Preparation method and application of Janus photo-thermo-piezoelectric hydrogel dressing for infected wound repair

Abstract

The application relates to a preparation method and application of a Janus structure photothermal-piezoelectric hydrogel dressing for infected wound repair, and the preparation method comprises the following steps: phenylboronic acid grafted methyl acrylamide gelatin and dopamine grafted hyaluronic acid are respectively dissolved in deionized water, MXene@CeO2 heterojunction is uniformly ultrasonically dispersed, after mixing, sodium hydroxide is used to adjust the pH to be slightly alkaline, and a Janus structure inner layer is formed; phenylboronic acid grafted gelatin and ortho-nitrobenzyl alcohol grafted hyaluronic acid are respectively dissolved in deionized water, BaTiO3 is uniformly ultrasonically dispersed, and a Janus structure outer layer is formed after mixing. The Janus structure photothermal-piezoelectric hydrogel dressing prepared by the application has the characteristics of space-time release to adapt to the wound repair process, in addition, has excellent photothermal conversion performance, kills bacteria on the infected wound through photothermal heating, effectively simulates physiological electrical signals through piezoelectric effect, regulates cell behavior, and can be used for infected wound repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials, in particular to a preparation method and application of a Janus structured photothermal-piezoelectric hydrogel dressing for infected wound repair. BACKGROUND

[0002] With the continuous improvement of medical level, infected wounds caused by trauma, diabetes, and postoperative surgery have become a thorny problem in clinical treatment. Infected wounds not only destroy the integrity of skin tissue, but also cause bacterial growth and persistent inflammatory reactions, leading to hindered healing process. According to statistics, patients with severe infected wounds are at risk of serious complications such as tissue necrosis and sepsis, and even endanger life; and patients with chronic infected wounds are long-term suffering from illness, with long treatment cycle and high medical expenses, which brings heavy burden to the patient family and the social medical system. Therefore, developing new infected wound repair materials and technologies that are efficient, safe and can accelerate healing has become a key requirement for clinical treatment.

[0003] Traditional wound repair dressings can only play a simple isolation and protection role, and their pore structure cannot effectively block bacterial invasion. At the same time, such dressings have poor water retention and air permeability, which can easily cause wound dryness and scabbing or effusion, hindering the growth of new tissues and delaying the healing process, and frequent replacement can cause secondary damage. In recent years, new wound dressings have emerged, among which hydrogel dressings stand out with unique advantages. They have high water content and good biocompatibility, providing a moist healing environment for wounds and promoting epithelial cell migration and proliferation. In addition, their porous structure can also load active substances such as drugs and growth factors, achieving local drug delivery for wounds, and becoming a research hotspot in the field of wound repair. However, the existing single-function hydrogel dressings still have obvious shortcomings. Antibacterial hydrogels can inhibit bacterial growth, but have limited promotion effect on wound healing. Promoting repair hydrogels can accelerate tissue regeneration, but are difficult to effectively control infection. In the process of infected wound repair, infection control and tissue repair are two interrelated and complementary links, and single-function hydrogel dressings cannot achieve multidimensional synergistic treatment of infected wounds, and are difficult to meet the dual needs of infection control and promoting repair, and new hydrogel dressings with multiple functions are urgently needed.

[0004] Photothermal effect utilizes the heat generated by photothermal materials under light irradiation to destroy the cell membrane structure and protein activity of bacteria at high temperature, achieving efficient sterilization with the advantages of non-contact, remote control, and difficulty in developing drug resistance. Piezoelectricity promotes repair technology simulates the physiological electrical signals of the human body by means of the weak electric field generated by piezoelectric materials under stress, which can effectively regulate cell behavior and promote cell adhesion, proliferation and differentiation, accelerating the regeneration of wound tissue. These two technologies have shown great application potential in wound repair and provide a new idea for the treatment of infected wounds. However, there are still many problems when applying photothermal and piezoelectric technologies separately to wound repair dressings. Although single photothermal antibacterial technology can quickly kill bacteria, it cannot actively promote tissue repair, and long-term high temperature may cause thermal damage to the surrounding normal tissues. Although single piezoelectricity promotes repair technology can effectively regulate cell function, it lacks effective control of infection and is difficult to create favorable conditions for wound healing in an infected environment. In addition, integrating photothermal and piezoelectric technologies into the same dressing faces technical difficulties such as poor material compatibility, mutual interference of functional modules, and difficulty in precisely controlling the synergistic effect of the two functions. Therefore, it is urgent to design an innovative structure to make the photothermal antibacterial and piezoelectricity promote repair functions work in order, achieve the use of photothermal effect to quickly kill bacteria and control infection in the early stage, and then the continuous effect of piezoelectricity to promote tissue repair, so as to meet the dual requirements of infection control and tissue repair in the repair of infected wounds.

