurbitacin@PDA solution to carboxymethyl chitosan in the carboxymethyl chitosan solution is 1:4-6.

[0031] According to a preferred embodiment of the present invention, in step (4), the reaction gelation conditions are a static reaction at room temperature for 6-8 hours.

[0032] In a second aspect, the present invention provides a natural polymer-based multifunctional hydrogel wound dressing, which is prepared by the above method.

[0033] In a third aspect, the present invention provides the application of the above-mentioned natural polymer-based multifunctional hydrogel wound dressing in a diabetic wound healing dressing.

[0034] The technical features and beneficial effects of this invention are as follows:

[0035] 1. This invention first oxidizes sodium alginate with sodium periodate to generate sodium alginate oxide with aldehyde groups; simultaneously, dopamine self-polymerizes to form a polydopamine network, then incorporating cucurbitacin nanoparticles into the polydopamine network to obtain polydopamine encapsulating cucurbitacin nanoparticles; next, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are used to link the sodium alginate oxide to the polydopamine encapsulating the cucurbitacin nanoparticles; finally, carboxymethyl chitosan is linked to the dopamine-linked sodium alginate oxide to form a natural polymer-based multifunctional hydrogel wound dressing. The preparation conditions of this invention are easy to achieve, and all raw materials used are natural polymer-based materials, making it green and environmentally friendly.

[0036] 2. In this invention, sodium alginate is first oxidized with sodium periodate to obtain a dialdehyde structure. Then, sodium periodate is removed by dialysis to avoid its oxidation of other monomers and its impact on gel performance. Next, dopamine is first self-polymerized to incorporate cucurbitacin nanoparticles into the polydopamine network, thus firmly fixing the cucurbitacin nanoparticles in the gel. Then, polydopamine is linked to oxidized sodium alginate, followed by dialysis to remove EDC and NHS, and finally linked with carboxymethyl chitosan to form a gel. The reaction sequence in this invention cannot be changed; altering the reaction sequence will not yield the dressing with the structure and properties of this invention. The reaction in this invention is carried out in four steps. Using any combination of steps will not achieve the superior effects of this invention. For example, the pH required for the self-polymerization of dopamine and the activation reactions of EDC and NHS differs. If carboxymethyl chitosan is added during the NHS and EDC activation reactions in a one-pot process, the amino groups of carboxymethyl chitosan may link to the carboxyl groups of oxidized sodium alginate, potentially leading to gelation midway and affecting the reaction between polydopamine and sodium alginate. Furthermore, the ratio of the raw materials in this invention must be appropriate; otherwise, the dressing with the structure and properties of this invention cannot be obtained.

[0037] 3. This invention uses sodium alginate and carboxymethyl chitosan as the main gel materials, and then adds dopamine and cuttlefish protein nanoparticles. All of these components are natural polymer-based materials. The resulting dressing is green and environmentally friendly, with excellent biocompatibility and good blood compatibility, making it more suitable for human skin.

[0038] 4. The addition of dopamine and cuttlefishene nanoparticles in this invention endows the hydrogel with excellent photothermal properties, thereby improving its antibacterial performance. The catechin groups carried on the surface of the dopamine and cuttlefishene nanoparticles possess antioxidant capabilities, capable of capturing free radicals and exhibiting antioxidant properties. The dopamine and cuttlefishene nanoparticles of this invention have similar structures and exert synergistic effects in photothermal, antioxidant, antibacterial, biocompatibility, hemostatic, and adhesive properties, while also synergizing with other components to further enhance these properties.

[0039] 5. The photothermal properties of the dressing of this invention are measured by an 808 laser (10 min, 2 W / cm²).-2 When irradiated, the temperature rises to about 60°C, which can effectively inhibit the growth of bacteria without harming skin tissue, thus demonstrating the excellent photothermal performance of this invention.

