Mixed hemostatic hydrogel and preparation method thereof

Abstract

The invention discloses mixed hemostatic hydrogel and a preparation method thereof, and relates to the technical field of wound repair. The exosome-coated oxygen nano-bubble hydrogel stent is composed of a hydrogel stent and exosome-coated oxygen nano-bubbles, wherein the hydrogel stent is beta cyclodextrin-sodium alginate / modified nano-zinc oxide / methacrylated gelatin mixed hydrogel; the problems that an existing hydrogel wound coating material cannot be absorbed due to excessive seepage, the infection risk is increased, and wound healing is affected are solved.

Description

Technical Field

[0001] The invention relates to the technical field of wound repair, in particular to a mixed hemostatic hydrogel and a preparation method thereof. Background Art

[0002] Poor wound healing after trauma and surgery constitutes a pressing global medical problem. Statistics show that inadequate wound healing affects 6.5 million patients in the United States. Among all wound types, surgical wounds are the most expensive. The main goals of wound treatment include rapid wound closure and obtaining scars that are both practical and aesthetic. However, during the normal healing process, inflammation and hypoxia deviate from their typical patterns and may trigger abnormal wound healing, thereby inducing delayed wound closure, keloid formation, and the development of hypertrophic scars. Wound dressings often use hydrogels as scaffolds to carry active ingredients to achieve the effect of healing wounds. However, due to its structural characteristics, the fluid absorption capacity of hydrogels is relatively limited and can only absorb a small amount of exudate. Therefore, for wounds with more exudate, hydrogel dressings may not be able to effectively respond, resulting in accumulation of exudate and increased risk of infection. Although traditional hydrogels such as chitosan and silver ion dressings have good sterilization and antibacterial effects, they are all toxic and have certain damage to skin and soft tissues, affecting the effect of wound healing. Summary of the invention

[0003] The purpose of the present invention is to provide a mixed hemostatic hydrogel and a preparation method thereof, so as to solve the problem that the existing hydrogel wound coating materials are easily unable to absorb excessive exudate, resulting in increased infection risk and affecting wound healing.

[0004] In order to solve the above problems, the present invention adopts the following technical means: A mixed hemostatic hydrogel consists of a hydrogel scaffold and exosome-coated oxygen nanobubbles, wherein the hydrogel scaffold is a β-cyclodextrin-sodium alginate / modified nano zinc oxide / methacrylylated gelatin mixed hydrogel.

[0005] Preferably, the shell of the oxygen nanobubble is composed of a glycosylated protein conjugate consisting of glucose-bound bovine serum albumin.

[0006] In addition, a method for preparing a mixed hemostatic hydrogel comprises the following steps: S100. Gelatin modification, using methacrylic anhydride to modify the gelatin matrix; S200. Sodium alginate modification, using β-cyclodextrin grafted modified sodium alginate; S300.Nano zinc oxide modified; S400. Prepare a hydrogel scaffold; S500. Prepare exosome-encapsulated oxygen nanobubbles and load them into the hydrogel scaffold.

[0007] Preferably, step S100 includes: S110. Dissolve gelatin under stirring at 37-60° C., and add methacrylic anhydride dropwise; S120. After stirring the solution until it is clear, dialyzing it in a dialysis membrane; S130. Dry at 37-60℃ for 12-48 hours, freeze-dry for 48 hours, and store at 4℃ away from light.

[0008] Furthermore, in step S110, the mass ratio of methacrylic anhydride to gelatin is 1:2.

[0009] Furthermore, the step S200 includes: S210. Dissolving sodium alginate in an aqueous sodium hydroxide solution, and then adding β-cyclodextrin; S220. stirring in an oil bath at 75°C until sodium alginate and β-cyclodextrin are completely dissolved, adding epichlorohydrin, and reacting under mechanical stirring; S230. The solution was adjusted to neutral by adding glacial acetic acid and precipitated with N,N-dimethylformamide; S240. Wash the precipitate with distilled water, filter it, and then freeze-dry it to obtain modified sodium alginate.