[0005] Janus structure hydrogel can be endowed with two or more different functional components on both sides of the structure due to its unique dual-difference properties, achieving the dual functions of "rapid antibacterial and sustained repair promotion". Compared with traditional single-structure hydrogel, Janus hydrogel realizes the spatial separation and synergy of photothermal and piezoelectric materials through precise interface regulation and microstructure design, avoiding the mutual interference of the two functional components and improving the overall performance. One side is anchored with photothermal materials, which can quickly generate high temperature to kill bacteria under light irradiation, and the other side is equipped with piezoelectric materials, which can generate micro-current through mechanical stimulation to regulate cell behavior and promote tissue repair. At the same time, the unique spatial distribution characteristics of Janus structure make it possible to release active ingredients in sequence. According to the different needs of wound repair at different stages, the photothermal antibacterial component is designed to be released quickly in the early stage of repair to control infection, and the piezoelectric material continues to play a role in promoting tissue regeneration after the infection is alleviated, achieving dynamic and step-by-step treatment of infected wounds. The application of Janus structure to wound repair dressings not only breaks through the limitations of traditional dressings with single function, but also innovatively realizes the synergy of functions through structural design, providing technical support for the development of new dressings with efficient antibacterial and repair-promoting capabilities, and is expected to bring new solutions for the repair of infected wounds. SUMMARY

[0006] In view of the deficiencies of the prior art, a first object of the present application is to provide a Janus structured photo-thermal-piezoelectric hydrogel dressing for infected wound repair, which has ROS response characteristics and is used for loading MXene@CeO2 heterojunction, and has good adhesion and is used for loading BaTiO3 with piezoelectric effect.

[0007] A second object of the present application is to provide a preparation method of the Janus structured hydrogel.

[0008] A third object of the present application is to provide an application of the Janus structured hydrogel, which has photo-thermal effect, inhibits bacterial growth of infected wounds, destroys biofilm generated by bacteria, and has good immunomodulatory effect. In addition, the Janus structured hydrogel has good adhesion and piezoelectric effect, has the ability to regulate cell behavior and promote angiogenesis, and can be used for repair of infected wounds.

[0009] To achieve the above objects, the present application provides the following technical solutions.

[0010] In a first aspect of the present application, a preparation method of a Janus structured photo-thermal-piezoelectric hydrogel dressing for infected wound repair is provided, which comprises the following steps:

[0011] (1) Dissolve benzene boronic acid grafted methyl acrylamide gelatin and dopamine grafted hyaluronic acid in deionized water respectively, and add MXene@CeO2 heterojunction to uniformly ultrasonic disperse, after mixing, adjust pH to slightly alkaline using sodium hydroxide to form a Janus structured inner layer;

[0012] (2) Dissolve benzene boronic acid grafted gelatin and o-nitrobenzyl alcohol grafted hyaluronic acid in deionized water respectively, and add BaTiO3 to uniformly ultrasonic disperse, after mixing, form a Janus structured outer layer.

[0013] Preferably, the MXene@CeO2 heterojunction is prepared by the following steps:

[0014] S1. Etch Ti3AlC2 MXene and LiF in HCl, wash to neutral, then ultrasonic strip, and then collect single-layer Ti3C2 MXene nanosheets by centrifugation;

[0015] S2. Ultrasonic disperse CeO2 and the single-layer Ti3C2 MXene nanosheets in pure water to obtain MXene@CeO2 heterojunction.

[0016] Preferably, in step S1, the Ti3AlC2 MXene: LiF: HCl = 1-2 mmol: 10-20 mmol: 3-10 mL, the concentration of HCl is 5-11 mol / L, the etching time is 24-36 hours, the ultrasonic peeling power is 200-400 W, the ultrasonic peeling time is 30-60 min, and the collection centrifugal speed is 3000-5000 rpm.

[0017] Preferably, in step S2, the mass ratio of CeO2 to the monolayer Ti3C2 MXene nanosheet is 1:1-1:5, and the ultrasonic dispersion time is 0.5-2 hours.

[0018] Preferably, in step S1, the concentration of the benzene boronic acid grafted methyl acrylamide gelatin is 10%-30%, the concentration of the dopamine grafted hyaluronic acid is 5%-15%, the concentration of the MXene@CeO2 heterojunction is 0.1-2.0 mg / mL, the concentration of sodium hydroxide is 0.5-2.0 M, and the pH range is 7-9.

[0019] Preferably, in step S2, the concentration of the benzene boronic acid grafted gelatin is 10%-30%, the concentration of the o-nitrobenzyl alcohol grafted hyaluronic acid is 1%-3%, and the concentration of BaTiO3 is 0.1-2.0 mg / mL.

[0020] In a second aspect of the present application, a hydrogel dressing prepared by the method is provided.

[0021] In a third aspect of the present application, the hydrogel dressing is provided for use in the preparation of a wound repair product.

[0022] Preferably, the wound includes any one or more of an infected wound, a diabetic wound, a burn wound, and a complex wound.

[0023] In a fourth aspect of the present application, the hydrogel dressing is provided for use in the preparation of a product having the following functions: antibacterial; active oxygen scavenging; anti-inflammatory; immune regulation.

