A nano-enzyme cross-linked polyprionic acid-based hydrogel adhesive as well as preparation and application thereof

Abstract

The application discloses a nano-enzyme cross-linked polysulfooxynic acid-based hydrogel adhesive as well as a preparation method and application thereof, wherein the preparation method comprises the following steps: dissolving caffeic acid in ethanol to obtain a caffeic acid solution, adding an AgNO3 solution into the caffeic acid solution, and stirring and reacting to prepare caffeic acid-silver nano-enzymes; adding sulfooxynic acid and silk fibroin into an aqueous solution of lithium hydroxide in sequence, and then adding the caffeic acid-silver nano-enzymes into the mixture, stirring and mixing to form a gel, and obtaining the hydrogel adhesive after cooling. The hydrogel provided by the application has excellent wet adhesion, remarkable antibacterial property, good mechanical property and biocompatibility, these properties enable the hydrogel to provide a good repair environment for a wound and provide effective antibacterial treatment, thereby being beneficial to the rapid healing of a traumatic wound, and having a very good application prospect in the field of wound repair.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, specifically to a nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive, its preparation method, and its application in wound repair. Background Technology

[0002] Uncontrolled traumatic bleeding, especially from deep, irregularly shaped, and incompressible wounds, remains a significant challenge in emergency and surgical care and a leading cause of death. However, common dressings such as sutures and gauze often fail to effectively close wounds and control bleeding in a timely manner, increasing the risk of infection and prolonging the healing process. Therefore, developing multifunctional hydrogels with strong tissue adhesion, rapid hemostasis, and antibacterial properties to promote wound healing is crucial.

[0003] Lipoic acid (TA) is a small molecule naturally found in mitochondria, exhibiting excellent biocompatibility and adhesive properties, and has attracted widespread attention in the field of wound closure. TA can be lithiated through thermally induced ring-opening polymerization to form poly(lipoic acid)Li. + (PTALi) improves its hydrophobicity and ionic conductivity, thereby promoting wound healing. However, supramolecular polythioctic acid (PTA)-based hydrogels have poor mechanical properties and are prone to depolymerization, limiting their application in wet adhesion. Recent studies have shown that constructing a double crosslinked network is a promising strategy to improve the strength and toughness of PTA-based hydrogels. For example, Zhang et al. reported that 1,3-diisopropylbenzene and Fe2+ are linked by hydrogen bonds. 3+ Adding to PTA hydrogels can enhance the hydrogel network strength. However, using toxic organic crosslinking agents or heavy metal ions to stabilize PTA hydrogels may hinder their in vivo application. Sericin is a natural adhesive glycoprotein known for its suitable elasticity, mechanical strength, and biocompatibility; therefore, regulating the conformation of sericin to form novel biomimetic dual networks holds promise for improving the mechanical properties of PTA-based hydrogels.

[0004] In addition to enhanced mechanical properties, hydrogel adhesives also need to possess multifunctional characteristics, such as wet adhesion and antibacterial properties. Inspired by marine mussels, caffeic acid (CA), a plant-derived catechol derivative, has good biocompatibility, antibacterial properties, and adhesiveness. Therefore, the inventors disclosed a mussel-inspired polythiooctanoic acid-based hydrogel adhesive in their previous research (patent application CN 119303151 A). This adhesive utilizes caffeic acid (CA) with a phenolic hydroxyl structure to improve the wet adhesion properties of the hydrogel tissue. However, it still cannot cope with more complex wound environments, and its properties, such as wet adhesion, need further improvement. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive, its preparation method and application, aiming to solve the technical problems of poor wet adhesion performance and insufficient antibacterial properties of PTA-based hydrogels.