[0010] Furthermore, the step S300 includes: S310. The nano zinc oxide powder is added to the mixed solvent and ultrasonically dispersed to prepare a suspension; S320. Prepare a pH adjusting solution having a pH of 7 to 8; S330. The pH adjusting solution is slowly dripped into the suspension within 2 hours; S340. After the reaction is completed, the suspension is washed and centrifuged.

[0011] Furthermore, the step S400 includes: S410. The modified sodium alginate solution prepared in S200 is dissolved in pure water to prepare a hydrogel solution; S420. adding the modified gelatin to the hydrogel solution and fully dissolving it at 70° C. to obtain a hydrogel dressing; S430. Disperse the modified nano zinc oxide in a CaCl2 solution, and immerse the hydrogel dressing in the calcium ion dispersion of zinc oxide to complete cross-linking.

[0012] Furthermore, the step S500 includes: S510. dispersing the exosome suspension in PBS buffer and adjusting the concentration; S520. Soaking the cross-linked hydrogel scaffold in PBS buffer; S530. Under the action of a shaking table, at a temperature range of 4 to 10°C, add the PBS buffer containing dispersed exosomes into the PBS buffer containing the hydrogel scaffold, mix in the dark, and obtain a finished product.

[0013] Furthermore, the exosome concentration of the PBS buffer dispersed in the exosome suspension is 5-150 μg / μL.

[0014] During use, the present invention has the following beneficial effects: By introducing β-cyclodextrin-sodium alginate / modified nano zinc oxide / methacrylated gelatin mixed hydrogel as a hydrogel scaffold, β-cyclodextrin-sodium alginate is better than traditional sodium alginate. Due to the good inclusion capacity of β-cyclodextrin, the grafted modified sodium alginate can be used as a new drug controlled release material to improve the stability and bioavailability of drugs. Traditional gelatin has poor mechanical properties, is fragile and difficult to shape, and methacrylated gelatin has better biocompatibility and mechanical properties. The hydrogel exhibits significant hemostatic synergy through combined therapeutic properties, especially for irregularly shaped and bleeding wounds. Exosome-coated oxygen nanobubbles are embedded in the hydrogel, thereby improving the delivery efficiency of exosomes and solving the problem of hypoxia. This combination can not only accelerate the wound healing process to achieve antibacterial and anti-inflammatory effects, but also effectively remove excess ROS in wound tissue, relieve hypoxia and reduce the risk of hypertrophic scars. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0016] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features of the embodiments may be combined with each other.

[0018] A mixed hemostatic hydrogel consists of a hydrogel scaffold and exosome-coated oxygen nanobubbles, wherein the hydrogel scaffold is a β-cyclodextrin-sodium alginate / modified nano zinc oxide / methacrylylated gelatin mixed hydrogel.

[0019] Preferably, the shell of the oxygen nanobubble is composed of a glycosylated protein conjugate consisting of glucose-bound bovine serum albumin.

[0020] In addition, a method for preparing a mixed hemostatic hydrogel comprises the following steps: S100. Gelatin modification, using methacrylic anhydride to modify the gelatin matrix; S200. Sodium alginate modification, using β-cyclodextrin grafted modified sodium alginate; S300.Nano zinc oxide modified; S400. Prepare a hydrogel scaffold; S500. Prepare exosome-encapsulated oxygen nanobubbles and load them into the hydrogel scaffold.

[0021] Preferably, step S100 includes: S110. Dissolve gelatin under stirring at 37-60° C., and add methacrylic anhydride dropwise; S120. After stirring the solution until it is clear, dialyzing it in a dialysis membrane; S130. Dry at 37-60℃ for 12-48 hours, freeze-dry for 48 hours, and store at 4℃ away from light.

[0022] Furthermore, in step S110, the mass ratio of methacrylic anhydride to gelatin is 1:2.

