Chinese mugwort essential oil loaded multifunctional hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of hydrogel materials, and particularly relates to Chinese mugwort essential oil loaded multifunctional hydrogel as well as a preparation method and application thereof. The Chinese mugwort essential oil-loaded multifunctional hydrogel provided by the invention comprises a hydrogel matrix and composite nanoparticles compounded in the hydrogel matrix, wherein the composite nanoparticles are black phosphorus nanosheets entrapped with Chinese mugwort essential oil. The hydrogel has multiple functions of stopping bleeding, resisting inflammation, resisting bacteria, resisting oxidation, promoting angiogenesis and the like, is high in moisture retention capacity, excellent in mechanical property, good in biocompatibility and high in biological safety, and can be used for preparing a hydrogel dressing for promoting wound healing. Besides, the multifunctional hydrogel has the characteristic of near-infrared light response, controlled release of the Chinese mugwort essential oil can be realized, and the released Chinese mugwort essential oil can cooperate with the photothermal effect of the black phosphorus nanosheet, so that the performance of the hydrogel is further improved.

Description

Technical Field

[0001] The present application belongs to the technical field of hydrogel materials, and more specifically, relates to a multifunctional hydrogel loaded with Qi Ai essential oil, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetic wound healing is a common clinical problem, in which factors such as persistent bacterial infection, hypoxia, and ischemia cause it to become a chronic and difficult-to-heal wound. In clinical practice, surgical debridement and the application of negative pressure wound dressings are usually used to treat diabetic wounds, but these strategies are often constrained by inherent limitations. For example, surgical debridement is an invasive operation that destroys the integrity of the skin and tissues, providing new invasion pathways for bacteria and other pathogens, and the wound after debridement may have a local excessive inflammatory response, leading to delayed wound healing; traditional wound dressings such as gauze and cotton are easy to adhere to the wound and have the disadvantages of large loss.

[0003] The literature (International Journal of Biological Macromolecules, 263 (2024), 130386) uses graphene oxide (GO) as an encapsulation carrier to encapsulate essential oils to obtain nanoparticles, and uses GelMA as a hydrogel skeleton to embed the nanoparticles into the GelMA hydrogel, which can promote wound healing. However, the encapsulation carrier graphene oxide used in the hydrogel has certain toxicity, which will increase the biosafety risk when used for a long time, and is not conducive to promotion and application. Summary of the invention

[0004] In view of the defects of the prior art, the purpose of the present application is to provide a multifunctional hydrogel loaded with Qi Ai essential oil, a preparation method and application thereof. The above-mentioned multifunctional hydrogel can polarize M1 macrophages to M2 type, has excellent anti-inflammatory effect, and also has excellent hemostatic, anti-inflammatory, antioxidant, and angiogenesis-promoting functions. The hydrogel has good biocompatibility, strong water retention capacity, and excellent mechanical properties. It is suitable for the preparation of hydrogel dressings that promote wound healing, especially for diabetic wound healing.

[0005] To achieve the above objectives, in a first aspect, the present application provides a multifunctional hydrogel loaded with Qi Ai essential oil, which includes a hydrogel matrix and composite nanoparticles composited therein; the composite nanoparticles include black phosphorus nanosheets and Qi Ai essential oil encapsulated therein.

[0006] Preferably, in the above-mentioned composite nanoparticles, the ratio of black phosphorus nanosheets to Qi Ai essential oil is (20~40) mg:1 mL.

[0007] Preferably, the hydrogel matrix is ​​obtained by cross-linking methacrylated gelatin and hyaluronic acid methacrylate.

[0008] Further preferably, the mass ratio of the methacrylated gelatin to the hyaluronic acid methacrylate is (4-6):1.

[0009] In a second aspect, the present application provides a method for preparing the multifunctional hydrogel, comprising the following steps: S1, mixing the black phosphorus nanosheet suspension and the Qi Ai essential oil, and then loading the black phosphorus nanosheet with the Qi Ai essential oil through an extrusion cycle to prepare a solution containing composite nanoparticles; S2, mixing methacrylated gelatin, hyaluronic acid methacrylate and buffer to prepare a hydrogel solution; S3. After mixing the hydrogel solution and the solution containing the composite nanoparticles, crosslinking and curing are performed under the condition of initiation by a photoinitiator or a light source to prepare a multifunctional hydrogel.

[0010] Preferably, in step S1, the concentration of the black phosphorus nanosheet suspension is 1-5 mg / mL.

[0011] Preferably, the volume ratio of the black phosphorus nanosheet suspension to the Artemisia argyi essential oil is (5-15):1.

[0012] Preferably, in step S1, the number of the extrusion cycles is 5 to 15 times.

