Preparation and application of pH / ROS stimuli-responsive hydrogel for diabetic wound repair

Abstract

The invention discloses preparation and application of pH / ROS stimuli-responsive hydrogel for diabetic wound repair. The preparation method comprises the following steps: step 1, providing collagen drug-loaded microspheres; step 2, providing an MXene (at) PDA composite nano particle; step 3, providing modified hyaluronic acid and modified recombinant collagen; step 4, mixing the sterilized collagen drug-loaded microspheres with a modified hyaluronic acid solution to obtain a system A; mixing the sterilized MXene (at) PDA composite nanoparticles with a modified recombinant collagen solution to obtain a system B; and stirring and mixing the system A, the system B and a polyvinyl alcohol solution to obtain the pH / ROS stimulation response hydrogel for diabetes wound repair. The hydrogel can be tightly attached to chronic diabetic wounds, can effectively absorb tissue seepage, can perform drug sustained release, has antibacterial and anti-inflammatory characteristics and angiogenesis capability, and can effectively promote the repair of the diabetic wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and is specifically used for the preparation and application of pH / ROS stimulation responsive hydrogel for diabetic wound repair. Background Art

[0002] Chronic wounds refer to wounds that cannot heal after more than 90 days, and chronic diabetic wounds, as one of the typical chronic wounds, have become one of the serious complications of diabetic patients. In addition, chronic diabetic wounds are difficult to recover through the normal wound repair process. If such chronic wounds are not treated in time, a series of special physiological microenvironments will aggravate the deterioration of the wound. Diabetes is a common disease worldwide. The incidence of diabetic foot among diabetic patients worldwide is about 6.4%, and 1% of diabetic patients have to undergo amputation or even die due to foot lesions. At this stage, treatment measures still have the defects of not being able to fully meet the complex pathological needs of diabetic wounds, relatively long repair time, and not having a good fit to the wound surface.

[0003] In the diabetic state, the lack of insulin will lead to the disorder of glucose and lipid metabolism, thus leading to the increase of oxidative stress level in the body. At the same time, hypoxia, abnormal angiogenesis, nerve damage, reduced secretion of growth factors, accumulation of extracellular matrix components and imbalance of matrix metalloproteinase remodeling affect each other, which together lead to slow wound healing in diabetic patients. The unique tissue microenvironment of diabetic wounds affects the cascade advancement of each stage of wound healing, and ultimately leads to delayed healing or non-healing of chronic diabetic wounds. The traditional treatment of diabetic wounds is to perform debridement first, and then use dry dressings such as bandages and gauze for dressing management to absorb wound exudate, while adding antibiotics to prevent infection. However, as a passive intervention strategy, dry dressings cannot fully adapt to the diabetic wound microenvironment, and the adhesion of dry dressings to the wound surface may cause secondary injury. Therefore, designing wound dressings that can highly promote the healing of chronic diabetic wounds is one of the problems that need to be solved in the current field of biomaterials. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide the preparation and application of pH / ROS stimulation responsive hydrogel for diabetic wound repair in view of the deficiencies of the above-mentioned prior art. The hydrogel of the present invention can fit closely to chronic diabetic wounds, can effectively absorb tissue exudate, and simultaneously release drugs slowly, and has both antibacterial and anti-inflammatory properties and angiogenesis ability, and the hydrogel can effectively promote diabetic wound repair.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing a pH / ROS stimulus-responsive hydrogel for diabetic wound repair, characterized in that it comprises:

[0006] Step 1: Providing collagen drug-loaded microspheres, specifically including:

[0007] Step 101, under a temperature condition of 40° C. to 45° C., stirring and mixing liquid paraffin and an emulsifier at a speed of 1000 rpm to 2000 rpm for 0.5 h to 3 h to obtain an emulsifier-containing system;

[0008] Step 102, uniformly dispersing mangiferin in the collagen solution, stirring at a temperature of 37° C. to 45° C. and a speed of 1000 rpm to 2000 rpm for 0.5 h to 3 h to obtain a suspension containing mangiferin;

[0009] Step 103, under the temperature condition of 37° C. to 45° C., the suspension containing mangiferin in step 102 is added dropwise to the emulsifier-containing system in step 101 at a rate of 0.5 mL / min to 3 mL / min, and stirred for 0.5 h to 2 h to obtain an emulsion system containing mangiferin;

[0010] Step 104, rapidly cooling the mangiferin-containing emulsion system to 0°C to 10°C, then rapidly adding the glutaraldehyde solution to the cooled mangiferin-containing emulsion system, stirring for 1h to 3h at a temperature of 0°C to 10°C to obtain microspheres, and washing the microspheres by centrifugation, rotary evaporation and freeze-drying to obtain collagen drug-loaded microspheres;

[0011] Step 2: Providing MXene@PDA composite nanoparticles, specifically including:

[0012] Step 201, etching titanium aluminum carbide with hydrofluoric acid for 12 hours to 96 hours, centrifuging, and drying to obtain multi-layer MXene nanosheets;

[0013] Step 202, exfoliating the MXene multilayer nanosheets described in step 201 with a tetramethylammonium hydroxide solution for 12 h to 48 h, centrifuging, and drying to obtain a few-layer MXene nanosheet;

[0014] Step 203, adding dopamine hydrochloride to deionized water to obtain a dopamine hydrochloride suspension, adjusting the pH to be alkaline to obtain an alkaline dopamine suspension, placing the few-layer MXene nanosheet in the alkaline dopamine suspension and stirring for 6 hours to 10 hours to obtain MXene@PDA composite nanoparticles;

[0015] Step 3: providing modified hyaluronic acid and modified recombinant collagen, specifically including:

[0016] Step 301, stirring and activating an aqueous solution containing sodium periodate and 3-aminophenylboronic acid at a temperature of 18° C. to 25° C. for 1 h to 2 h to obtain an activated modifier A solution;

[0017] Step 302, mixing the activated modifier A solution and the hyaluronic acid aqueous solution in step 301, stirring and reacting for 3 hours to 6 hours in a dark environment, dialyzing the stirred reaction system, and freeze-drying to obtain modified hyaluronic acid;

[0018] Step 303, stirring and activating the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at a temperature of 18° C. to 37° C. for 1 h to 3 h to obtain an activated modifier B solution;

[0019] In step 304, the activated modifier B solution and the recombinant collagen aqueous solution are mixed, stirred and reacted at a temperature of 25° C. to 37° C. for 2 h to 6 h, and the stirred reaction system is dialyzed and freeze-dried to obtain modified recombinant collagen;

[0020] Step 4: Providing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, specifically comprising:

[0021] Step 401, sterilizing the collagen drug-loaded microspheres and the MXene@PDA composite nanoparticles by Co60 irradiation, respectively, to obtain sterilized collagen drug-loaded microspheres and sterilized MXene@PDA composite nanoparticles;

[0022] Step 402, dissolving the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid solution;

[0023] Step 403, mixing the sterilized collagen drug-loaded microspheres in step 401 with the modified hyaluronic acid solution in step 402, and stirring for 5 min to 10 min to obtain system A;

[0024] Step 404, dissolving the modified recombinant collagen in deionized water to obtain a recombinant collagen solution;

[0025] Step 405, mixing the sterilized MXene@PDA composite nanoparticles in step 401 with the modified recombinant collagen solution in step 404, and stirring for 5 min to 10 min to obtain system B;

[0026] Step 406, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution;

[0027] Step 407, stirring and mixing the system A of step 403, the system B of step 405 and the polyvinyl alcohol solution of step 406 in a mass ratio of (1-2):(1-3):(1-2) to obtain a pH / ROS stimulation responsive hydrogel for diabetic wound repair.