[0024] Compared with the prior art, the present application has the following beneficial and unique effects:

[0025] The Janus structure photothermal-piezoelectric hydrogel of the application combines the inner layer and the outer layer through hydrogen bonding to form a Janus structure, has a time and space release of active ingredients, the Janus inner layer directly contacts the wound surface to rapidly decompose and release MXene@CeO2 heterojunction, realizes rapid antibiosis, the Janus outer layer has adhesion effect and piezoelectric effect, continuously regulates cell behavior, promotes angiogenesis, and then accelerates tissue repair, and has the dual functions of antibiosis and repair promotion; the prepared MXene@CeO2 heterojunction has a sheet structure and good photothermal antibacterial performance, and also has good photothermal stability, blood compatibility, low cytotoxicity and immunomodulation advantages; BaTiO3 has significant piezoelectric effect, promotes cell proliferation and differentiation, regulates vascular endothelial cell behavior, and can be used for wound infection repair products. In addition, the method of the application also has the advantages of easy availability of raw materials, simple operation and excellent effect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a photothermal effect diagram of MXene@CeO2 heterojunction. (A) Photothermal temperature rising curve of MXene@CeO2 heterojunction with different concentrations; (B) Photothermal temperature rising curve of MXene@CeO2 heterojunction with different powers; (C) Photothermal cycle curve of MXene@CeO2 heterojunction; (D) Temperature rising and falling curve of MXene@CeO2 heterojunction.

[0027] Figure 2 It is an antibacterial effect diagram of Janus structure hydrogel. (A) Bacterial growth; (B) Scanning electron microscope image; (C) Biofilm removal experiment result.

[0028] Figure 3 It is a repair effect diagram of Janus structure hydrogel and physical blending hydrogel on infected wounds. (A) General diagram of infected wound repair; (B) H&E staining of wound tissue; (C) Masson staining of wound tissue; (D) CD31 immunofluorescence staining of wound tissue. DETAILED DESCRIPTION

[0029] The application will be further described below in combination with specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] Example 1

[0032] A Janus structure photothermal-piezoelectric hydrogel dressing for infected wound repair is prepared by the following steps:

[0033] 1. Preparation of MXene@CeO2 heterojunction

[0034] (1) 1.5 mmol of Ti3AlC2 MXene (commercial product, purchased from Forsman Technology (Beijing) Co., Ltd.) and 15 mmol of LiF (lithium fluoride, Shanghai Maierye Biotech Co., Ltd.) were dissolved in 5 mL of 9M HCl (hydrochloric acid) and reacted at room temperature for 24 hours for etching.

[0035] (2) The etched liquid was centrifuged to remove the upper acid solution, and then washed with deionized water until neutral. Then, ultrasonic stripping was performed at a power of 200 W for 30 min.

[0036] (3) The stripped mixed solution was centrifuged at a speed of 3500 rpm, and then mixed again after centrifugation. This operation was repeated until the upper liquid was black, and then the single-layer Ti3C2 MXene nanosheet in the upper clear liquid was collected.

[0037] (4) 5 mg of CeO2 (cerium dioxide) and 5 mg of single-layer Ti3C2 MXene nanosheet were ultrasonically dispersed in pure water for 0.5 hours to obtain MXene@CeO2 heterojunction.

[0038] 2. Preparation of Janus structured hydrogel

[0039] (1) 200 mg of GelMA-PBA (benzenboronic acid grafted methacrylamide gelatin, Guangzhou Chuangai Biological Medical Material Co., Ltd.) and 1 mg of LAP (light crosslinking agent phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate salt, Shanghai Yinchang New Material Co., Ltd.) were dissolved in 1 mL of deionized water, 1 mg of MXene@CeO2 heterojunction was added and ultrasonically dispersed uniformly, and a microsphere loaded with MXene@CeO2 heterojunction was prepared by microfluidic technology; 100 mg of HA-DA (dopamine grafted hyaluronic acid, Guangzhou Chuangai Biological Medical Material Co., Ltd.) was dissolved in 1 mL of deionized water to obtain a HA-DA solution.

[0040] (2) 100 mg of the microsphere loaded with MXene@CeO2 heterojunction was mixed with 100 μL of the HA-DA solution, and the pH of the system was adjusted to 7-9 using 1 M sodium hydroxide. The microgel after gelation was injected into a mold to form a Janus inner layer.

[0041] (3) 200 mg of Gel-PBA (benzene boronic acid grafted gelatin, Guangzhou Chuang Sai Biological Medical Material Co., Ltd.) was dissolved in 1 mL of deionized water, 1 mg of BaTiO3 (barium titanate) was added and uniformly dispersed by ultrasonic, to obtain a Gel-PBA solution; 20 mg of HA-NB (o-nitrobenzyl alcohol grafted hyaluronic acid, Shanghai Lingjue Medical Technology Co., Ltd.) was dissolved in 1 mL of deionized water to obtain an HA-NB solution.

[0042] (4) The Gel-PBA solution and the HA-NB solution were mixed at a volume ratio of 1:1 to form a gel, and the hydrogel after gelation was injected into a mold to form a Janus outer layer. The Janus inner layer and the Janus outer layer were combined by hydrogen bonds to form a Janus structure.