[0023] Furthermore, the step S200 includes: S210. Dissolving sodium alginate in an aqueous sodium hydroxide solution, and then adding β-cyclodextrin; S220. stirring in an oil bath at 75°C until sodium alginate and β-cyclodextrin are completely dissolved, adding epichlorohydrin, and reacting under mechanical stirring; S230. The solution was adjusted to neutral by adding glacial acetic acid and precipitated with N,N-dimethylformamide; S240. Wash the precipitate with distilled water, filter it, and then freeze-dry it to obtain modified sodium alginate.

[0024] Furthermore, the step S300 includes: S310. The nano zinc oxide powder is added to the mixed solvent and ultrasonically dispersed to prepare a suspension; S320. Prepare a pH adjusting solution having a pH of 7 to 8; S330. The pH adjusting solution is slowly dripped into the suspension within 2 hours; S340. After the reaction is completed, the suspension is washed and centrifuged.

[0025] Furthermore, the step S400 includes: S410. The modified sodium alginate solution prepared in S200 is dissolved in pure water to prepare a hydrogel solution; S420. adding the modified gelatin to the hydrogel solution and fully dissolving it at 70° C. to obtain a hydrogel dressing; S430. Disperse the modified nano zinc oxide in a CaCl2 solution, and immerse the hydrogel dressing in the calcium ion dispersion of zinc oxide to complete cross-linking.

[0026] Furthermore, the step S500 includes: S510. dispersing the exosome suspension in PBS buffer and adjusting the concentration; S520. Soaking the cross-linked hydrogel scaffold in PBS buffer; S530. Under the action of a shaking table, at a temperature range of 4 to 10°C, add the PBS buffer containing dispersed exosomes into the PBS buffer containing the hydrogel scaffold, mix in the dark, and obtain a finished product.

[0027] Furthermore, the exosome concentration of the PBS buffer dispersed in the exosome suspension is 5-150 μg / μL.

[0028] During use, the present invention has the following beneficial effects: By introducing β-cyclodextrin-sodium alginate / modified nano zinc oxide / methacrylated gelatin mixed hydrogel as a hydrogel scaffold, β-cyclodextrin-sodium alginate is better than traditional sodium alginate. Due to the good inclusion capacity of β-cyclodextrin, the grafted modified sodium alginate can be used as a new drug controlled release material to improve the stability and bioavailability of drugs. Traditional gelatin has poor mechanical properties, is fragile and difficult to shape, and methacrylated gelatin has better biocompatibility and mechanical properties. The hydrogel exhibits significant hemostatic synergy through combined therapeutic properties, especially for irregularly shaped and bleeding wounds. Exosome-coated oxygen nanobubbles are embedded in the hydrogel, thereby improving the delivery efficiency of exosomes and solving the problem of hypoxia. This combination can not only accelerate the wound healing process to achieve antibacterial and anti-inflammatory effects, but also effectively remove excess ROS in wound tissue, relieve hypoxia and reduce the risk of hypertrophic scars.

[0029] The technical solution of the present invention is described below in conjunction with specific embodiments.

[0030] Step 100: Gelatin modification: dissolve gelatin in deionized water, stir and dissolve, raise the solution temperature to 37-60°C, add methacrylic anhydride dropwise, stir, dialyze the obtained clear solution in a dialysis membrane, dry at 37-60°C for 12-48 hours, take out and freeze-dry for 48 hours to obtain methacrylated gelatin (GelMA), and store it at 4°C away from light; the mass ratio of methacrylic anhydride to gelatin is 1:2; Step 2: Modification of sodium alginate: 1.21 g of sodium alginate was weighed using an electronic balance and dissolved in 15 ml of sodium hydroxide aqueous solution, and then 0.8 g of β-cyclodextrin was added. After complete dissolution in a 75°C oil bath, 0.5 mL of epichlorohydrin was added and stirred electrically. After the reaction was completed, the solution was neutralized to neutrality with glacial acetic acid, precipitated with N,N-dimethylformamide, washed with distilled water, filtered, and freeze-dried to obtain modified β-cyclodextrin-sodium alginate.