[0028] The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair is characterized in that, in step 101, the emulsifier is Span80, and the volume of the liquid paraffin is 30 to 60 times the volume of the emulsifier.

[0029] The method for preparing the pH / ROS stimuli-responsive hydrogel for diabetic wound repair is characterized in that in step 102, the mass of mangiferin in the mangiferin-containing suspension is 2×10 of the mass of collagen. -3 Times ~ 8×10 -3 times; in step 102, the collagen solution is a collagen solution with a mass percentage of 10% to 20% formed by dissolving collagen in water at a temperature of 37°C to 45°C; in step 102, the collagen is one or more of pig skin collagen, recombinant collagen and recombinant collagen doped with natural collagen.

[0030] The above-mentioned method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair is characterized in that, in step 103, the volume of the mangiferin suspension is 5 to 10 times the volume of the emulsifier in the emulsifier-containing system; in step 104, the mass percentage of the glutaraldehyde solution is 10wt% to 25wt%; in step 104, the volume of paraffin is 30 to 60 times the volume of the glutaraldehyde solution.

[0031] The above-mentioned method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair is characterized in that, in step 201, the mass of the hydrofluoric acid is 5 to 10 times the mass of titanium aluminum carbide, and the mass percentage of the hydrofluoric acid is 30% to 45%; in step 202, the mass of the tetramethylammonium hydroxide solution is 5 to 10 times the mass of titanium aluminum carbide, and the mass percentage of the tetramethylammonium hydroxide is 25% to 50%; in step 203, the mass percentage of the dopamine hydrochloride suspension is 1% to 5%, the pH of the alkaline dopamine suspension is 8 to 10, and the mass of the dopamine hydrochloride is 1 to 2 times the mass of the few-layer MXene nanosheets.

[0032] The above-mentioned method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair is characterized in that, in step 301, in the aqueous solution containing sodium periodate and 3-aminophenylboronic acid, the concentration of sodium periodate is 1% to 5%; in step 302, the amount of sodium periodate substance is 0.2 times to 0.5 times the amount of hyaluronic acid substance, and the amount of 3-aminophenylboronic acid substance is 0.5 times to 1 times the amount of hyaluronic acid substance; in step 302, the mass percentage of hyaluronic acid in the hyaluronic acid aqueous solution is 1% to 5%.

[0033] The preparation method of the pH / ROS stimulus-responsive hydrogel for diabetic wound repair is characterized in that in step 303, the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is prepared by dissolving polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in 2-morpholineethanesulfonic acid buffer solution. The buffer solution of hydroxysuccinimide, the pH value of the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 5.5-6.5, the mass of the polylysine is 10%-15% of the mass of the 2-morpholineethanesulfonic acid buffer solution, and the molecular weight of the polylysine is 2000-5000; in step 303, the molar ratio of polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is (1-2):1:1.

[0034] The above-mentioned method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair is characterized in that, in step 304, the amount of polylysine substance in the activated modifier B solution is 15% to 20% of the amount of recombinant collagen substance, and the mass percentage of recombinant collagen in the recombinant collagen aqueous solution is 10% to 15%.

[0035] The above-mentioned method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair is characterized in that, in step 402, the mass percentage of the modified hyaluronic acid solution is 1% to 5%; in step 403, the mass of the modified hyaluronic acid is 0.1 times to 0.5 times the mass of the collagen drug-loaded microspheres; in step 404, the mass percentage of the modified recombinant collagen solution is 10% to 15%; in step 405, the mass of the modified recombinant collagen is 100 times to 150 times the mass of the MXene@PDA composite nanoparticles; in step 406, the mass percentage of the polyvinyl alcohol solution is 10wt% to 15wt%, and the molecular weight of the polyvinyl alcohol is 89,000 to 98,000.

[0036] In addition, the present invention also provides an application of the hydrogel prepared by the method for preparing the pH / ROS stimulation responsive hydrogel for diabetic wound repair in preparing a gel for diabetic wound repair.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] 1. The present invention provides a method for preparing a pH / ROS stimulus-responsive hydrogel for diabetic wound repair. The method uses a modified hyaluronic acid system containing collagen drug-loaded microspheres, a modified recombinant collagen system containing MXene@PDA composite nanoparticles, and a polyvinyl alcohol solution as main raw materials, and obtains a pH / ROS stimulus-responsive hydrogel for diabetic wound repair by stirring and mixing. The hydrogel can fit tightly to chronic diabetic wounds, effectively absorb tissue exudate, and simultaneously perform sustained-release of drugs, as well as have both antibacterial and anti-inflammatory properties and angiogenesis ability. The hydrogel can effectively promote diabetic wound repair.

[0039] 2. The preparation method of the pH / ROS stimulus-responsive hydrogel for diabetic wound repair of the present invention comprises cross-linking a collagen suspension containing mangiferin with an oil phase under glutaraldehyde solution conditions to obtain collagen drug-loaded microspheres, and introducing the collagen drug-loaded microspheres obtained by combining a Schiff base chemical bond and a borate ester bond through double dynamic chemical bonds into a hydrogel system, which can effectively improve the pH and ROS response of the hydrogel and promote the sequential release of the encapsulated substance.

[0040] 3. The preparation method of the pH / ROS stimulus-responsive hydrogel for diabetic wound repair of the present invention comprises co-loading collagen drug-loaded microspheres loaded with mangiferin and nanoparticles MXene@PDA into a double-cross-linked hydrogel, which can achieve the rapid release of MXene@PDA and the sustained release of mangiferin, and endow the hydrogel with the biological properties of rapid antibacterial and anti-inflammatory effects and sustained promotion of angiogenesis.

[0041] 4. The preparation method of the pH / ROS stimulation-responsive hydrogel for diabetic wound repair of the present invention is simple, has good reproducibility, and has good application prospects.

[0042] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 a is a schematic diagram of the process of preparing collagen drug-loaded microspheres in Example 1;

[0044] Figure 1 b is the SEM image of collagen drug-loaded microspheres in Example 1;

[0045] Figure 2 a is the XRD spectrum of the few-layer MXene nanosheets prepared in step 202 of Example 1 and the MXene@PDA composite nanoparticles prepared in step 2;

[0046] Figure 2 b is the SEM image and mapping element distribution diagram of the MXene@PDA composite nanoparticles of Example 1, wherein the scale bar of the SEM image is 200 μm;

[0047] Figure 2 c is the SEM image of the MXene@PDA composite nanoparticles of Example 1, wherein the scale bar of the SEM image is 200 nm;

[0048] Figure 2 d is the SEM image of the few-layer nanosheet MXene of Example 1 at a scale of 1 μm;

[0049] Figure 2 e is the SEM image of the MXene@PDA composite nanoparticles of Example 1 at a scale of 1 μm;

[0050] Figure 3 a is an infrared spectrum of the modified hyaluronic acid in Example 1, wherein HA is hyaluronic acid, OHA is oxidized hyaluronic acid, and POA is modified hyaluronic acid;

[0051] Figure 3 b is the FTIR spectrum of the modified recombinant collagen of Example 1, CF-1552 is the recombinant collagen, and EPL-CF-1552 is the modified recombinant collagen;

[0052] Figure 3 c is a morphology image of the pH / ROS stimulation-responsive hydrogel used for diabetic wound repair in Example 1;

[0053] Figure 3 d is a SEM image of the pH / ROS stimulation-responsive hydrogel for diabetic wound repair in Example 1;

[0054] Figure 4 a is the cytotoxicity test result of the pH / ROS stimulation-responsive hydrogel for diabetic wound repair in Example 1;

[0055] Figure 4 b is the test result of the cell migration promoting ability of the pH / ROS stimulation responsive hydrogel used for diabetic wound repair in Example 1. DETAILED DESCRIPTION