[0043] Example 2

[0044] A physical mixed photo-thermo-piezoelectric hydrogel dressing for infected wound repair, the preparation steps are as follows:

[0045] 1. Preparation of MXene@CeO2 heterojunction

[0046] (1) 1.5 mmol of Ti3AlC2 MXene and 15 mmol of LiF (lithium fluoride) were dissolved in 5 mL of 9M HCl (hydrochloric acid), and the etching was carried out at room temperature for 24 hours.

[0047] (2) The etched liquid was centrifuged to remove the upper acid solution, and deionized water was used to wash to neutral, then ultrasonic stripping was carried out at a power of 200 W for 30 min.

[0048] (3) The mixed solution after stripping was centrifuged at a speed of 3500 rpm, and after centrifugation, it was mixed again, and this operation was repeated until the upper liquid was black, and then the single-layer Ti3C2 MXene nanosheet in the upper clear liquid was collected.

[0049] (4) 5 mg of CeO2 (cerium dioxide) and 5 mg of single-layer Ti3C2 MXene nanosheet prepared in step (3) were ultrasonically dispersed in pure water to obtain MXene@CeO2 heterojunction.

[0050] 2. Preparation of hydrogel

[0051] (1) 200 mg of GelMA-PBA and 1 mg of LAP were dissolved in 1 mL of deionized water, 1 mg of MXene@CeO2 heterojunction was added and uniformly dispersed by ultrasonic, and a microsphere (GelMA-PBA microsphere) loaded with MXene@CeO2 heterojunction was prepared by microfluidic technology; 100 mg of HA-DA was dissolved in 1 mL of deionized water to obtain an HA-DA solution.

[0052] (2) 200 mg of Gel-PBA was dissolved in 1 mL of deionized water, 1 mg of BaTiO3 was added and ultrasonically dispersed to obtain a Gel-PBA solution; 20 mg of HA-NB was dissolved in 1 mL of deionized water to obtain an HA-NB solution.

[0053] (3) 100 mg of the microspheres loaded with MXene@CeO2 heterojunction, 100 μL of HA-DA solution, 100 μL of Gel-PBA solution and 100 μL of HA-NB solution were mixed uniformly, 1 M sodium hydroxide was used to adjust the pH of the system to between 7 and 8, and the formed hydrogel was injected into a mold to form a composite hydrogel.

[0054] Comparative Example 1

[0055] The MXene@CeO2 heterojunction solution was prepared as follows:

[0056] (1) 1.5 mmol of Ti3AlC2 MXene and 15 mmol of LiF were dissolved in 5 mL of 9M HCl, and etching was carried out at room temperature for 24 hours;

[0057] (2) After etching, the upper acid solution was removed by centrifugation, and deionized water was used to wash to neutral, then ultrasonic stripping was carried out at a power of 200W for 30 min;

[0058] (3) The mixed solution after stripping was centrifuged at a speed of 3500 rpm, and after centrifugation, it was mixed again, and this operation was repeated until the upper liquid was black, and then the single-layer Ti3C2 MXene nanosheet in the upper clear liquid was collected;

[0059] (4) 5 mg of CeO2 and 5 mg of single-layer Ti3C2 MXene nanosheet prepared in step (3) were ultrasonically dispersed in pure water to obtain MXene@CeO2 heterojunction.

[0060] (5) 1 mg of MXene@CeO2 heterojunction was ultrasonically dispersed in 1 mL of deionized water to obtain a MXene@CeO2 heterojunction solution.

[0061] Comparative Example 2

[0062] The MXene@CeO2 heterojunction solution was prepared as follows:

[0063] (1) 1.0 mmol of Ti3AlC2 MXene and 10 mmol of LiF were dissolved in 5 mL of 9M HCl, and etching was carried out at room temperature for 24 hours;

[0064] (2) After etching, the liquid is centrifuged to remove the upper acid solution, washed with deionized water until neutral, and then ultrasonically peeled at 200W for 30 min.

[0065] (3) Centrifuge the stripped mixed solution at 3500 rpm, remix after centrifugation, repeat this operation until the upper liquid is black, and then collect the monolayer Ti3C2MXene nanosheets in the upper clear liquid.

[0066] (4) 5 mg CeO2 and 5 mg monolayer Ti3C2MXene nanosheets prepared in step (3) were ultrasonically dispersed in pure water to obtain MXene@CeO2 heterojunction.

[0067] (5) Disperse 1 mg of MXene@CeO2 heterojunction in 1 mL of deionized water by ultrasonication to obtain MXene@CeO2 heterojunction solution.

[0068] Comparative Example 3

[0069] Prepare the MXene@CeO2 heterojunction solution using the following steps:

[0070] (1) Dissolve 2.0 mmol Ti3AlC2MXene and 20 mmol LiF in 5 mL of 9M HCl and etch at room temperature for 24 hours;

[0071] (2) After etching, the liquid is centrifuged to remove the upper acid solution, washed with deionized water until neutral, and then ultrasonically peeled at 200W for 30 min.

[0072] (3) Centrifuge the stripped mixed solution at 3500 rpm, remix after centrifugation, repeat this operation until the upper liquid is black, and then collect the monolayer Ti3C2MXene nanosheets in the upper clear liquid.