[0013] Preferably, in step S2, the concentration of methacryloyl gelatin in the hydrogel solution is 50-150 mg / mL.

[0014] Preferably, in step S2, the mass ratio of the methacrylated gelatin to the hyaluronic acid methacrylate is (4-6):1.

[0015] Preferably, in step S3, the cross-linking and curing time is 5 to 10 minutes.

[0016] Preferably, the photoinitiator is selected from one or more of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, 1,2-diketone, Irgacure 2959, and Alg-2959.

[0017] In a third aspect, the present application provides the use of the multifunctional hydrogel as or in the preparation of an anti-inflammatory drug, wherein the multifunctional hydrogel can polarize M1 macrophages to M2 macrophages.

[0018] In a fourth aspect, the present application provides the use of the multifunctional hydrogel as or in the preparation of a hydrogel auxiliary material for promoting wound healing.

[0019] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art: (1) This application prepares a hydrogel with multiple functions by uniformly loading black phosphorus nanosheets loaded with Qi Ai essential oil in a hydrogel matrix and utilizing the synergistic effect between the components, including hemostasis, anti-inflammatory, antibacterial, antioxidant, and angiogenesis-promoting functions. The hydrogel has strong water retention capacity, excellent mechanical properties, good biocompatibility, and high biosafety. In addition, the multifunctional hydrogel provided in this application has the characteristics of near-infrared light response. When the multifunctional hydrogel is irradiated with near-infrared light, it can effectively release Qi Ai essential oil. The released Qi Ai essential oil cooperates with the efficient photothermal effect of black phosphorus nanosheets, which can further improve the performance of the hydrogel and effectively promote wound healing. It can be used to prepare hydrogel dressings that promote wound healing.

[0020] (2) The method for preparing the multifunctional hydrogel loaded with Qi Ai essential oil provided in the present application has the advantages of simple preparation steps, wide sources of raw materials and low prices. In practical applications, the components constituting the hydrogel matrix and the ratio between the components, the size of the black phosphorus nanosheets and the amount of Qi Ai essential oil loaded thereon, and the cross-linking time can be adjusted according to actual needs. At the same time, different molds can be used to quickly prepare multifunctional hydrogels with different shapes and thicknesses. The method has broad application prospects in the field of wound healing, especially for diabetic wound healing.