[0006] In a first aspect, the present invention provides a method for preparing a nanoenzyme-crosslinked polythiooctanoic acid-based hydrogel adhesive, comprising the following steps: S1. Caffeic acid was dissolved in ethanol to obtain a caffeic acid solution. AgNO3 solution (with water as solvent) was added to the caffeic acid solution, and the reaction was stirred at room temperature to prepare caffeic acid-silver (CAA) nanozyme. S2. Add thioctic acid and sericin sequentially to the aqueous solution of lithium hydroxide, then add caffeic acid-silver nanozyme to the mixture, stir and mix until gel is formed, and after cooling, PTALi-SCAA hydrogel adhesive with CAA nanozyme crosslinked polythioctic acid-sericin dual network (PTALi-S) is obtained.

[0007] Preferably, in step S1, the concentration of the caffeic acid solution is 1-3 mg / mL, the concentration of the AgNO3 solution is 2-4 mg / mL, and the volume ratio of the caffeic acid solution to the AgNO3 solution is 2:1.

[0008] Preferably, in step S2, the concentration of the aqueous solution of lithium hydroxide is 1.5-3.5 mg / mL, and the mass ratio of lithium hydroxide: lipoic acid: sericin: caffeic acid-silver nanozyme is (0.03-0.07):(1-3):(0.5-1.5):(0.05-0.15).

[0009] Preferably, the reaction temperature in step S2 is 40-80℃.

[0010] More preferably, step S2 specifically includes the following steps: S21. Disperse lithium hydroxide in deionized water and heat to 40-80℃; S22. Add thioctic acid to the solution obtained in step S21 and stir until completely dissolved; S23. Add sericin to the mixture obtained in step S22 and stir at 40-80℃ for 20-40 min. S24. Add caffeic acid-silver nanozyme to the mixture obtained in step S23, stir at 40-80℃ until gel is formed, and then cool to obtain hydrogel adhesive.

[0011] In step S2, the present invention regulates the pH and controls the temperature by adjusting lithium hydroxide to modulate the transformation of sericin from a disordered state to a β-form, which can enhance the mechanical strength and cohesion of the hydrogel, thereby improving wet adhesion properties and avoiding the problem of conformational transformation that was not easy to occur due to the grafting of caffeic acid in previous studies.

[0012] Secondly, the present invention provides a nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive obtained based on the above preparation method, which has excellent wet adhesion properties, significant antibacterial properties, good mechanical properties and biocompatibility.

[0013] Thirdly, the present invention provides the application of the above-mentioned nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive in the preparation of wound repair products, which can be pharmaceuticals or medical devices.

[0014] Furthermore, given the properties of hydrogel adhesives, the wound repair products that can be prepared include at least the following types: A1) Products that promote wound hemostasis; A2) Products for sealing wounds; A3) Products that promote wound healing.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention first prepared CAA nanozyme and constructed a double-network PTALi-SCAA hydrogel based on the nanozyme. The hydrogel has excellent wet adhesion properties, significant antibacterial properties, good mechanical properties and biocompatibility. These properties enable the hydrogel to provide a good repair environment for wounds and provide effective antibacterial treatment, which is conducive to the rapid healing of traumatic wounds and has a very good application prospect in the field of wound management.

[0016] (2) In the PTALi-SCAA multifunctional hydrogel system of the present invention, SCAA provides wet adhesion and rapid hemostasis, the disulfide bond in polythioctic acid endows the hydrogel with self-healing properties, and lithiation of thioctic acid provides conductivity; moreover, through electron transfer inside the CAA nanozyme, the system can, on the one hand, prevent the oxidation of catechol through redox reaction, improve the wet adhesion and hemostatic properties of the hydrogel, and on the other hand, generate ROS through the catalytic properties of nanozyme, further improving the antibacterial ability. Attached Figure Description

[0017] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0018] Figure 1 This is a scanning electron microscope image of the PTALi-SCAA composite hydrogel in this invention; Figure 2The image shows a scanning electron microscope (SEM) image of the PTALi-S composite hydrogel obtained in Comparative Example 1. Figure 3 This is a diagram illustrating the effectiveness of the PTALi-SCAA composite hydrogel against Escherichia coli and Staphylococcus aureus in this invention. Figure 4 The image shows the wet adhesion performance test results of the PTALi-SCAA composite hydrogel in this invention. Figure 5 The image shows the test results of the hemostatic performance of the PTALi-SCAA composite hydrogel in this invention. Detailed Implementation

[0019] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention. The terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion.