[0031] Step 3: Modification of nano zinc oxide: First, slowly add 0.2 g of nano zinc oxide powder weighed accurately to a solvent mixed with 12 ml of deionized water and ethanol in a volume ratio of 8:2. Subsequently, the mixture was homogenized for 0.5 hours using an ultrasonic device to ensure that the nano zinc oxide particles were fully dispersed and agglomeration was reduced. Preparation of pH adjusting solution: Take another container, measure 10 ml of a deionized water / ethanol mixed solution of the same ratio (8:2 v / v), and dissolve an appropriate amount of glacial acetic acid therein. By adding sodium hydroxide (NaOH) solution dropwise, the pH value of the solution was finely adjusted to between 7 and 8 to ensure that the subsequent reaction was carried out in a suitable acid-base environment. Mixing reaction: While maintaining the temperature of the constant temperature magnetic stirring oil bath set at 40°C and the speed at 800 rpm, the pH adjusting solution prepared in step 2 was slowly and continuously added to the nano zinc oxide suspension completed in step 1. This process continued while ensuring that the substances in the suspension were fully mixed and reacted, and the total duration was 2 hours. Washing and centrifugal separation: After the reaction is completed, the reaction product is washed several times with ethanol to remove unreacted impurities and by-products. Subsequently, the washed product is transferred to a centrifuge and centrifuged at 5000 rpm for 20 minutes to achieve solid-liquid separation. Vacuum drying: Finally, the solid material obtained after centrifugation is placed in a vacuum drying oven at 70°C for 12 hours of drying. This step is intended to completely remove the residual solvent in the sample, thereby obtaining the modified nano zinc oxide product and ensuring its stability and purity.

[0032] Step 4: Preparation of hydrogel component: Add modified sodium alginate to a beaker and add 30 mL of laboratory pure water with a measuring cylinder. Stir on a magnetic stirrer for 2.5 hours to ensure that it is fully dissolved to obtain a hydrogel solution. We weigh a certain amount of methacrylated gelatin with a balance and place it in the above-prepared hydrogel solution; Then use a glass rod to stir quickly so that most of it can be dissolved in the above solution; After sealing with a sealing film, place it in a 70 ° C water bath and fully dissolve it; After thorough dissolution, the preparation of the hydrogel component is completed. The modified nano zinc oxide is mixed in a certain proportion, and a certain amount of CaCl2 is weighed and dissolved in laboratory pure water to prepare a 0.6% (w / v) calcium ion solution. The prepared hydrogel wound dressing is soaked in a 0.6% (w / v) calcium chloride solution for 2 hours of cross-linking. After cross-linking is completed, the calcium chloride solution on the surface of the dressing is fully cleaned with deionized water.

[0033] Step 5: First, suspend the exosomes in PBS buffer and adjust the concentration range to 5 to 150 micrograms per microliter to prepare an exosome suspension. Subsequently, soak the aforementioned cross-linked hydrogel scaffold in PBS buffer. Next, gradually add the exosome suspension to the hydrogel scaffold soaking solution at a volume ratio of 1:3 to 150, and use a shaker to mix in the dark at a temperature range of 4°C to 40°C for a duration ranging from 30 seconds to 1 hour. The finished product formed after mixing needs to be stored at -80°C.

[0034] To embed exosome-coated oxygen nanobubbles into the hydrogel, we used nanoparticle tracking analysis (NTA) to accurately measure the concentration and size of exosomes. Further, scanning transmission electron microscopy (TEM) images were captured and negatively stained with uranyl acetate (1% w / w) to enhance image contrast.

[0035] In order to comprehensively evaluate the biological properties of the composite material, we conducted a series of biological characterization experiments, including but not limited to in vitro degradation experiments, hemolysis tests, cell viability assessment, cell proliferation and migration analysis, exosome uptake efficiency detection, immunofluorescence staining observation, and the establishment and study of an in vivo wound healing model. These experiments aim to reveal the biocompatibility, functional activity, and application potential of the GelMA-exosome complex in the biomedical field from multiple dimensions.