[0056] Example 1

[0057] This embodiment provides a method for preparing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, comprising:

[0058] Step 1: Provide collagen drug-loaded microspheres. The process is as follows: Figure 1 a, specifically including:

[0059] Step 101, at a temperature of 40° C., 30 mL of liquid paraffin and 0.5 mL of an emulsifier were stirred and mixed at a rate of 1000 rpm for 0.5 h to obtain an emulsifier-containing system; the emulsifier was Span80; the model of the paraffin was 2649, purchased from Tianjin Damao Chemical Reagent Factory;

[0060] Step 102, uniformly dispersing mangiferin in the collagen solution, stirring at 37°C for 0.5h, to obtain a suspension containing mangiferin; the mass of mangiferin in the suspension containing mangiferin is 2×10 of the mass of collagen. -3 times; the collagen solution is a collagen solution with a mass percentage of 15% formed by dissolving collagen in water at a temperature of 37°C; the collagen is pig skin collagen; the collagen can also be one or more of other natural collagens, recombinant collagens and recombinant collagens doped with natural collagens, other natural collagens such as bovine type I collagen, recombinant collagens can be recombinant collagens with a model of CF-1552, recombinant collagens doped with natural collagens such as high Bloom gelatin, and the recombinant collagens and recombinant collagens doped with natural collagens are purchased from Xi'an Juzi Biogene Technology Co., Ltd.; the stirring rate is 1500rpm;

[0061] Step 103, at a temperature of 40° C., 5 mL of the mangiferin suspension described in step 102 was added dropwise to the emulsifier-containing system described in step 101 at a dropping rate of 1 mL / min, and stirred for 1 h to obtain a mangiferin-containing emulsion system;

[0062] Step 104, rapidly cooling the mangiferin-containing emulsion system to 4°C, then rapidly adding 1 mL of glutaraldehyde solution to the cooled mangiferin-containing emulsion system, stirring at 4°C for 2 hours to obtain microspheres, and subjecting the microspheres to centrifugal washing, rotary evaporation and freeze-drying to obtain collagen drug-loaded microspheres; the mass percentage of the glutaraldehyde solution is 15wt%; the particle size of the collagen drug-loaded microspheres is 8μm to 20μm; the centrifugal washing is washing with isopropanol, ethanol and distilled water alternately in a 4500rpm centrifuge, and each washing time is 3min;

[0063] Step 2: Providing MXene@PDA composite nanoparticles, specifically including:

[0064] Step 201, etching titanium aluminum carbide (Ti3AlC2) with hydrofluoric acid for 48 hours, centrifuging, and drying to obtain multi-layer MXene multi-layer nanosheets; the mass of the hydrofluoric acid is 5 times the mass of the titanium aluminum carbide, and the mass percentage of the hydrofluoric acid is 30%;

[0065] Step 202, exfoliating the MXene multilayer nanosheets in step 201 with a tetramethylammonium hydroxide solution for 24 hours, centrifuging, and drying to obtain a few-layer MXene nanosheet; the mass of the tetramethylammonium hydroxide solution is 8 times the mass of titanium aluminum carbide, and the mass percentage of the tetramethylammonium hydroxide is 25%;

[0066] Step 203, adding dopamine hydrochloride (PDA) to deionized water to obtain a dopamine hydrochloride suspension, adjusting the pH to alkaline to obtain an alkaline dopamine suspension, placing the few-layer MXene nanosheets in the alkaline dopamine suspension and stirring for 8 hours to obtain MXene@PDA composite nanoparticles; the mass percentage of the dopamine hydrochloride suspension is 1%, the pH of the alkaline dopamine suspension is 8, and the mass of the dopamine hydrochloride is 1 times the mass of the MXene;

[0067] Step 3: providing modified hyaluronic acid and modified recombinant collagen, specifically including:

[0068] Step 301, stirring and activating an aqueous solution containing sodium periodate and 3-aminophenylboronic acid at 18° C. for 1 hour to obtain an activated modifier A solution; in the aqueous solution containing sodium periodate and 3-aminophenylboronic acid, the concentration of sodium periodate is 1%;

[0069] Step 302, mixing the activated modifier A solution and the hyaluronic acid aqueous solution described in step 301, stirring and reacting for 3 hours in a dark environment, placing the stirred reaction system in a dialysis bag with a molecular weight cutoff of 8000 and dialyzing with distilled water for 3 days, changing the water 5 times a day during the period, and then freeze-drying to obtain modified hyaluronic acid; the amount of sodium periodate substance is 0.2 times the amount of hyaluronic acid substance, and the amount of 3-aminophenylboronic acid substance is 0.5 times the amount of hyaluronic acid substance; the mass percentage of hyaluronic acid in the hyaluronic acid aqueous solution is 1%;

[0070] Step 303, stirring and activating a buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at 18° C. for 1 hour to obtain an activated modifier B solution; the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is a polylysine-containing solution prepared by dissolving polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a 2-morpholineethanesulfonic acid buffer solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide buffer solution; the pH of the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 5.5, the mass of the polylysine is 10% of the mass of the 2-morpholineethanesulfonic acid buffer solution, and the molecular weight of the polylysine is 2000-2200; the molar ratio of polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:1:1;

[0071] In step 304, the activated modifier B solution and the recombinant collagen aqueous solution described in step 303 are mixed, stirred and reacted at a temperature of 25° C. for 2 hours, and the stirred reaction system is placed in a dialysis bag with a molecular weight cutoff of 8000 and dialyzed with distilled water for 3 days, during which the water is changed 5 times a day, and then freeze-dried to obtain modified recombinant collagen; the amount of polylysine substance in the activated modifier B solution is 15% of the amount of recombinant collagen substance, and the mass percentage of recombinant collagen in the recombinant collagen aqueous solution is 10%; the recombinant collagen is CF-1552 recombinant collagen purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0072] Step 4: Providing a pH / ROS stimulus-responsive hydrogel (PPCMPF hydrogel) for diabetic wound repair, specifically comprising:

[0073] Step 401, sterilize 0.1 g of the collagen drug-loaded microspheres and 1 mg of MXene@PDA composite nanoparticles by Co60 irradiation, respectively, to obtain sterilized collagen drug-loaded microspheres and sterilized MXene@PDA composite nanoparticles;

[0074] Step 402, dissolving the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid solution with a mass fraction of 1%;

[0075] Step 403, mixing the sterilized collagen drug-loaded microspheres described in step 401 with 1 mL of the modified hyaluronic acid solution described in step 402, and stirring for 5 minutes to obtain system A;

[0076] Step 404, dissolving the modified recombinant collagen in deionized water to obtain a modified recombinant collagen solution with a mass fraction of 10%;

[0077] Step 405, mixing the sterilized MXene@PDA composite nanoparticles in step 401 with 1 mL of the modified recombinant collagen solution in step 404, and stirring for 5 minutes to obtain system B;

[0078] Step 406, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution with a mass percentage of 10 wt%; the molecular weight of the polyvinyl alcohol is 89000-90000;

[0079] Step 407, under manual stirring conditions, the system A of step 403, the system B of step 405 and the polyvinyl alcohol solution of step 406 are stirred and mixed at a mass ratio of 1:2:1 for 2 minutes to obtain a pH / ROS stimulation responsive hydrogel for diabetic wound repair.