[0021] Hydrogel materials used for wound repair must possess good mechanical strength and biocompatibility, as well as excellent wet adhesion and antibacterial properties. The inventors' previously developed mussel-inspired polythioctic acid (PTA)-based hydrogel adhesive effectively improved the mechanical and wet adhesion properties of the hydrogel, but shortcomings remained. Furthermore, the grafting efficiency of sericin with caffeic acid was low, and the catechol groups of caffeic acid were easily oxidized, reducing the overall performance of the hydrogel. To address these shortcomings, this invention provides a nanoenzyme-crosslinked PTA-based hydrogel adhesive, its preparation method, and its applications. By crosslinking a PTA-serin dual network (PTALi-S) with CAA nanoenzymes, a novel supramolecular self-assembled biomimetic hydrogel adhesive with excellent wet adhesion, hemostasis, and antibacterial capabilities is prepared, thereby achieving rapid hemostasis, wound sealing, and wound healing promotion, providing a more efficient solution for wound management.

[0022] This invention provides a nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive, the preparation method of which includes the following steps: (1) Dissolve caffeic acid in ethanol to obtain caffeic acid solution, add AgNO3 aqueous solution to caffeic acid solution, and stir the reaction at room temperature to obtain CAA nanozyme; wherein, the concentration of caffeic acid solution is 1-3 mg / mL, the concentration of AgNO3 solution is 2-4 mg / mL, and the volume ratio of caffeic acid solution to AgNO3 solution is 2:1.

[0023] (2) At 40-80℃, thioctic acid and sericin were added sequentially to an aqueous solution of lithium hydroxide to obtain the PTALi-S complex. Then, CAA nanozyme was added to the complex and stirred until it formed a gel. After cooling, the PTALi-SCAA hydrogel adhesive was obtained. The concentration of the aqueous solution of lithium hydroxide was 1.5-3.5 mg / mL, and the mass ratio of lithium hydroxide: thioctic acid: sericin: caffeic acid-silver nanozyme was (0.03-0.07):(1-3):(0.5-1.5):(0.05-0.15).

[0024] In some embodiments of the present invention, the concentration of the caffeic acid solution is 2 mg / mL, the concentration of the AgNO3 solution is 2.4 mg / mL, and the volume ratio of the caffeic acid solution to the AgNO3 solution is 2:1.

[0025] In some embodiments of the present invention, the concentration of the aqueous solution of lithium hydroxide is 2.5 mg / mL, and the mass ratio of lithium hydroxide: lipoic acid: sericin: caffeic acid-silver nanozyme is 0.05:2:1:0.1.

[0026] In the preparation method of this invention, firstly, CAA nanozymes are synthesized using CA as a reducing agent and stabilizer; secondly, the conformation of sericin is changed by adjusting the pH and temperature with lithium hydroxide, so that TA and sericin undergo self-assembly, and the carboxyl group of TA can form stable hydrogen bonds with the amino group on sericin, resulting in a PTALi-S double network structure; finally, a novel PTALi-CAA hydrogel adhesive is formed by reacting the catechol group on the nanozyme with the carboxyl or amino group of PTALi-S; in addition, the nanozyme can also form covalent bonds with polythioctic acid to prevent the depolymerization of polythioctic acid.

[0027] In existing technologies, caffeic acid (CA) is rarely used in nanozymes because the catechol groups of CA are easily over-oxidized to quinones under alkaline conditions, negatively impacting the function of the hydrogel. Studies on mussel-induced adhesion show that dynamically controlling the redox balance between catechol and quinone groups is crucial for maintaining the long-term stability of CA. Research indicates that nanozymes based on noble metals, transition metals, and carbon nanomaterials exhibit enzyme-like catalytic properties through electron transfer effects. In particular, silver nanoparticles, due to their surface plasmon resonance, can form silver-based nanozymes with polyphenols to reduce the oxidation reaction of o-phenylenediamine. In the presence of quinone electron acceptors, the quinone groups in the polyphenols are converted back to catechol groups via electron transfer. Furthermore, the partially positive charge induced on the Ag surface enhances its catalytic activity, thereby generating reactive oxygen species (ROS). Therefore, this invention improves the wet adhesion and antibacterial properties of PTA-based hydrogels by preparing novel CAA nanozymes.