[0040] 6. The preparation method of the present invention, as a whole, enables the dressing obtained by the present invention to have excellent antioxidant and antibacterial properties, good compatibility, adhesion and coagulation properties, and excellent photothermal properties, which can effectively inhibit bacterial growth without damaging skin tissue. Attached Figure Description

[0041] Figure 1 Image a shows the infrared spectra of sodium alginate before and after oxidation in S1; image b shows the infrared spectra of PDA, cuttlefishene nanoparticles, and cuttlefishene@PDA in S2; image c shows the infrared spectra of CMCS, OSA, OSA-cuttlefishene@PDA, and CMCS-OSA-cuttlefishene@PDA in S3 and S4; image d shows the infrared spectra of sodium alginate before and after oxidation in S1. 1 H NMR spectrum;

[0042] Figure 2 Temperature curves of different gels under 808 laser irradiation in Experiment Example 1;

[0043] Figure 3 This is a graph showing the free radical scavenging rate of different gels tested using DPPH as an indicator in Experiment Example 2.

[0044] Figure 4 The hemolysis rate of the gel in Experiment Example 3 is shown in the graph.

[0045] Figure 5 The coagulation index graph of the gel in Experiment Example 4;

[0046] Figure 6 The graph shows the inhibition rate of the gel against Staphylococcus aureus and Escherichia coli in Experiment Example 5. Detailed Implementation

[0047] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. It should be noted that the specific embodiments described are explanations of the present invention and not limitations thereof.

[0049] In the embodiments, unless otherwise specified, all raw materials used are commercially available; and unless otherwise specified, all methods used are based on existing technologies.

[0050] In the examples, the cuttlefish spore nanoparticles were prepared according to the method described in the literature “Superior Performance of Polyurethane Based on Natural Melanin Nanoparticles, Biomacromolecules 2016, 17, 3782-3789”.

[0051] Example 1:

[0052] A method for preparing a natural polymer-based multifunctional hydrogel wound dressing includes the following steps:

[0053] S1. Dissolve 1g of sodium alginate (SA) in 100ml of water, add 1.07g of sodium periodate, and stir at room temperature in the dark for 5 hours. Then add 2mL of ethylene glycol and stir at room temperature for 1 hour. Transfer the product to a dialysis bag (Mw = 8000Da), dialyze for 3 days, changing the water every 6 hours during dialysis, and freeze-dry to obtain oxidized sodium alginate (OSA).

[0054] S2. Dissolve 0.15g of dopamine (DA) in 20ml of NaOH (pH=11) aqueous solution and stir at room temperature for 20min to form polydopamine (PDA). Then add 0.3g of cuttlefish oleoresin nanoparticles (particle size 150-200nm) and stir at room temperature for 30min to obtain a solution containing polydopamine (cuttlefish oleoresin@PDA) encapsulated with cuttlefish oleoresin nanoparticles.

[0055] S3. Dissolve 0.5 g of sodium alginate in 30 ml of water, add 0.484 g of EDC and 0.291 g of NHS, and activate by stirring at 37 °C for 30 min under a nitrogen atmosphere. Then add the cuttlefish protein@PDA solution prepared in S2. React by stirring at 37 °C under a nitrogen atmosphere for 12 h. Transfer the product to a dialysis bag (Mw = 8000 Da) and dialyze for 3 days, changing the water every 6 h during dialysis. Freeze-dry to obtain OSA-cuttlefish protein@PDA.

[0056] S4. Dissolve OSA-cuttlefish extract@PDA in PBS buffer solution at pH 7.4 to prepare a wt% solution; dissolve carboxymethyl chitosan (CMCS) (Maclean) in PBS solution at pH 7.4 to prepare a wt% solution; mix the OSA-cuttlefish extract@PDA solution and the carboxymethyl chitosan solution, stir at room temperature for 30 s, wherein the mass ratio of OSA-cuttlefish extract@PDA to carboxymethyl chitosan is 1:5, and let stand at room temperature for 7 h to form a gel, to obtain a natural polymer-based multifunctional hydrogel wound dressing, namely CMCS-OSA-cuttlefish extract@PDA hydrogel, denoted as CMCS-OSA-cuttlefish extract (0.3 g)@PDA (gel 3).

[0057] Figure 1 a is the FT-IR spectrum of SA and OSA, where 1610 cm⁻¹ is the FT-IR spectrum of OSA. -1 The absorption peak for the asymmetric stretching vibration of -COO- is 1421 cm⁻¹. -1 Symmetric stretching absorption peak belonging to -COO-, 1028 cm⁻¹ -1 It is a stretching vibration of -COC-. At 3435cm -1 The relatively broad peak at 1734 cm⁻¹ belongs to the stretching vibration of -OH groups. This peak narrows after oxidation, indicating a decrease in the number of -OH groups. Furthermore, the OSA obtained after oxidation with NaIO₄ shows a peak at 1734 cm⁻¹. -1 A new characteristic absorption peak appeared, which is the vibrational absorption peak of the C=O bond on the aldehyde group. The results show that the experiment successfully used NaIO4 to oxidize the adjacent hydroxyl groups at the C-2 and C-3 necks of the SA monomer to aldehyde groups, and the SA spectrum is similar to other parts of the OSA spectrum, indicating that most other functional groups remained unchanged during the reaction.