[0080] Example 2

[0081] This embodiment provides a method for preparing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, comprising:

[0082] Step 1: Providing collagen drug-loaded microspheres, specifically including:

[0083] Step 101, at a temperature of 40° C., 30 mL of liquid paraffin and 0.5 mL of an emulsifier were stirred and mixed at a rate of 1500 rpm for 1 hour to obtain an emulsifier-containing system; the emulsifier was Span80;

[0084] Step 102, uniformly dispersing mangiferin in the collagen solution, stirring at 37°C for 1 hour, to obtain a suspension containing mangiferin; the mass of mangiferin in the suspension containing mangiferin is 4×10 of the mass of collagen. -3 times, the collagen solution is a collagen solution with a mass percentage of 10% formed by dissolving collagen in water at a temperature of 37°C; the collagen is pig skin collagen; the collagen can also be one or more of other natural collagen, recombinant collagen and recombinant collagen doped with natural collagen; the stirring rate is 1000rpm;

[0085] Step 103, at a temperature of 40° C., 5 mL of the mangiferin suspension described in step 102 was added dropwise to the emulsifier-containing system described in step 101 at a dropping rate of 1 mL / min, and stirred for 1.5 h to obtain a mangiferin-containing emulsion system;

[0086] Step 104, rapidly cooling the mangiferin-containing emulsion system to 8°C, then rapidly adding 0.5 mL of glutaraldehyde solution into the cooled mangiferin-containing emulsion system, stirring at 8°C for 1 hour to obtain microspheres, and subjecting the microspheres to centrifugal washing, rotary evaporation and freeze-drying to obtain collagen drug-loaded microspheres; the mass percentage of the glutaraldehyde solution is 10wt%; the particle size of the collagen drug-loaded microspheres is 8μm to 20μm; the centrifugal washing is washing with isopropanol, ethanol and distilled water alternately in a 4500rpm centrifuge, and each washing time is 3min;

[0087] Step 2: Providing MXene@PDA composite nanoparticles, specifically including:

[0088] Step 201, etching titanium aluminum carbide with hydrofluoric acid for 12 hours, centrifuging, and drying to obtain multi-layer MXene multi-layer nanosheets; the mass of the hydrofluoric acid is 10 times the mass of the titanium aluminum carbide, and the mass percentage of the hydrofluoric acid is 45%;

[0089] Step 202, exfoliating the MXene multilayer nanosheets in step 201 with a tetramethylammonium hydroxide solution for 12 hours, centrifuging, and drying to obtain a few-layer MXene nanosheet; the mass of the tetramethylammonium hydroxide solution is 5 times the mass of titanium aluminum carbide, and the mass percentage of the tetramethylammonium hydroxide is 25%;

[0090] Step 203, adding dopamine hydrochloride to deionized water to obtain a dopamine hydrochloride suspension, adjusting the pH to alkaline to obtain an alkaline dopamine suspension, placing the few-layer MXene nanosheets in the alkaline dopamine suspension and stirring for 6 hours to obtain MXene@PDA composite nanoparticles; the mass percentage of the dopamine hydrochloride suspension is 1%, the pH of the alkaline dopamine suspension is 8, and the mass of the dopamine hydrochloride is 1 times the mass of the MXene;

[0091] Step 3: providing modified hyaluronic acid and modified recombinant collagen, specifically including:

[0092] Step 301, stirring and activating an aqueous solution containing sodium periodate and 3-aminophenylboronic acid at 20° C. for 1.5 hours to obtain an activated modifier A solution; in the aqueous solution containing sodium periodate and 3-aminophenylboronic acid, the concentration of sodium periodate is 2%;

[0093] Step 302, mixing the activated modifier A solution and the hyaluronic acid aqueous solution described in step 301, stirring and reacting for 5 hours in a dark environment, placing the stirred reaction system in a dialysis bag with a molecular weight cutoff of 8000 and dialyzing with distilled water for 3 days, changing the water 5 times a day during the period, and then freeze-drying to obtain modified hyaluronic acid; the amount of sodium periodate substance is 0.3 times the amount of hyaluronic acid substance, and the amount of 3-aminophenylboronic acid substance is 0.8 times the amount of hyaluronic acid substance; the mass percentage of hyaluronic acid in the hyaluronic acid aqueous solution is 1%;

[0094] Step 303, stirring and activating a buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at 25° C. for 1.5 h to obtain an activated modifier B solution; the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is a polylysine-containing solution prepared by dissolving polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a 2-morpholineethanesulfonic acid buffer solution. , 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide buffer solution; the pH of the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 5.5, the mass of the polylysine is 12% of the mass of the 2-morpholineethanesulfonic acid buffer solution, and the molecular weight of the polylysine is 4500-5000; the molar ratio of polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 2:1:1;

[0095] In step 304, the activated modifier B solution and the recombinant collagen aqueous solution described in step 303 are mixed, stirred and reacted at a temperature of 30° C. for 3 hours, and the stirred reaction system is placed in a dialysis bag with a molecular weight cutoff of 8000 and dialyzed with distilled water for 3 days, during which the water is changed 5 times a day, and then freeze-dried to obtain modified recombinant collagen; the amount of polylysine substance in the activated modifier B solution is 20% of the amount of recombinant collagen substance, and the mass percentage of recombinant collagen in the recombinant collagen aqueous solution is 15%; the recombinant collagen is CF-1552 recombinant collagen purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0096] Step 4: Providing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, specifically comprising:

[0097] Step 401, sterilize 0.1 g of the collagen drug-loaded microspheres and 1 mg of MXene@PDA composite nanoparticles by Co60 irradiation, respectively, to obtain sterilized collagen drug-loaded microspheres and sterilized MXene@PDA composite nanoparticles;

[0098] Step 402, dissolving the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid solution with a mass fraction of 3%;

[0099] Step 403, mixing the sterilized collagen drug-loaded microspheres described in step 401 with 1 mL of the modified hyaluronic acid solution described in step 402, and stirring for 5 minutes to obtain system A;

[0100] Step 404, dissolving the modified recombinant collagen in deionized water to obtain a modified recombinant collagen solution with a mass fraction of 15%;

[0101] Step 405, mixing the sterilized MXene@PDA composite nanoparticles in step 401 with 1 mL of the modified recombinant collagen solution in step 404, and stirring for 5 minutes to obtain system B;

[0102] Step 406, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution with a mass percentage of 15 wt%; the molecular weight of the polyvinyl alcohol is 89000-90000;

[0103] Step 407, under manual stirring conditions, the system A of step 403, the system B of step 405 and the polyvinyl alcohol solution of step 406 are stirred and mixed at a mass ratio of 1:3:1 for 2 minutes to obtain a pH / ROS stimulus-responsive hydrogel for diabetic wound repair.

[0104] The performance of the pH / ROS stimulation responsive hydrogel for diabetic wound repair in this example is basically the same as that in Example 1.

[0105] Example 3

[0106] This embodiment provides a method for preparing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, comprising:

[0107] Step 1: Providing collagen drug-loaded microspheres, specifically including:

[0108] Step 101, at a temperature of 40° C., 30 mL of liquid paraffin and 0.6 mL of an emulsifier are stirred and mixed at a rate of 1000 rpm for 3 hours to obtain an emulsifier-containing system; the emulsifier is Span80;

[0109] Step 102, uniformly dispersing mangiferin in the collagen solution, stirring at 37°C for 3h, to obtain a suspension containing mangiferin; the mass of mangiferin in the suspension containing mangiferin is 6×10 of the mass of collagen. -3 times, the collagen solution is a collagen solution with a mass percentage of 20% formed by dissolving collagen in water at a temperature of 37°C; the collagen is pig skin collagen; the collagen can also be one or more of other natural collagen, recombinant collagen and recombinant collagen doped with natural collagen; the stirring speed is 2000rpm;

[0110] Step 103, at a temperature of 45° C., 5 mL of the mangiferin suspension described in step 102 was added dropwise to the emulsifier-containing system described in step 101 at a dropping rate of 0.5 mL / min, and stirred for 1 h to obtain a mangiferin-containing emulsion system;

[0111] Step 104, rapidly cooling the mangiferin-containing emulsion system to 6°C, then rapidly adding 0.6 mL of glutaraldehyde solution to the cooled mangiferin-containing emulsion system, stirring at 6°C for 2 h to obtain microspheres, and subjecting the microspheres to centrifugal washing, rotary evaporation and freeze-drying to obtain collagen drug-loaded microspheres; the mass percentage of the glutaraldehyde solution is 20 wt%; the particle size of the collagen drug-loaded microspheres is 8 μm to 20 μm; the centrifugal washing is washing with isopropanol, ethanol and distilled water alternately in a 4500 rpm centrifuge, and each washing time is 3 min.