[0073] (4) 5 mg CeO2 and 5 mg monolayer Ti3C2MXene nanosheets prepared in step (3) were ultrasonically dispersed in pure water to obtain MXene@CeO2 heterojunction.

[0074] (5) Disperse 1 mg of MXene@CeO2 heterojunction in 1 mL of deionized water by ultrasonication to obtain MXene@CeO2 heterojunction solution.

[0075] Comparative Example 4

[0076] Prepare the MXene@CeO2 heterojunction solution using the following steps:

[0077] (1) 1.5 mmol Ti3AlC2 MXene and 15 mmol LiF were dissolved in 5 mL of 9M HCl, and etching was performed at room temperature for 24 hours;

[0078] (2) After etching, the liquid was centrifuged to remove the upper acid solution, and then washed with deionized water until neutral. Subsequently, ultrasonic exfoliation was performed at a power of 200 W for 30 min;

[0079] (3) The exfoliated mixed solution was centrifuged at a speed of 3500 rpm, and after centrifugation, the mixture was mixed again. This operation was repeated until the upper liquid was black, and then single-layer Ti3C2 MXene nanosheets in the upper clear liquid were collected;

[0080] (4) 5 mg of CeO2 and 25 mg of single-layer Ti3C2 MXene nanosheets prepared in step (3) were ultrasonically dispersed in pure water to obtain MXene@CeO2 heterojunctions.

[0081] (5) 1 mg of MXene@CeO2 heterojunctions were ultrasonically dispersed in 1 mL of deionized water to obtain a MXene@CeO2 heterojunction solution.

[0082] Comparative Example 5

[0083] The composite hydrogel was prepared according to the following steps:

[0084] (1) 200 mg of GelMA-PBA and 1 mg of LAP were dissolved in 1 mL of deionized water to prepare GelMA-PBA microspheres by microfluidic technology; 100 mg of HA-DA was dissolved in 1 mL of deionized water to obtain an HA-DA solution.

[0085] (2) 200 mg of Gel-PBA was dissolved in 1 mL of deionized water to obtain a Gel-PBA solution; 20 mg of HA-NB was dissolved in 1 mL of deionized water to obtain an HA-NB solution.

[0086] (3) 100 mg of GelMA-PBA microspheres, 100 μL of HA-DA solution, 100 μL of Gel-PBA solution, and 100 μL of HA-NB solution were mixed uniformly, and the pH value of the system was adjusted to between 7 and 8 using 1 M sodium hydroxide. The gelled hydrogel was injected into a mold to form a composite hydrogel.

[0087] Comparative Example 6

[0088] The Janus structure hydrogel was prepared according to the following steps:

[0089] (1) Dissolve 200 mg GelMA-PBA and 1 mg LAP in 1 mL of deionized water to prepare GelMA-PBA microspheres by microfluidic technology; dissolve 100 mg HA-DA in 1 mL of deionized water to obtain HA-DA solution.

[0090] (2) Mix 100 mg GelMA-PBA microspheres with 100 μL HA-DA solution, adjust the pH of the system to between 7 and 9 using 1 M sodium hydroxide, and inject the gelled microgel into the mold to form the Janus inner layer.

[0091] (3) Dissolve 200 mg Gel-PBA in 1 mL of deionized water to obtain Gel-PBA solution; dissolve 20 mg HA-NB in ​​1 mL of deionized water to obtain HA-NB solution.

[0092] (4) Mix the Gel-PBA solution and HA-NB solution in a volume ratio of 1:1 to form a gel. Inject the gelled hydrogel into a mold to form the Janus outer layer. The two layers are bonded together by hydrogen bonds to form the Janus structure.

[0093] Comparative Example 7

[0094] Prepare Janus-structured hydrogels using the following steps:

[0095] (1) Dissolve 200 mg GelMA-PBA and 1 mg LAP in 1 mL of deionized water, add 1 mg MXene@CeO2 heterojunction and ultrasonically disperse evenly, and prepare microspheres loaded with MXene@CeO2 heterojunction by microfluidic technology; dissolve 100 mg HA-DA in 1 mL of deionized water to obtain HA-DA solution.

[0096] (2) Mix 100 mg of MXene@CeO2 heterojunction microspheres with 100 μL HA-DA solution, adjust the pH of the system to between 7 and 9 with 1 M sodium hydroxide, and inject the gelled microgel into the mold to form the Janus inner layer.

[0097] (3) Dissolve 200 mg Gel-PBA in 1 mL of deionized water to obtain Gel-PBA solution; dissolve 20 mg HA-NB in ​​1 mL of deionized water to obtain HA-NB solution.

[0098] (4) Mix the Gel-PBA solution and HA-NB solution in a volume ratio of 1:1 to form a gel. Inject the gelled hydrogel into a mold to form the Janus outer layer. The two layers are bonded together by hydrogen bonds to form the Janus structure.