[0058] pass 1 H NMR spectrum ( Figure 1 d) It can be seen that the δ values ​​of SA and OSA in the range of 5.0–3.5 belong to the proton signal of the alginate backbone, while OSA… 1 In the 1H NMR spectrum, two additional characteristic signal peaks were observed at 5.4 ppm and 5.6 ppm compared to SA. This is due to the formation of a hemiacetal between the aldehyde group and the adjacent hydroxyl group. The proton of the hemiacetal produces these characteristic signal peaks, which also results in a peak at 1734 cm⁻¹ in the infrared spectrum. -1 The absorption peak is not obvious. (By...) 1 1H NMR and FT-IR spectra confirmed that OSA was successfully prepared using sodium periodate.

[0059] Depend on Figure 1 As shown in b, no new peaks were found in the infrared spectrum of Cucurbitacin@PDA, indicating that the encapsulation of Cucurbitacin nanoparticles by polydopamine is a physical encapsulation.

[0060] from Figure 1 As can be seen from the FT-IR spectrum of c, compared to OSA, the OSA-cucurbitacin@PDA FT-IR spectrum shows a 1560 cm⁻¹. -1 and 1288cm -1 The absorption peaks of amide II and amide III bands were observed. These results indicate that an amide reaction occurred between OSA and cuttlefish olein@PDA. Compared with the CMCS spectrum, CMCS-OSA-cuttlefish olein@PDA showed absorption peaks at 1617 cm⁻¹. -1 An absorption peak for the stretching vibration of the imine group appeared at 1430 cm⁻¹. -1The amino absorption peak at 1734 cm⁻¹ weakened, while compared with the OSA spectrum, the peak at 1734 cm⁻¹ was stronger. -1 The disappearance of the aldehyde absorption peak indicates that the amino group on the CMCS molecular chain reacted with the aldehyde group of OSA, and the CMCS-OSA-cuttlefish olein@PDA hydrogel was successfully prepared.

[0061] Example 2

[0062] A method for preparing a natural polymer-based multifunctional hydrogel wound dressing is described in Example 1, except that in step S2, the amount of cuttlefish oleoresin nanoparticles used is 0.2g; other steps and conditions are the same as in Example 1. The resulting dressing is designated as CMCS-OSA-cuttlefish oleoresin (0.2g)@PDA (gel 2).

[0063] Example 3

[0064] A method for preparing a natural polymer-based multifunctional hydrogel wound dressing is described in Example 1, except that in step S2, the amount of cuttlefish oleoresin nanoparticles used is 0.4g; other steps and conditions are the same as in Example 1. The resulting dressing is designated as CMCS-OSA-cuttlefish oleoresin (0.4g)@PDA (gel 4).

[0065] Comparative Example 1

[0066] A method for preparing a hydrogel wound dressing, comprising the following steps:

[0067] S1. Dissolve 1g of sodium alginate (SA) in 100ml of water, add 1.07g of sodium periodate, and stir at room temperature in the dark for 5 hours. Then add 2mL of ethylene glycol and stir at room temperature for 1 hour. Transfer the product to a dialysis bag (Mw = 8000Da), dialyze for 3 days, changing the water every 6 hours during dialysis, and freeze-dry to obtain oxidized sodium alginate (OSA).

[0068] S2. Dissolve OSA in PBS buffer solution at pH 7.4 to prepare a wt% solution; dissolve carboxymethyl chitosan (CMCS) (Maclean) in PBS buffer solution at pH 7.4 to prepare a wt% solution; mix the OSA solution and the carboxymethyl chitosan solution, stir at room temperature for 30 seconds, wherein the mass ratio of OSA to carboxymethyl chitosan is 1:5, and let stand at room temperature for 7 hours to obtain a hydrogel wound dressing, namely CMCS-OSA (gel 0).