[0112] Step 2: Providing MXene@PDA composite nanoparticles, specifically including:

[0113] Step 201, etching titanium aluminum carbide with hydrofluoric acid for 96 hours, centrifuging, and drying to obtain multi-layer MXene multi-layer nanosheets; the mass of the hydrofluoric acid is 10 times the mass of the titanium aluminum carbide, and the mass percentage of the hydrofluoric acid is 45%;

[0114] Step 202, exfoliating the MXene multilayer nanosheets in step 201 with a tetramethylammonium hydroxide solution for 48 hours, centrifuging, and drying to obtain a few-layer MXene nanosheet; the mass of the tetramethylammonium hydroxide solution is 10 times the mass of titanium aluminum carbide, and the mass percentage of the tetramethylammonium hydroxide is 50%;

[0115] Step 203, adding dopamine hydrochloride to deionized water to obtain a dopamine hydrochloride suspension, adjusting the pH to alkaline to obtain an alkaline dopamine suspension, placing the few-layer MXene nanosheets in the alkaline dopamine suspension and stirring for 10 hours to obtain MXene@PDA composite nanoparticles; the mass percentage of the dopamine hydrochloride suspension is 5%, the pH of the alkaline dopamine suspension is 10, and the mass of the dopamine hydrochloride is twice the mass of the MXene;

[0116] Step 3: providing modified hyaluronic acid and modified recombinant collagen, specifically including:

[0117] Step 301, stirring and activating an aqueous solution containing sodium periodate and 3-aminophenylboronic acid at 18° C. for 2 hours to obtain an activated modifier A solution; in the aqueous solution containing sodium periodate and 3-aminophenylboronic acid, the concentration of sodium periodate is 2%;

[0118] Step 302, mixing the activated modifier A solution and the hyaluronic acid aqueous solution described in step 301, stirring and reacting for 3 hours in a dark environment, placing the stirred reaction system in a dialysis bag with a molecular weight cutoff of 8000 and dialyzing with distilled water for 3 days, changing the water 5 times a day during the period, and then freeze-drying to obtain modified hyaluronic acid; the amount of sodium periodate substance is 0.5 times the amount of hyaluronic acid substance, and the amount of 3-aminophenylboronic acid substance is 0.5 times the amount of hyaluronic acid substance; the mass percentage of hyaluronic acid in the hyaluronic acid aqueous solution is 3%;

[0119] Step 303, stirring and activating a buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at 25° C. for 2 hours to obtain an activated modifier B solution; the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is a polylysine-containing solution prepared by dissolving polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a 2-morpholineethanesulfonic acid buffer solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide buffer solution; the pH of the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 6.5, the mass of the polylysine is 10% of the mass of the 2-morpholineethanesulfonic acid buffer solution, and the molecular weight of the polylysine is 3000-3300; the molar ratio of polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 2:1:1;

[0120] In step 304, the activated modifier B solution and the recombinant collagen aqueous solution described in step 303 are mixed, stirred and reacted at a temperature of 37° C. for 2 hours, and the stirred reaction system is placed in a dialysis bag with a molecular weight cutoff of 8000 and dialyzed with distilled water for 3 days, during which the water is changed 5 times a day, and then freeze-dried to obtain modified recombinant collagen; the amount of polylysine substance in the activated modifier B solution is 15% of the amount of recombinant collagen substance, and the mass percentage of recombinant collagen in the recombinant collagen aqueous solution is 10%; the recombinant collagen is CF-1552 recombinant collagen purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0121] Step 4: Providing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, specifically comprising:

[0122] Step 401, sterilize 0.1 g of the collagen drug-loaded microspheres and 1 mg of MXene@PDA composite nanoparticles by Co60 irradiation, respectively, to obtain sterilized collagen drug-loaded microspheres and sterilized MXene@PDA composite nanoparticles;

[0123] Step 402, dissolving the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid solution with a mass fraction of 3%;

[0124] Step 403, mixing the sterilized collagen drug-loaded microspheres described in step 401 with 1 mL of the modified hyaluronic acid solution described in step 402, and stirring for 8 minutes to obtain system A;

[0125] Step 404, dissolving the modified recombinant collagen in deionized water to obtain a modified recombinant collagen solution with a mass fraction of 13%;

[0126] Step 405, mixing the sterilized MXene@PDA composite nanoparticles in step 401 with 1 mL of the modified recombinant collagen solution in step 404, and stirring for 8 minutes to obtain system B;

[0127] Step 406, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution with a mass percentage of 12 wt%; the molecular weight of the polyvinyl alcohol is 90,000 to 98,000;

[0128] Step 407, under manual stirring conditions, the system A of step 403, the system B of step 405 and the polyvinyl alcohol solution of step 406 are stirred and mixed at a mass ratio of 2:2:1 for 2 minutes to obtain a pH / ROS stimulation responsive hydrogel for diabetic wound repair.

[0129] The performance of the pH / ROS stimulation responsive hydrogel for diabetic wound repair in this example is basically the same as that in Example 1.

[0130] Example 4

[0131] This embodiment provides a method for preparing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, comprising:

[0132] Step 1: Providing collagen drug-loaded microspheres, specifically including:

[0133] Step 101, at a temperature of 45° C., 30 mL of liquid paraffin and 0.8 mL of an emulsifier are stirred and mixed at a rate of 2000 rpm for 2 h to obtain an emulsifier-containing system; the emulsifier is Span80;

[0134] Step 102, uniformly dispersing mangiferin in the collagen solution, stirring at 45°C for 0.5h, to obtain a suspension containing mangiferin; the mass of mangiferin in the suspension containing mangiferin is 8×10 of the mass of collagen. -3 times, the collagen solution is a collagen solution with a mass percentage of 20% formed by dissolving collagen in water at a temperature of 45°C; the collagen is pig skin collagen; the collagen can also be one or more of other natural collagen, recombinant collagen and recombinant collagen doped with natural collagen; the stirring rate is 1000rpm;

[0135] Step 103, at a temperature of 37° C., 5 mL of the mangiferin suspension described in step 102 was added dropwise to the emulsifier-containing system described in step 101 at a dropping rate of 2 mL / min, and stirred for 0.5 h to obtain a mangiferin-containing emulsion system;

[0136] Step 104, rapidly cooling the mangiferin-containing emulsion system to 0°C, then rapidly adding 1 mL of glutaraldehyde solution into the cooled mangiferin-containing emulsion system, stirring at 0°C for 2 hours to obtain microspheres, and subjecting the microspheres to centrifugal washing, rotary evaporation and freeze-drying to obtain collagen drug-loaded microspheres; the mass percentage of the glutaraldehyde solution is 15wt%; the particle size of the collagen drug-loaded microspheres is 8μm to 20μm; the centrifugal washing is washing with isopropanol, ethanol and distilled water alternately in a 4500rpm centrifuge, and each washing time is 3min;

[0137] Step 2: Providing MXene@PDA composite nanoparticles, specifically including:

[0138] Step 201, etching titanium aluminum carbide with hydrofluoric acid for 48 hours, centrifuging, and drying to obtain multi-layer MXene multi-layer nanosheets; the mass of the hydrofluoric acid is 10 times the mass of the titanium aluminum carbide, and the mass percentage of the hydrofluoric acid is 45%;

[0139] Step 202, exfoliating the MXene multilayer nanosheets in step 201 with a tetramethylammonium hydroxide solution for 48 hours, centrifuging, and drying to obtain a few-layer MXene nanosheet; the mass of the tetramethylammonium hydroxide solution is 5 times the mass of titanium aluminum carbide, and the mass percentage of the tetramethylammonium hydroxide is 40%;

[0140] Step 203, adding dopamine hydrochloride to deionized water to obtain a dopamine hydrochloride suspension, adjusting the pH to alkaline to obtain an alkaline dopamine suspension, placing the few-layer MXene nanosheets in the alkaline dopamine suspension and stirring for 10 hours to obtain MXene@PDA composite nanoparticles; the mass percentage of the dopamine hydrochloride suspension is 1%, the pH of the alkaline dopamine suspension is 10, and the mass of the dopamine hydrochloride is twice the mass of the MXene;

[0141] Step 3: providing modified hyaluronic acid and modified recombinant collagen, specifically including:

[0142] Step 301, stirring and activating an aqueous solution containing sodium periodate and 3-aminophenylboronic acid at 22° C. for 2 hours to obtain an activated modifier A solution; in the aqueous solution containing sodium periodate and 3-aminophenylboronic acid, the concentration of sodium periodate is 5%;

[0143] Step 302, mixing the activated modifier A solution and the hyaluronic acid aqueous solution described in step 301, stirring and reacting for 3 hours in a dark environment, placing the stirred reaction system in a dialysis bag with a molecular weight cutoff of 8000 and dialyzing with distilled water for 3 days, changing the water 5 times a day during the period, and then freeze-drying to obtain modified hyaluronic acid; the amount of sodium periodate substance is 0.5 times the amount of hyaluronic acid substance, and the amount of 3-aminophenylboronic acid substance is 0.8 times the amount of hyaluronic acid substance; the mass percentage of hyaluronic acid in the hyaluronic acid aqueous solution is 5%;

[0144] Step 303, stirring and activating a buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at 37° C. for 3 hours to obtain an activated modifier B solution; the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is a polylysine-containing solution prepared by dissolving polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a 2-morpholineethanesulfonic acid buffer solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide buffer solution; the pH of the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 5.5; the mass of the polylysine is 15% of the mass of the 2-morpholineethanesulfonic acid buffer solution, and the molecular weight of the polylysine is 4500-5000; the molar ratio of polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 2:1:1;

[0145] In step 304, the activated modifier B solution and the recombinant collagen aqueous solution described in step 303 are mixed, stirred and reacted at a temperature of 25° C. for 6 hours, and the stirred reaction system is placed in a dialysis bag with a molecular weight cutoff of 8000 and dialyzed with distilled water for 3 days, during which the water is changed 5 times a day, and then freeze-dried to obtain modified recombinant collagen; the amount of polylysine substance in the activated modifier B solution is 20% of the amount of recombinant collagen substance, and the mass percentage of recombinant collagen in the recombinant collagen aqueous solution is 10%; the recombinant collagen is CF-1552 recombinant collagen purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0146] Step 4: Providing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, specifically comprising:

[0147] Step 401, sterilize 0.1 g of the collagen drug-loaded microspheres and 1 mg of MXene@PDA composite nanoparticles by Co60 irradiation, respectively, to obtain sterilized collagen drug-loaded microspheres and sterilized MXene@PDA composite nanoparticles;

[0148] Step 402, dissolving the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid solution with a mass fraction of 1%;

[0149] Step 403, mixing the sterilized collagen drug-loaded microspheres described in step 401 with 1 mL of the modified hyaluronic acid solution described in step 402, and stirring for 10 minutes to obtain system A;

[0150] Step 404, dissolving the modified recombinant collagen in deionized water to obtain a modified recombinant collagen solution with a mass fraction of 10%;

[0151] Step 405, mixing the sterilized MXene@PDA composite nanoparticles in step 401 with 1 mL of the modified recombinant collagen solution in step 404, and stirring for 10 minutes to obtain system B;

[0152] Step 406, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution with a mass percentage of 10 wt%; the molecular weight of the polyvinyl alcohol is 90,000 to 98,000;

[0153] Step 407, under manual stirring conditions, the system A of step 403, the system B of step 405 and the polyvinyl alcohol solution of step 406 are stirred and mixed at a mass ratio of 2:1:2 for 2 minutes to obtain a pH / ROS stimulation responsive hydrogel for diabetic wound repair.

[0154] The performance of the pH / ROS stimulation responsive hydrogel for diabetic wound repair in this example is basically the same as that in Example 1.

[0155] Example 5

[0156] This embodiment provides a method for preparing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, comprising:

[0157] Step 1: Providing collagen drug-loaded microspheres, specifically including:

[0158] Step 101, at a temperature of 45° C., 30 mL of liquid paraffin and 1 mL of an emulsifier are stirred and mixed at a rate of 2000 rpm for 3 hours to obtain an emulsifier-containing system; the emulsifier is Span80;

[0159] Step 102, uniformly dispersing mangiferin in the collagen solution, stirring at 45°C for 2h, to obtain a suspension containing mangiferin; the mass of mangiferin in the suspension containing mangiferin is 8×10 of the mass of collagen. -3 times, the collagen solution is a collagen solution with a mass percentage of 20% formed by dissolving collagen in water at a temperature of 45°C; the collagen is pig skin collagen; the collagen can also be one or more of other natural collagen, recombinant collagen and recombinant collagen doped with natural collagen; the stirring speed is 2000rpm;

[0160] Step 103, at a temperature of 45° C., 5 mL of the mangiferin suspension described in step 102 was added dropwise to the emulsifier-containing system described in step 101 at a dropping rate of 3 mL / min, and stirred for 2 h to obtain a mangiferin-containing emulsion system;

[0161] Step 104, rapidly cooling the mangiferin-containing emulsion system to 10° C., then rapidly adding 0.5 mL of glutaraldehyde solution into the cooled mangiferin-containing emulsion system, stirring at 10° C. for 3 h to obtain microspheres, and subjecting the microspheres to centrifugal washing, rotary evaporation and freeze-drying to obtain collagen drug-loaded microspheres; the mass percentage of the glutaraldehyde solution is 25 wt %; the particle size of the collagen drug-loaded microspheres is 8 μm to 20 μm; the centrifugal washing is washing with isopropanol, ethanol and distilled water alternately in a 4500 rpm centrifuge, and each washing time is 3 min;

[0162] Step 2: Providing MXene@PDA composite nanoparticles, specifically including:

[0163] Step 201, etching titanium aluminum carbide with hydrofluoric acid for 56 hours, centrifuging, and drying to obtain multi-layer MXene multi-layer nanosheets; the mass of the hydrofluoric acid is 10 times the mass of the titanium aluminum carbide, and the mass percentage of the hydrofluoric acid is 45%;

[0164] Step 202, exfoliating the MXene multilayer nanosheets in step 201 with a tetramethylammonium hydroxide solution for 24 hours, centrifuging, and drying to obtain a few-layer MXene nanosheet; the mass of the tetramethylammonium hydroxide solution is 10 times the mass of titanium aluminum carbide, and the mass percentage of the tetramethylammonium hydroxide is 25%;