[0028] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0029] Example 1 This example provides a nanoenzyme-crosslinked polythiooctanoic acid-based hydrogel adhesive, the preparation of which includes the following steps: (1) Preparation of CAA nanozyme.

[0030] Accurately weigh caffeic acid powder and add it to ethanol. Stir magnetically at room temperature until completely dissolved to prepare a 2 mg / mL caffeic acid solution. Then, add a 2.4 mg / mL AgNO3 aqueous solution to the above caffeic acid solution, with a volume ratio of caffeic acid solution to AgNO3 aqueous solution of 2:1. Stir the reaction at room temperature for 30 min. After the reaction is complete, centrifuge the reaction solution and wash the precipitate to obtain caffeic acid-silver nanozyme.

[0031] (2) Preparation of hydrogels.

[0032] 0.05 g of lithium hydroxide was dispersed in 20 mL of deionized water and heated to 60 °C. Then, 2 g of lipoic acid was added, and the mixture was stirred continuously until completely dissolved to obtain the PTALi complex. Next, 2 g of sericin was added to the above solution, and the mixture was stirred at 60 °C for 30 minutes to obtain the PTALi-S complex. Finally, 0.1 g of SCAA nanozyme was added, and the mixture was stirred for 2 h to form a gel. The resulting product was poured into a mold and cooled to 25 °C to obtain the PTALi-SCAA composite hydrogel.

[0033] Figure 1 The image shows a scanning electron microscope (SEM) image of the PTALi-SCAA composite hydrogel. As can be seen from the image, the hydrogel adhesive has a three-dimensional network structure, microporous features, and high porosity.

[0034] Comparative Example 1 Unlike Example 1, the hydrogel provided in this example does not contain caffeic acid-silver nanozyme, and its preparation process is as follows: Disperse 0.05 g of lithium hydroxide in 20 mL of deionized water, heat to 60 °C, then add 2 g of thioctic acid and stir continuously until completely dissolved. Add 2 g of sericin to the above solution and stir continuously at 60 °C until gel is formed. Pour the resulting product into a mold and cool to 25 °C to obtain PTALi-S composite hydrogel.

[0035] Figure 2The image shows a scanning electron microscope (SEM) image of the PTALi-S composite hydrogel. As can be seen from the image, the internal structure of the PTALi-S composite hydrogel has a small number of micropores, but the dispersion is uneven, which is not conducive to the efficient transport of nutrients.

[0036] Example 2 Taking *Escherichia coli* and *Staphylococcus aureus* as examples, this study tested the antibacterial properties of the nanozyme-crosslinked polythioctic acid hydrogel adhesive provided by this invention. The specific experimental procedure is as follows: Add the hydrogel to a 24-well cell culture plate and sterilize with ultraviolet light for 24 hours; add the bacterial suspension (10... 8 CFU / mL was added to the sterile hydrogel, and a blank control group was set up, i.e., bacterial suspension (10 CFU / mL) was added to a 24-cell culture plate without hydrogel. 8 (CFU / mL); then, incubate for 2 hours in a shaking incubator at 100 rpm / min and 37°C. Take 100 μL of bacterial suspension from the 24 cell culture plate and spread it evenly on an agar plate, then incubate for another 12 hours in a constant temperature shaking incubator. Finally, analyze the antibacterial properties of the material by taking pictures and counting the colonies on the agar plate.

[0037] The colony count on the agar plate is shown below. Figure 3 As shown, Experiment 1 used the hydrogel prepared in Comparative Example 1, while Experiment 2 used the hydrogel prepared in Example 1. From... Figure 3 It is known that the nanoenzyme crosslinked polythioctic acid-based hydrogel adhesive provided by the present invention has excellent antibacterial properties.