[0099] Comparative Example 8

[0100] The Janus structure hydrogel is prepared by the following steps:

[0101] (1) 200 mg of GelMA-PBA and 1 mg of LAP are dissolved in 1 mL of deionized water to prepare GelMA-PBA microspheres by microfluidic technology; 100 mg of HA-DA is dissolved in 1 mL of deionized water to obtain an HA-DA solution.

[0102] (2) 100 mg of GelMA-PBA microspheres are mixed with 100 μL of the HA-DA solution, and the pH value of the system is adjusted to 7-9 using 1 M sodium hydroxide; the microgel after gelation is injected into a mold to form a Janus inner layer.

[0103] (3) 200 mg of Gel-PBA is dissolved in 1 mL of deionized water, 1 mg of BaTiO3 is added and uniformly dispersed by ultrasonic, to obtain a Gel-PBA solution; 20 mg of HA-NB is dissolved in 1 mL of deionized water to obtain an HA-NB solution.

[0104] (4) The Gel-PBA solution and the HA-NB solution are mixed at a volume ratio of 1:1 to form a gel, and the hydrogel after gelation is injected into a mold to form a Janus outer layer, and the two layers are combined by hydrogen bonds to form a Janus structure.

[0105] Test Example

[0106] 1. Photothermal characterization

[0107] Test method: Prepare MXene@CeO2 heterojunction solutions with different concentrations (0.25, 0.50, 0.75 and 1.00 mg / mL), irradiate with an 808 nm NIR laser for 10 minutes, the power is 1.0 W / cm 2 , use a thermocouple thermometer to record the real-time temperature, record the real-time temperature every 20 s, take time as the abscissa and temperature as the ordinate, and plot the obtained data into a temperature rise curve.

[0108] The test results are shown in Figure 1 , it can be seen that the photothermal effect is more significant as the concentration increases, but after a certain concentration, it tends to be stable, and the difference between 0.75 mg / mL and 1 mg / mL is not large, and the MXene@CeO2 heterojunction synthesized by the embodiment of the present application has good photothermal cycle performance.

[0109] 2. Antibacterial performance

[0110] Test method: Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureusAfter recovery, the bacteria were cultured to logarithmic growth phase, centrifuged, collected and resuspended in physiological saline, and the final concentration of the bacterial solution was 1 x 10 8 CFU / mL. In a 24-well plate, 1 mL of the samples of Example 1-2 and Comparative Example 1-8 were added, followed by the addition of 100 μL of the diluted bacterial suspension and 1 mL of sterile physiological saline in each well, and irradiation with an 808 nm NIR laser for 10 minutes at a power of 1.0 W / cm 2 , incubation at 37°C for 24 hours, gradient dilution, plating on LB agar plates, counting, and the Control group was the physiological saline group.

[0111] For scanning electron microscopy (SEM), 1 mL of the cultured bacterial solution was taken in a 1.5 mL centrifuge tube, centrifuged at 3500 rpm for 10 minutes to remove the supernatant and take the precipitate, and the bacterial precipitate obtained by centrifugation was dispersed with 1 mL of 2.5% glutaraldehyde for 2-3 hours of fixation. The fixed bacteria were centrifuged at 3500 rpm to take the precipitate, washed and dispersed in deionized water, and 10-20 μL of the bacteria dispersed in deionized water in the previous step were dropped onto a single-crystal silicon wafer and air-dried. The naturally dried sample was attached to a copper platform with conductive glue, gold-coated for 90-120 seconds, and photographed.

[0112] For biofilm removal, after the culture was completed, the biofilm was fixed with 500 μL of anhydrous methanol for 1 hour, 200 μL of 1% crystal violet staining solution was added, and the staining was performed in the dark for 30 min in an incubator, followed by multiple washes with sterile water. The culture plate was inverted on filter paper to remove residual water, dried at room temperature, and photographed.

[0113] The test results are shown in Table 1 and Figure 2 Figure 2

[0114] Table 1: Bacteriostatic rate of examples and comparative examples

[0115]

[0116] From Table 1 and Figure 2It can be seen that the Janus structured hydrogel of embodiment 1 of the present application has good antibacterial effect, which is mainly realized by CeO2. Further under NIR irradiation, the MXene@CeO2 heterojunction of the present application can also absorb photons, convert light energy into heat energy, increase the temperature of the infected wound site, and cause the death of bacteria, effectively destroying the biofilm produced by bacteria. This is related to the rapid release and contact surface of the Janus structure. The Janus structured hydrogel is more fully contacted with bacteria, and after NIR irradiation, the oxygen vacancies capture photo-generated electrons and enhance the photo-thermal conversion, and the photo-thermal conversion efficiency can be significantly increased. The bacteriostatic rate of embodiment 2 is slightly lower than that of comparative examples 1-4, mainly because comparative examples 1-4 are directly cultured with the heterojunction, and embodiment 2 loads the heterojunction in the physically mixed hydrogel. The antibacterial effect of pure heterojunction is stronger than that of the antibacterial effect of the gel loaded in the gel, so the bacteriostatic rate of embodiment 2 is slightly reduced. Comparative examples 5 and 6 are blank gels and do not contain antibacterial substances, so the bacteriostatic rate is low. Comparative example 8 only loads piezoelectric material BaTiO3, so the antibacterial effect is very weak.