[0165] Step 203, adding dopamine hydrochloride to deionized water to obtain a dopamine hydrochloride suspension, adjusting the pH to alkaline to obtain an alkaline dopamine suspension, placing the few-layer MXene nanosheets in the alkaline dopamine suspension and stirring for 8 hours to obtain MXene@PDA composite nanoparticles; the mass percentage of the dopamine hydrochloride suspension is 3%, the pH of the alkaline dopamine suspension is 10, and the mass of the dopamine hydrochloride is 1 times the mass of the MXene;

[0166] Step 3: providing modified hyaluronic acid and modified recombinant collagen, specifically including:

[0167] Step 301, stirring and activating an aqueous solution containing sodium periodate and 3-aminophenylboronic acid at 25° C. for 1 hour to obtain an activated modifier A solution; in the aqueous solution containing sodium periodate and 3-aminophenylboronic acid, the concentration of sodium periodate is 5%;

[0168] Step 302, mixing the activated modifier A solution and the hyaluronic acid aqueous solution described in step 301, stirring and reacting for 6 hours in a dark environment, placing the stirred reaction system in a dialysis bag with a molecular weight cutoff of 8000 and dialyzing with distilled water for 3 days, changing the water 5 times a day during the period, and then freeze-drying to obtain modified hyaluronic acid; the amount of sodium periodate substance is 0.5 times the amount of hyaluronic acid substance, and the amount of 3-aminophenylboronic acid substance is 1 times the amount of hyaluronic acid substance; the mass percentage of hyaluronic acid in the hyaluronic acid aqueous solution is 5%;

[0169] Step 303, stirring and activating a buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at 18° C. for 1 hour to obtain an activated modifier B solution; the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is a polylysine-containing solution prepared by dissolving polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a 2-morpholineethanesulfonic acid buffer solution. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide buffer solution; the pH of the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 5.5; the mass of the polylysine is 15% of the mass of the 2-morpholineethanesulfonic acid buffer solution; the molecular weight of the polylysine is 2000-2200; the molar ratio of polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 2:1:1;

[0170] In step 304, the activated modifier B solution and the recombinant collagen aqueous solution described in step 303 are mixed, stirred and reacted at a temperature of 25° C. for 2 hours, and the stirred reaction system is placed in a dialysis bag with a molecular weight cutoff of 8000 and dialyzed with distilled water for 3 days, during which the water is changed 5 times a day, and then freeze-dried to obtain modified recombinant collagen; the amount of polylysine substance in the activated modifier B solution is 20% of the amount of recombinant collagen substance, and the mass percentage of recombinant collagen in the recombinant collagen aqueous solution is 15%; the recombinant collagen is CF-1552 recombinant collagen purchased from Xi'an Juzi Biogene Technology Co., Ltd.;

[0171] Step 4: Providing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, specifically comprising:

[0172] Step 401, sterilize 0.1 g of the collagen drug-loaded microspheres and 1 mg of MXene@PDA composite nanoparticles by Co60 irradiation, respectively, to obtain sterilized collagen drug-loaded microspheres and sterilized MXene@PDA composite nanoparticles;

[0173] Step 402, dissolving the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid solution with a mass fraction of 5%;

[0174] Step 403, mixing the sterilized collagen drug-loaded microspheres described in step 401 with 1 mL of the modified hyaluronic acid solution described in step 402, and stirring for 10 minutes to obtain system A;

[0175] Step 404, dissolving the modified recombinant collagen in deionized water to obtain a modified recombinant collagen solution with a mass fraction of 15%;

[0176] Step 405, mixing the sterilized MXene@PDA composite nanoparticles in step 401 with 1 mL of the modified recombinant collagen solution in step 404, and stirring for 10 minutes to obtain system B;

[0177] Step 406, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution with a mass percentage of 12 wt%; the molecular weight of the polyvinyl alcohol is 90,000 to 98,000;

[0178] Step 407, under manual stirring conditions, the system A of step 403, the system B of step 405 and the polyvinyl alcohol solution of step 406 are stirred and mixed at a mass ratio of 2:3:2 for 2 minutes to obtain a pH / ROS stimulation responsive hydrogel for diabetic wound repair.

[0179] The performance of the pH / ROS stimulation responsive hydrogel for diabetic wound repair in this example is basically the same as that in Example 1.

[0180] Performance Evaluation

[0181] Figure 1 a is a schematic diagram of the process of preparing collagen drug-loaded microspheres in Example 1, Figure 1 b is the SEM image of collagen drug-loaded microspheres in Example 1. Figure 1 b It can be seen that the collagen drug-loaded microspheres are spherical in shape with a smooth surface, and the drug mangiferin is successfully loaded into the microspheres.

[0182] Figure 2 a is the XRD spectrum of the few-layer MXene nanosheets in step 202 of Example 1 and the MXene@PDA composite nanoparticles prepared in step 2, according to Figure 2a As can be seen from the figure, the MXene peak in the MXene@PDA composite nanoparticles shifts to the left, and the characteristic peak of PDA appears, indicating the successful synthesis of MXene@PDA. Figure 2 b is the SEM image and mapping element distribution diagram of the MXene@PDA composite nanoparticles of Example 1. Figure 2 b As can be seen, the PDA-specific N element distribution appears on MXene@PDA, indicating that PDA is successfully attached to the MXene surface. Figure 2 c is the SEM image of the MXene@PDA composite nanoparticles of Example 1 at a scale of 200 nm. Figure 2 d is the SEM image of the few-layer nanosheet MXene of Example 1 at a scale of 1 μm, Figure 2 e is the SEM image of the MXene@PDA composite nanoparticles of Example 1 at a scale of 1 μm. Figure 2 From c to 2e, we can see that the interlayer spacing of MXene@PDA composite nanoparticles becomes larger, indicating that PDA is successfully intercalated between MXene layers and attached to the surface.

[0183] Figure 3 a is the infrared spectrum of the modified hyaluronic acid in Example 1. It can be seen from the figure that at 1750cm -1 The characteristic peak of C=O belonging to the aldehyde group appeared at 2782 cm -1 A characteristic peak attributed to CH appeared, which proved that the hydroxyl group in the HA molecule was oxidized to aldehyde group, and the peak at 1653 cm -1 With 1827cm -1 The stretching vibration peaks of C=N and BO appeared at the 400 nm region, respectively, proving the successful grafting of phenylboronic acid. The table shows the successful preparation of modified hyaluronic acid. Figure 3 b is the FTIR spectrum of the modified recombinant collagen in Example 1, where the -1 The characteristic absorption peak at 3291 cm -1 The peak at derives from the stretching vibration of the NH of the primary amine in EPL, indicating the successful preparation of the modified recombinant collagen. Figure 3 c is a morphological diagram of the pH / ROS stimulation responsive hydrogel for diabetic wound repair in Example 1, Figure 3 d is a SEM image of the pH / ROS stimulus-responsive hydrogel for diabetic wound repair in Example 1, Figure 3 c and Figure 3 d shows that the pH / ROS stimulation-responsive hydrogel used for diabetic wound repair is moist and soft, with uniform pores inside, which can absorb tissue fluid exuded from the wound.

[0184] Figure 4a is the cytotoxicity test result of the pH / ROS stimulation-responsive hydrogel for diabetic wound repair in Example 1. The test method is MTT colorimetry. The results show that the hydrogel of the present invention has a cell proliferation-promoting effect.

[0185] Figure 4 b is the test result of the cell migration promoting ability of the hydrogel based on pH / ROS stimulation response for diabetic wound repair in Example 1. The test method is the scratch wound simulation method. The results show that the hydrogel of the present invention has the effect of promoting wound healing.

[0186] Figure 4 Both a and 4b show that the hydrogel has excellent biocompatibility.