[0069] Comparative Example 2

[0070] A method for preparing a hydrogel wound dressing, comprising the following steps:

[0071] S1. Dissolve 1g of sodium alginate (SA) in 100ml of water, add 1.07g of sodium periodate, and stir at room temperature in the dark for 5 hours. Then add 2mL of ethylene glycol and stir at room temperature for 1 hour. Transfer the product to a dialysis bag (Mw = 8000Da), dialyze for 3 days, changing the water every 6 hours during dialysis, and freeze-dry to obtain oxidized sodium alginate (OSA).

[0072] S2. Dissolve 0.15g of dopamine (DA) in 20ml of NaOH (pH=11) aqueous solution and stir at room temperature for 20min to form polydopamine (PDA) and obtain PDA solution.

[0073] S3. Dissolve 0.5g of sodium alginate oxide in 30ml of water, add 0.484g of EDC and 0.291g of NHS, and activate by stirring at 37°C for 30min under a nitrogen atmosphere. Then add the PDA solution prepared in S2. React by stirring at 37°C under a N2 atmosphere for 12h. Transfer the product to a dialysis bag (Mw = 8000Da), dialyze for 3 days, changing the water every 6h during dialysis, and freeze-dry to obtain OSA@PDA.

[0074] S4. Dissolve OSA@PDA in PBS buffer solution at pH 7.4 to prepare a wt% solution; dissolve carboxymethyl chitosan (CMCS) (Maclean) in PBS buffer solution at pH 7.4 to prepare a wt% solution; mix the OSA@PDA solution and the carboxymethyl chitosan solution, stir at room temperature for 30 s, wherein the mass ratio of OSA@PDA to carboxymethyl chitosan is 1:5, and let stand at room temperature for 7 h to form a gel, thus obtaining a hydrogel wound dressing, namely CMCS-OSA-PDA (gel 1).

[0075] Test Example 1

[0076] Photothermal capability test

[0077] Test samples: hydrogels prepared in Examples 1-3 and Comparative Examples 1-2.

[0078] Test method: Take equal volumes of hydrogel and place them in centrifuge tubes. Insert a thermocouple probe into the gel and measure the temperature using an 808nm laser (2W·cm²). -2 Under irradiation, the temperature change of the gel was recorded every 1 minute for a total of 10 minutes, and temperature rise curves of different materials over time were plotted. Figure 2 ).

[0079] Depend on Figure 2 It can be seen that the photothermal temperature of the gel continuously increases with the addition of polydopamine and cuttlefish oleoresin nanoparticles. Among them, gel 3 exhibits the best photothermal performance.

[0080] Experimental Example 2

[0081] In vitro antioxidant capacity test

[0082] Test samples: hydrogels prepared in Examples 1-3 and Comparative Examples 1-2.

[0083] Test Method: The antioxidant capacity of cuttlefish scavenging nanoparticles and hydrogels was determined using the DPPH free radical scavenging method to assess their ability to scavenge free radicals. A DPPH / ethanol solution (0.1 mM) was prepared; the sample was then immersed in the DPPH solution in the dark at 37°C for 0.5 h. The absorbance of the mixed reaction was measured at 517 nm. The percentage of DPPH scavenging capacity was calculated using the following formula:

[0084] Inhibition rate = (1-A / A) i )×100%

[0085] Where A i A represents the initial absorbance of the DPPH solution. t The absorbance is the result of mixing the DPPH solution with the sample for a certain period of time.

[0086] Depend on Figure 3 It can be seen that the free radical scavenging ability of the gel gradually increases with the addition of polydopamine and cuttlefish pigment nanoparticles, and the higher the content of polydopamine and cuttlefish pigment nanoparticles, the stronger the free radical scavenging ability.

[0087] Experimental Example 3

[0088] hemolysis experiment

[0089] Test samples: hydrogels prepared in Example 1 and Comparative Examples 1-2.