[0117] 3. ROS scavenging in vitro

[0118] Test method: H2O2 scavenging is to mix the sample with 1 mM H2O2, avoid light reaction for different time (0.5, 1, 3, 5, 8 hours), and then use H2O2 kit to detect the remaining H2O2 concentration, and use ultraviolet-visible spectrum analysis to detect the absorbance curve at 340-600 nm wavelength.

[0119] According to the hydroxyl radical (·OH) kit, the sample is reacted with the precursor liquid in the kit for different time (0.5, 1, 2, 4, 6, 8 hours), and the absorbance curve at 400-650 nm wavelength is detected by ultraviolet-visible spectrum analysis. Take 0.5 mL of 0.05 mol / L Tris-HCl buffer, add 0.4 mL of sample to it, react in a 25°C water bath for 20 min, then add 10 mmol / L pyrogallol solution 0.1 mL, mix well, and react in a 25°C water bath for different time (0.5, 1, 2, 4, 6, 8 hours), then add 0.1 mL of 10 M HCl to terminate the reaction, and detect the absorbance curve at 260-500 nm wavelength by ultraviolet-visible spectrum analysis.

[0120] The test results are shown in Table 2.

[0121] Table 2: ROS scavenging efficiency of examples and comparative examples

[0122]

[0123] Table 2 shows the scavenging effect of Janus structure hydrogel on H2O2. After co-incubation of Janus structure hydrogel with H2O2 for different time, the concentration of residual H2O2 was detected by H2O2 kit, and full spectrum scanning was carried out by ultraviolet visible spectrophotometer. The results show that the concentration of residual H2O2 is reduced, which also means that more H2O2 is scavenged.

[0124] Table 2 shows the scavenging effect of Janus structure hydrogel on ·OH and O 2- . After co-incubation of Janus structure hydrogel with the precursor solution of ·OH and O 2- , full spectrum scanning was carried out on the incubated liquid, and the results show that the concentration of residual ·OH and O 2- is low, showing good active oxygen scavenging effect, which is mainly due to the crystal structure and variable oxidation state of MXene@CeO2 in Janus structure hydrogel, which realizes the catalytic conversion of active oxygen through the cycle of Ce 4+ and Ce 3+ redox pair.

[0125] 4. Cell evaluation

[0126] Test method: The cultured L929 cells, HUVEC cells and macrophages were digested and suspended with trypsin, inoculated into 48-well plates, and after 12 hours of culture, the original culture solution was removed, 1 mL hydrogel sample was added to 10 mL cell culture solution, and the extraction liquid was obtained by extraction at 37℃ for 24 h, and the extraction liquid was added for continuous culture for 24, 48 and 72 hours. The culture medium was aspirated and washed with PBS for 3 times, CCK-8 staining agent was added, PBS was washed, and then the image was taken by fluorescence microscope.

[0127] For in vitro macrophages, RAW264.7 was inoculated into a 6-well plate with cell slides at a density of 1×10 6 cells / well, and after overnight culture, 5 μg / mL of lipopolysaccharide (LPS) was used for induction overnight, and then Janus structure hydrogel extraction liquid was added for continuous culture for 24 hours. After PBS washing, 1% paraformaldehyde was added for fixation for 15 min, PBS was washed for 1-2 times, membrane breaking solution was added for treatment for 10 min, PBS was washed for 1-2 times, 1% BSA blocking solution was added for blocking for 30 min, then rabbit anti-iNOS and mouse anti-CD206 fluorescent antibody diluent (dilution ratio 1:200) were added, and incubated at room temperature for 1 hour. After incubation, PBS was washed twice, FITC goat anti-rabbit IgG and Cy3 goat anti-mouse IgG fluorescent secondary antibody were added, and incubated at room temperature for 1 hour. After incubation, the slides were taken out, mounted with DAPI anti-fluorescence quencher, and observed and photographed under fluorescence microscope for analysis.

[0128] Test results: As shown in Tables 3 and 4, it can be seen that the Janus structured hydrogel prepared in Example 1 has good biocompatibility, promotes the polarization of macrophages to M2, and exhibits good immunomodulatory effect in vitro, promotes the release of anti-inflammatory factors TGF-β1 and IL-4, and at the same time inhibits the secretion of pro-inflammatory factors TNF-α and IFN-γ.

[0129] Table 3: Cell survival rate of examples and comparative examples

[0130]

[0131] Table 4: Relative expression amount of inflammatory factors of examples and comparative examples

[0132]

[0133] 5. Repair effect of infected wound

[0134] Test method: After the rats were anesthetized, the hair on the back skin of the rats was shaved, and the exposed skin was disinfected, and a full-thickness circular skin defect with a diameter of 12 mm was caused by surgical scissors. Subsequently, a mixture of Escherichia coli and Staphylococcus aureus (1x10 8 CFU / mL) was injected into the skin defect site, and fixed with 3M Tegaderm waterproof agent for 24 hours to cause infection. The infected rats were randomly divided into groups, and the Janus structured hydrogel of Example 1 and the physical mixed hydrogel (mixed hydrogel) of Example 2 were placed at the wound site as experimental groups, and the wound healing was observed at 3, 7, 10, 14 and 21 days after the operation. Among them, the Control group: normal saline group.