[0187] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a pH / ROS stimulus-responsive hydrogel for diabetic wound repair, characterized in that: include: Step 1: Providing collagen drug-loaded microspheres, specifically including: Step 101, under a temperature condition of 40° C. to 45° C., stirring and mixing liquid paraffin and an emulsifier at a speed of 1000 rpm to 2000 rpm for 0.5 h to 3 h to obtain an emulsifier-containing system; Step 102, uniformly dispersing mangiferin in the collagen solution, stirring at a temperature of 37° C. to 45° C. and a speed of 1000 rpm to 2000 rpm for 0.5 h to 3 h to obtain a suspension containing mangiferin; Step 103, under the temperature condition of 37° C. to 45° C., the suspension containing mangiferin in step 102 is added dropwise to the emulsifier-containing system in step 101 at a rate of 0.5 mL / min to 3 mL / min, and stirred for 0.5 h to 2 h to obtain an emulsion system containing mangiferin; Step 104, rapidly cooling the mangiferin-containing emulsion system to 0°C to 10°C, then rapidly adding the glutaraldehyde solution to the cooled mangiferin-containing emulsion system, stirring for 1h to 3h at a temperature of 0°C to 10°C to obtain microspheres, and washing the microspheres by centrifugation, rotary evaporation and freeze-drying to obtain collagen drug-loaded microspheres; Step 2: Providing MXene@PDA composite nanoparticles, specifically including: Step 201, etching titanium aluminum carbide with hydrofluoric acid for 12 hours to 96 hours, centrifuging, and drying to obtain multi-layer MXene nanosheets; Step 202, exfoliating the MXene multilayer nanosheets described in step 201 with a tetramethylammonium hydroxide solution for 12 h to 48 h, centrifuging, and drying to obtain a few-layer MXene nanosheet; Step 203, adding dopamine hydrochloride to deionized water to obtain a dopamine hydrochloride suspension, adjusting the pH to be alkaline to obtain an alkaline dopamine suspension, placing the few-layer MXene nanosheet in the alkaline dopamine suspension and stirring for 6 hours to 10 hours to obtain MXene@PDA composite nanoparticles; Step 3: providing modified hyaluronic acid and modified recombinant collagen, specifically including: Step 301, stirring and activating an aqueous solution containing sodium periodate and 3-aminophenylboronic acid at a temperature of 18° C. to 25° C. for 1 h to 2 h to obtain an activated modifier A solution; Step 302, mixing the activated modifier A solution and the hyaluronic acid aqueous solution in step 301, stirring and reacting for 3 hours to 6 hours in a dark environment, dialyzing the stirred reaction system, and freeze-drying to obtain modified hyaluronic acid; Step 303, stirring and activating the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at a temperature of 18° C. to 37° C. for 1 h to 3 h to obtain an activated modifier B solution; Step 304, mixing the activated modifier B solution and the recombinant collagen aqueous solution in step 303, stirring and reacting at a temperature of 25° C. to 37° C. for 2 h to 6 h, dialyzing the stirred reaction system, and freeze-drying to obtain modified recombinant collagen; Step 4: Providing a pH / ROS stimulation-responsive hydrogel for diabetic wound repair, specifically comprising: Step 401, sterilizing the collagen drug-loaded microspheres and the MXene@PDA composite nanoparticles by Co60 irradiation, respectively, to obtain sterilized collagen drug-loaded microspheres and sterilized MXene@PDA composite nanoparticles; Step 402, dissolving the modified hyaluronic acid in deionized water to obtain a modified hyaluronic acid solution; Step 403, mixing the sterilized collagen drug-loaded microspheres in step 401 with the modified hyaluronic acid solution in step 402, and stirring for 5 min to 10 min to obtain system A; Step 404, dissolving the modified recombinant collagen in deionized water to obtain a recombinant collagen solution; Step 405, mixing the sterilized MXene@PDA composite nanoparticles in step 401 with the modified recombinant collagen solution in step 404, and stirring for 5 min to 10 min to obtain system B; Step 406, dissolving polyvinyl alcohol in water to obtain a polyvinyl alcohol solution; Step 407, stirring and mixing the system A of step 403, the system B of step 405 and the polyvinyl alcohol solution of step 406 in a mass ratio of (1-2):(1-3):(1-2) to obtain a pH / ROS stimulation responsive hydrogel for diabetic wound repair.

2. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 101, the emulsifier is Span80, and the volume of the liquid paraffin is 30 to 60 times the volume of the emulsifier.

3. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 102, the mass of mangiferin in the mangiferin-containing suspension is 2×10 -3 Times ~ 8×10 -3 times; in step 102, the collagen solution is a collagen solution with a mass percentage of 10% to 20% formed by dissolving collagen in water at a temperature of 37°C to 45°C; in step 102, the collagen is one or more of pig skin collagen, recombinant collagen and recombinant collagen doped with natural collagen.

4. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 103, the volume of the mangiferin suspension is 5 to 10 times the volume of the emulsifier in the emulsifier-containing system; in step 104, the mass percentage of the glutaraldehyde solution is 10wt% to 25wt%; in step 104, the volume of paraffin is 30 to 60 times the volume of the glutaraldehyde solution.

5. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 201, the mass of the hydrofluoric acid is 5 to 10 times the mass of titanium aluminum carbide, and the mass percentage of the hydrofluoric acid is 30% to 45%; in step 202, the mass of the tetramethylammonium hydroxide solution is 5 to 10 times the mass of titanium aluminum carbide, and the mass percentage of the tetramethylammonium hydroxide is 25% to 50%; in step 203, the mass percentage of the dopamine hydrochloride suspension is 1% to 5%, the pH of the alkaline dopamine suspension is 8 to 10, and the mass of the dopamine hydrochloride is 1 to 2 times the mass of the few-layer MXene nanosheets.

6. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 301, in the aqueous solution containing sodium periodate and 3-aminophenylboronic acid, the concentration of sodium periodate is 1% to 5%; in step 302, the amount of sodium periodate is 0.2 times to 0.5 times the amount of hyaluronic acid, and the amount of 3-aminophenylboronic acid is 0.5 times to 1 times the amount of hyaluronic acid; in step 302, the mass percentage of hyaluronic acid in the hyaluronic acid aqueous solution is 1% to 5%.

7. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 303, the buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is a buffer solution containing polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide prepared by dissolving polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a 2-morpholineethanesulfonic acid buffer solution. The pH value of the buffer solution of 2-morpholineethanesulfonic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 5.5-6.5, the mass of the polylysine is 10%-15% of the mass of the 2-morpholineethanesulfonic acid buffer solution, and the molecular weight of the polylysine is 2000-5000; in step 303, the molar ratio of the polylysine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is (1-2):1:

1.

8. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 304, the amount of polylysine in the activated modifier B solution is 15% to 20% of the amount of recombinant collagen, and the mass percentage of recombinant collagen in the recombinant collagen aqueous solution is 10% to 15%.

9. The method for preparing the pH / ROS stimulus-responsive hydrogel for diabetic wound repair according to claim 1, characterized in that: In step 402, the mass percentage of the modified hyaluronic acid solution is 1% to 5%; in step 403, the mass of the modified hyaluronic acid is 0.1 to 0.5 times the mass of the collagen drug-loaded microspheres; in step 404, the mass percentage of the modified recombinant collagen solution is 10% to 15%; in step 405, the mass of the modified recombinant collagen is 100 to 150 times the mass of the MXene@PDA composite nanoparticles; in step 406, the mass percentage of the polyvinyl alcohol solution is 10wt% to 15wt%, and the molecular weight of the polyvinyl alcohol is 89,000 to 98,000.

10. Use of a hydrogel prepared by the method for preparing a pH / ROS stimuli-responsive hydrogel for diabetic wound repair according to claim 1 in preparing a gel for diabetic wound repair.

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