[0038] Example 3 This example evaluates the wet adhesion properties of hydrogels using a shear test on a universal testing machine. The specific procedure is as follows: The hydrogel was applied to two substrates of two different sizes (50 mm long × 20 mm wide) with an adhesion area of ​​20 × 20 mm; then, the samples were immersed in PBS solution; finally, the samples were measured at a speed of 10 mm / min.

[0039] Test results are as follows Figure 4 As shown, Experiment 1 used the hydrogel prepared in Comparative Example 1, while Experiment 2 used the hydrogel prepared in Example 1. From... Figure 4 It can be seen that the nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive provided in Example 1 has excellent wet adhesion performance, while the hydrogel in Comparative Example 1 exhibits lower wet adhesion strength.

[0040] Example 4 This example tested the hemostatic properties of nanozyme-crosslinked polythiooctanoic acid hydrogel adhesives. The experimental procedure is as follows: Hydrogel was deposited in 96-well plates; then, 50 μL of recalcitrant rat blood was added to the wells; after a specified time, the wells containing the mixture were washed several times with 0.9 wt% NaCl solution. After the experiment, the final clotting time was recorded, and the thrombus was imaged using a digital camera.

[0041] Test results are as follows Figure 5 As shown in the figure, experimental group 1 used the hydrogel prepared in Comparative Example 1, and experimental group 2 used the hydrogel prepared in Example 1. The figure shows that the clotting time of the blank group was approximately 8 minutes, while the clotting time of experimental group 1 was approximately 1 minute, and the clotting time of experimental group 2 was 30 seconds.

[0042] In summary, this invention effectively improves the wet adhesion and antibacterial properties of hydrogels by employing a biomimetic dual-network and enzyme-like catalysis strategy, enabling rapid and timely sealing of wounds and quick hemostasis, thereby promoting wound repair.

[0043] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for preparing a nanoenzyme-crosslinked polythiooctanoic acid-based hydrogel adhesive, characterized in that, Includes the following steps: S1. Caffeic acid is dissolved in ethanol to obtain a caffeic acid solution. AgNO3 solution is added to the caffeic acid solution, and the reaction is stirred to obtain caffeic acid-silver nanozyme. S2. Add lipoic acid and sericin sequentially to an aqueous solution of lithium hydroxide, then add caffeic acid-silver nanozyme to the mixture, stir and mix until a gel is formed, and cool to obtain a hydrogel adhesive; the mass ratio of lithium hydroxide:lipoic acid:sericin:caffeic acid-silver nanozyme is (0.03-0.07):(1-3):(0.5-1.5):(0.05-0.15); Step S2 specifically includes the following steps: S21. Disperse lithium hydroxide in deionized water and heat to 40-80℃; S22. Add thioctic acid to the solution obtained in step S21 and stir until completely dissolved; S23. Add sericin to the mixture obtained in step S22 and stir at 40-80℃ for 20-40 min. S24. Add caffeic acid-silver nanozyme to the mixture obtained in step S23, stir at 40-80℃ until gel is formed, and then cool to obtain hydrogel adhesive.

2. The preparation method according to claim 1, characterized in that, The concentration of the caffeic acid solution is 1-3 mg / mL, the concentration of the AgNO3 solution is 2-4 mg / mL, and the volume ratio of the caffeic acid solution to the AgNO3 solution is 2:

1.

3. The preparation method according to claim 1, characterized in that, The concentration of the aqueous solution of lithium hydroxide is 1.5-3.5 mg / mL.

4. The nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive obtained by the preparation method according to any one of claims 1-3.

5. The application of the nanoenzyme crosslinked polythiooctanoic acid-based hydrogel adhesive as described in claim 4 in the preparation of wound repair products.

6. The application according to claim 5, characterized in that, The wound healing products include the following: A1) Products that promote wound hemostasis; A2) Products for sealing wounds; A3) Products that promote wound healing.

7. The application according to claim 5, characterized in that, The wound repair products include medicines or medical devices used for wound repair.

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