[0135] Test results: As shown in Table 5, the infected wounds of rats in different groups were photographed and recorded, and the healing traces of each wound were simulated and statistically analyzed. As can be seen from the results, the Janus structured hydrogel treated for 7 days, the wound has been scabbed and healed obviously, and the wound area of the Janus structured hydrogel treatment group is significantly lower than that of the blank group after 10 days of treatment, and the result is better than that of the physical mixed hydrogel. Figure 3

[0136] Subsequently, the wound tissue was subjected to HE staining and Masson staining, and the results showed that the wound width was significantly reduced after treatment with the Janus structured hydrogel, and there was more collagen deposition, and the repair effect was better.

[0137] Finally, the wound tissue was subjected to immunofluorescence staining, and CD31 is a marker of neovascularization, and it can be clearly seen that the Janus structured hydrogel can effectively promote the angiogenesis of the wound tissue.

[0138] ​Any technical features in the above-described embodiments can be combined in any manner, and for the sake of brevity, not all possible combinations are described, however, it is understood that the scope of the present disclosure encompasses all possible combinations. It should be noted that, for those of ordinary skill in the art, some modifications and improvements can be made to the present application without departing from the concept of the present application, and these should all fall within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. A method for preparing a Janus structured photothermal-piezoelectric hydrogel dressing for repairing infected wounds, characterized in that, Includes the following steps: (1) Phenylboronic acid-grafted methacrylamide gelatin was dissolved in deionized water, and MXene@CeO2 heterojunction was added and ultrasonically dispersed evenly. Microspheres loaded with MXene@CeO2 heterojunction were prepared by microfluidic technology. Dopamine-grafted hyaluronic acid was dissolved in deionized water to obtain a dopamine-grafted hyaluronic acid solution. (2) The microspheres loaded with MXene@CeO2 heterostructures were mixed with a dopamine-grafted hyaluronic acid solution, and the pH of the system was adjusted to 7-9 using sodium hydroxide. The gelled microgel was then injected into a mold to form the Janus inner layer. (3) Dissolve phenylboronic acid-grafted gelatin in deionized water, add barium titanate and ultrasonically disperse evenly to obtain phenylboronic acid-grafted gelatin solution; dissolve o-nitrobenzyl alcohol-grafted hyaluronic acid in deionized water to obtain o-nitrobenzyl alcohol-grafted hyaluronic acid solution. (4) Mix the phenylboronic acid grafted gelatin solution and the o-nitrobenzyl alcohol grafted hyaluronic acid solution in a volume ratio of 1:1 to form a gel. Inject the gelled hydrogel into a mold to form the Janus outer layer. The Janus inner layer and the Janus outer layer are bonded together by hydrogen bonds to form the Janus structure. In step (1), the concentration of phenylboronic acid-grafted methacrylamide gelatin is 10%-30%, the concentration of dopamine-grafted hyaluronic acid is 5%-15%, and the concentration of MXene@CeO2 heterojunction is 0.1-2.0 mg / mL; In step (2), the concentration of sodium hydroxide is 0.5-2.0 M; In step (3), the concentration of phenylboronic acid grafted gelatin is 10%-30%, the concentration of o-nitrobenzyl alcohol grafted hyaluronic acid is 1%-3%, and the concentration of BaTiO3 is 0.1-2.0 mg / mL.

2. The preparation method according to claim 1, characterized in that, The MXene@CeO2 heterojunction was prepared by the following steps: S1. Ti3AlC2 MXene and LiF were dissolved in HCl for etching, washed until neutral, and then exfoliated by ultrasonication. The monolayer Ti3C2 MXene nanosheets were then collected by centrifugation. S2. CeO2 and the monolayer Ti3C2 MXene nanosheets were ultrasonically dispersed in pure water to obtain MXene@CeO2 heterojunctions.

3. The preparation method according to claim 2, characterized in that, In step S1, the Ti3AlC2 MXene:LiF:HCl ratio is 1-2 mmol:10-20 mmol:3-10 mL, the HCl concentration is 5-11 mol / L, the etching time is 24-36 hours, the ultrasonic stripping power is 200-400 W, the ultrasonic stripping time is 30-60 min, and the collection centrifugation speed is 3000-5000 rpm.

4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of CeO2 to the monolayer Ti3C2 MXene nanosheets is 1:1 to 1:5, and the ultrasonic dispersion time is 0.5 to 2 hours.

5. The hydrogel dressing prepared by the method according to any one of claims 1-4.

6. The application of the hydrogel dressing as described in claim 5 in the preparation of wound repair products.

7. The application according to claim 6, characterized in that, The wound includes any one or more of the following: infected wound, diabetic wound, burn wound, and complex wound.

8. The use of the hydrogel dressing as described in claim 5 in the preparation of products having the following effects: antibacterial; reactive oxygen species scavenging; anti-inflammatory; and immunomodulatory.

Images