[0090] Test Method: The blood compatibility of the hydrogel was evaluated using sheep red blood cells. Simply put, sheep blood was diluted to a concentration of 5% (v / v) with PBS (pH=7.4). At 37°C, an equal volume of hydrogel and an equal volume of sheep blood were added to a centrifuge tube, incubated for 1 hour, and then centrifuged at 3000 rpm for 5 minutes. The supernatant was collected, and the absorbance at 540 nm was measured using a UV spectrophotometer. Additionally, 0.1% Triton X-100 was used as a positive control, and PBS as a negative control. Each experiment was repeated three times. The percentage of hemolysis was calculated using the formula:

[0091] Hemolysis rate (%) = [(Ap-Ab) / (At-Ab)] × 100%, where Ap is the absorbance value of the hydrogel group; At is the absorbance value of the positive control Triton X-100; and Ab is the absorbance value of PBS.

[0092] Depend on Figure 4As can be seen, the 0.1% Triton X-100 supernatant, used as the positive control group, was bright red, indicating red blood cell rupture and hemoglobin leakage. The PBS group and hydrogel group were colorless, with a hemolysis rate of <5%, indicating good blood compatibility.

[0093] Test Example 4

[0094] In vitro coagulation ability test

[0095] Test samples: hydrogels prepared in Example 1 and Comparative Examples 1-2.

[0096] Test Method: The coagulation properties of the hydrogel were evaluated using sheep red blood cells. Simply put, at 37°C, the same volume of hydrogel and the same volume of reactivated sheep blood (250 μL whole blood plus 50 μL of 25 mM CaCl2 aqueous solution) were added to a centrifuge tube. Gauze was used as a control. 50 μL of untreated recalcified whole blood was incubated at the same time (negative control) (Control), with absorbance reference value used as a control. 50 μL of unincubated recalcified whole blood was used as a blank. The whole blood coagulation properties of the hydrogel were evaluated using the following formula:

[0097] Blood coagulation index (BCI) = [(As-Ao) / (Ab-Ao)] × 100%

[0098] Where “As” represents the absorbance of the supernatant of each group of samples;

[0099] “Ab” indicates the absorbance of the supernatant of the blank control;

[0100] “Ao” indicates the absorbance of deionized water.

[0101] All of the above experiments were repeated three times.

[0102] Depend on Figure 5 It can be seen that the addition of polydopamine and cuttlefishene nanoparticles can improve the coagulation ability of the gel. The amino groups in the dynamic Schiff base and the catechol structure of the cuttlefishene nanoparticles may promote blood coagulation.

[0103] Experimental Example 5

[0104] Antibacterial performance test

[0105] Test sample: Hydrogel prepared in Example 1.

[0106] Test Method: The antibacterial properties of the gel were evaluated using *Escherichia coli* and *S. aureus*. First, a suitable concentration of bacteria was cultured in PBS (pH 7.4) at 37°C for 24 hours as a blank control. Simultaneously, the same concentration of bacteria was added to the gel and cultured at 37°C for 24 hours. During the culture period, the gel was irradiated with an 808 nm laser every 8 hours for 10 minutes each time. After 24 hours, the gel was plated and cultured at 37°C for another 24 hours. The bacterial density was estimated using the CFU (Cellular Fusion Unit) counting method.

[0107] Depend on Figure 6 It can be seen that the gel exhibits excellent antibacterial and bactericidal capabilities under laser irradiation. In the figure, sample PBS+NIR refers to PBS co-cultured with bacteria while being irradiated with an 808 laser using the method described above; sample gel+NIR refers to gel 3 co-cultured with bacteria while being irradiated with an 808 laser using the method described above; sample gel refers to gel 3 co-cultured with bacteria without laser irradiation.

Claims

1. A method for preparing a natural polymer-based multifunctional hydrogel wound dressing, comprising the following steps: (1) Sodium alginate (SA) was dissolved in water, sodium periodate was added, and an oxidation reaction was carried out; then ethylene glycol was added, and after reaction, dialysis and freeze drying, oxidized sodium alginate (OSA) was obtained. (2) Dopamine (DA) was dissolved in NaOH aqueous solution and polymerized to obtain polydopamine (PDA); cuttlefish olein nanoparticles were added and reacted to obtain polydopamine (cuttlefish olein@PDA) encapsulated with cuttlefish olein nanoparticles. (3) Dissolve sodium oxidized alginate (OSA) in water, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate it; then add polydopamine (cucurbitacin@PDA) encapsulating cucurbitacin nanoparticles, and obtain OSA-cucurbitacin@PDA through reaction, dialysis and freeze drying; (4) Mix OSA-cuttlefish extract@PDA solution with carboxymethyl chitosan solution evenly, and then react to form a gel to obtain a natural polymer-based multifunctional hydrogel wound dressing.