[0036] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A mixed hemostatic hydrogel, characterized in that: It consists of a hydrogel scaffold and exosome-coated oxygen nanobubbles, wherein the hydrogel scaffold is a mixed hydrogel of β-cyclodextrin-sodium alginate / modified nano zinc oxide / methacrylylated gelatin.

2. A mixed hemostatic hydrogel according to claim 1, characterized in that: The shell of the oxygen nanobubble is composed of a glycosylated protein conjugate composed of glucose-bound bovine serum albumin.

3. A method for preparing a mixed hemostatic hydrogel, characterized in that: The following steps are involved: S100. Gelatin modification, using methacrylic anhydride to modify the gelatin matrix; S200. Sodium alginate modification, using β-cyclodextrin grafted modified sodium alginate; S300.Nano zinc oxide modified; S400. Prepare a hydrogel scaffold; S500. Prepare exosome-encapsulated oxygen nanobubbles and load them into the hydrogel scaffold.

4. The method for preparing a mixed hemostatic hydrogel according to claim 3, characterized in that: Step S100 includes: S110. Dissolve gelatin under stirring at 37-60° C., and add methacrylic anhydride dropwise; S120. After stirring the solution until it is clear, dialyzing it in a dialysis membrane; S130. Dry at 37-60℃ for 12-48 hours, freeze-dry for 48 hours, and store at 4℃ away from light.

5. The method for preparing a mixed hemostatic hydrogel according to claim 4, characterized in that: In step S110, the mass ratio of methacrylic anhydride to gelatin is 1:

2.

6. The method for preparing a mixed hemostatic hydrogel according to claim 3, characterized in that: The step S200 includes: S210. Dissolving sodium alginate in an aqueous sodium hydroxide solution, and then adding β-cyclodextrin; S220. stirring in an oil bath at 75°C until sodium alginate and β-cyclodextrin are completely dissolved, adding epichlorohydrin, and reacting under mechanical stirring; S230. The solution was adjusted to neutral by adding glacial acetic acid and precipitated with N,N-dimethylformamide; S240. Wash the precipitate with distilled water, filter it, and then freeze-dry it to obtain modified sodium alginate.

7. The method for preparing a mixed hemostatic hydrogel according to claim 3, characterized in that: The step S300 includes: S310. The nano zinc oxide powder is added to the mixed solvent and ultrasonically dispersed to prepare a suspension; S320. Prepare a pH adjusting solution having a pH of 7 to 8; S330. The pH adjusting solution is slowly dripped into the suspension within 2 hours; S340. After the reaction is completed, the suspension is washed and centrifuged.

8. The method for preparing a mixed hemostatic hydrogel according to claim 3, characterized in that: The step S400 includes: S410. The modified sodium alginate solution prepared in S200 is dissolved in pure water to prepare a hydrogel solution; S420. adding the modified gelatin to the hydrogel solution and fully dissolving it at 70° C. to obtain a hydrogel dressing; S430. Disperse the modified nano zinc oxide in a CaCl2 solution, and immerse the hydrogel dressing in the calcium ion dispersion of zinc oxide to complete cross-linking.

9. The method for preparing a mixed hemostatic hydrogel according to claim 3, characterized in that: The step S500 includes: S510. dispersing the exosome suspension in PBS buffer and adjusting the concentration; S520. Soaking the cross-linked hydrogel scaffold in PBS buffer; S530. Under the action of a shaking table, at a temperature range of 4 to 10°C, add the PBS buffer containing dispersed exosomes into the PBS buffer containing the hydrogel scaffold, mix in the dark, and obtain a finished product.

10. The method for preparing a mixed hemostatic hydrogel according to claim 9, characterized in that: The exosome concentration of the PBS buffer dispersed in the exosome suspension is 5-150 μg / μL.