2. The method for preparing the natural polymer-based multifunctional hydrogel wound dressing according to claim 1, characterized in that, Step (1) includes one or more of the following conditions: i. The mass ratio of sodium alginate to water is 0.001-0.1 g / mL; ii. The mass ratio of sodium alginate (SA) to sodium periodate is 1:1-1.5; iii. The oxidation reaction temperature is room temperature, the oxidation reaction time is 3-8 hours, and the oxidation reaction is carried out under light-proof and stirring conditions; iv. The mass ratio of sodium alginate (SA) to ethylene glycol is 0.1-1 g / mL; v. The reaction temperature after adding ethylene glycol is room temperature, the reaction time is 0.5-2 hours, and the reaction is carried out under stirring conditions; vi. Dialysis uses dialysis bags with a molecular weight cutoff of Mw = 8000 Da. The dialysis time is 2-4 days, and the water is changed every 5-7 hours during the dialysis process.

3. The method for preparing the natural polymer-based multifunctional hydrogel wound dressing according to claim 1, characterized in that, Step (2) includes one or more of the following conditions: i. The pH of the NaOH aqueous solution is 10-12; the mass ratio of dopamine (PDA) to the volume ratio of the NaOH aqueous solution is 0.001-0.01 g / mL; ii. The polymerization reaction temperature is room temperature, the polymerization reaction time is 10-30 min, and the polymerization reaction is carried out under stirring conditions.

4. The method for preparing the natural polymer-based multifunctional hydrogel wound dressing according to claim 1, characterized in that, In step (2), the cuttlefish spore nanoparticles have a particle size of 100-200 nm.

5. The method for preparing the natural polymer-based multifunctional hydrogel wound dressing according to claim 1, characterized in that, In step (2), the mass ratio of dopamine (DA) to cuttlefish oleoresin nanoparticles is 1:1-3.

6. The method for preparing the natural polymer-based multifunctional hydrogel wound dressing according to claim 1, characterized in that, In step (2), the reaction temperature after adding cuttlefish nanoparticles is room temperature, the reaction time is 20-40 min, and the reaction is carried out under stirring conditions.

7. The method for preparing the natural polymer-based multifunctional hydrogel wound dressing according to claim 1, characterized in that, Step (3) includes one or more of the following conditions: i. The mass ratio of oxidized sodium alginate (OSA) to water is 0.01-0.1 g / mL; ii. The mass ratio of sodium oxidized alginate (OSA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is 1:0.5-1.5:0.5-0.6; iii. The activation temperature is 30-40℃, the activation time is 20-40 min, and the activation is carried out under inert gas protection and stirring conditions; preferably, the inert gas is nitrogen or argon. iv. The mass ratio of oxidized sodium alginate (OSA) to dopamine in step (2) is 1:0.2-0.4; v. The reaction temperature after adding polydopamine (cucurbitacin@PDA) encapsulating cucurbitacin nanoparticles is 30-40℃, the reaction time is 10-14h, and the reaction is carried out under inert gas protection and stirring conditions; preferably, the inert gas is nitrogen or argon. vi. Dialysis uses dialysis bags with a molecular weight cutoff of Mw = 8000 Da. The dialysis time is 2-4 days, and the water is changed every 5-7 hours during the dialysis process.

8. The method for preparing the natural polymer-based multifunctional hydrogel wound dressing according to claim 1, characterized in that, Step (4) includes one or more of the following conditions: i. The OSA-cucurbitacin@PDA solution is a PBS solution of OSA-cucurbitacin@PDA with a mass concentration of 2-6%; the carboxymethyl chitosan solution is a PBS solution of carboxymethyl chitosan with a mass concentration of 2-6%; the pH of the PBS buffer used as the solvent is 7.

4. ii. The mass ratio of OSA-cucurbitacin@PDA in the OSA-cucurbitacin@PDA solution to carboxymethyl chitosan in the carboxymethyl chitosan solution is 1:4-6; iii. The gelation reaction conditions are room temperature and static reaction for 6-8 hours.

9. A natural polymer-based multifunctional hydrogel wound dressing, prepared by any one of claims 1-8.

10. The application of the natural polymer-based multifunctional hydrogel wound dressing as described in claim 9 in diabetic wound healing dressings.

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