[0021] (3) The present application provides the use of a multifunctional hydrogel loaded with Qi Ai essential oil for use as or in the preparation of an anti-inflammatory drug. Experiments have found that the multifunctional hydrogel can effectively inhibit the expression of M1 macrophage marker CD86, promote the expression of M2 macrophage marker CD206, downregulate the expression of CD86 mRNA and IL-1β mRNA, and upregulate the expression of CD206 mRNA and IL-10 mRNA, thereby polarizing M1 macrophages to M2 macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the characteristic of the black phosphorus nanosheet provided in the embodiment of the present application, wherein content A is a TEM image, content B is a particle size distribution, content C is an AFM, and content D is a thickness profile along the white line; Figure 2 It is the characteristic of WEO@BP nanoparticles provided in the examples of the present application, wherein content A is the SEM image of WEO, content B is the SEM image of WEO@BP nanoparticles, and content C is the particle size distribution of WEO@BP nanoparticles; Figure 3It is the physical properties of the hydrogel provided in the examples of the present application, wherein content A is an optical image of the hydrogel, content B is a SEM image of the hydrogel at different magnifications, content C is a SEM image of other hydrogels, content D is a FTIR spectrum of WEO@BP / GH hydrogel, and content E is a Raman spectrum of WEO@BP / GH hydrogel; Figure 4 The properties of the WEO@BP / GH hydrogel provided in the embodiment of the present application, wherein content A is the hygroscopic property, content B is the water retention property, content C is the adhesion, content D is the mechanical property, content E is the gelation threshold, content F is the elasticity, and content G is the self-healing ability; Figure 5 The near-infrared photothermal performance of the WEO@BP / GH hydrogel provided in the embodiment of the present application, wherein Content A, Content B, and Content C are the infrared thermal image, temperature rise curve, and photothermal stability of the WEO@BP / GH hydrogel precursor, respectively; Figure 6 The cumulative release of WEO in the WEO@BP / GH hydrogel at different times provided in the examples of the present application, wherein content A is 0-30 min, and content B is 0-120 h; Figure 7 The effect of WEO@BP / GH hydrogel polarizing M1 macrophages to M1 type provided in the embodiment of the present application, wherein content A is a fluorescence intensity microscopic image of CD206 and CD86 in the cells, content B and content C are relative fluorescence quantitative analysis of CD206 and CD86 in the cells, respectively, and content D, content E, content F, and content G are the relative expression levels of CD206 mRNA, IL-10 mRNA, CD86 mRNA, and IL-1β mRNA in the cells, respectively; Figure 8 The in vitro antibacterial properties of the WEO@BP / GH hydrogel provided in the examples of the present application, wherein content A is a plate cloning image of S.aureus and E.coli, content B is a live / dead cell staining fluorescence image of the plates of S.aureus and E.coli, content C is the survival rate of S.aureus, content D is the survival rate of E.coli, content E is the PI fluorescence value of S.aureus, and content F is the PI fluorescence value of E.coli; Fig. 9 The antioxidant properties of the WEO@BP / GH hydrogel provided in the examples of the present application, wherein content A is a Calcein-AM / PI staining fluorescence image of HaCaT cells after culture with 500 μM H2O2 and different treatment groups, content B is a reactive oxygen species (ROS) staining fluorescence image, content C is the percentage of dead cells, content D is the activity of the treated HaCaT cells, and content E is the relative DCF fluorescence intensity; Fig.10 The hemostatic performance of the WEO@BP / GH hydrogel provided in the embodiment of the present application, wherein content A is the hemostatic optical image, and content B is the amount of bleeding; Fig.11 The angiogenesis-promoting performance of the WEO@BP / GH hydrogel provided in the examples of the present application, wherein content A and content B are the healing of cell scratches and the formation of tubular structures of HaCaT cells cultured in different treatment groups at 0h and 24h of incubation, respectively, content C is the number of branch points, and content D is the tubular length; Fig.12 The in vivo antibacterial effect of the WEO@BP / GH hydrogel provided in the examples of the present application, wherein content A is a plate cloning diagram of the secretions of diabetic wounds in different treatment groups after 2 days of treatment, and content B is the survival rate of cells in the secretions; Fig.13 Optical images of diabetic wounds treated with the WEO@BP / GH hydrogel provided in the examples of the present application for different time periods; Fig.14 The results of pathological analysis of wound tissue after 14 days of treatment with the WEO@BP / GH hydrogel provided in the examples of the present application, wherein content A is an H&E staining image, content B is a Masson trichrome staining image, content C is scar width, and content D is collagen deposition; Fig.15 The immunohistochemistry and fluorescence staining of the wound tissue of diabetic rats treated with the WEO@BP / GH hydrogel provided in the embodiment of the present application for 14 days, wherein content A is the immunofluorescence staining of CD86, content B is the immunofluorescence staining of CD206, content C is the immunohistochemistry staining of VEGF, content D is the fluorescence staining of DHE for detecting ROS, content E is the expression level of CD86, content F is the expression level of CD206, content G is the expression level of VEGF, and content H is the expression level of DHE; Fig.16 The in vitro hemolytic performance of the WEO@BP / GH hydrogel provided in the embodiment of the present application, wherein content A is the optical image of the blood sample after being treated by different treatment groups, and content B is the hemolysis rate of the different treatment groups; Fig.17 The in vitro safety of the WEO@BP / GH hydrogel provided in the examples of the present application, wherein content A, content B, and content C are respectively the Calcein-AM / PI staining fluorescence images, Calcein-AM fluorescence expression levels, and cell viability of HaCaT cells after treatment with different groups; Fig.18 HE staining images of different organs of diabetic rat wounds treated with WEO@BP / GH hydrogel provided in the examples of the present application for 14 days; Fig.19 The results of blood biochemical analysis of diabetic rat wounds treated with the WEO@BP / GH hydrogel provided in the examples of the present application for 14 days, wherein content A is HGB, content B is ALT, content C is AST, content D is BUN, content E is CRE, content F is WBC, content G is PLT, and content H is RBC; In all figures, the same reference numerals are used to indicate the same level of significance, where ns represents P>0.05, represents P < 0.05, represents P < 0.01, Represents P < 0.001. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0024] The present application provides a multifunctional hydrogel loaded with Qi Ai essential oil, which comprises a hydrogel matrix and composite nanoparticles composited therein; the composite nanoparticles comprise black phosphorus nanosheets and Qi Ai essential oil encapsulated therein.

[0025] In some embodiments, in the above-mentioned composite nanoparticles, the ratio of black phosphorus nanosheets to Qi Ai essential oil is (20~40) mg:1 mL.

[0026] In a preferred embodiment, in the above-mentioned composite nanoparticles, the ratio of black phosphorus nanosheets to Qi Ai essential oil is (25~35) mg:1 mL.

[0027] In some embodiments, the black phosphorus nanosheets have a particle size of 200-600 nm and a thickness of 2.5-7.5 nm.

[0028] In some embodiments, the hydrogel matrix is ​​obtained by cross-linking methacryloyl gelatin and hyaluronic acid methacrylate. Furthermore, the mass ratio of the methacryloyl gelatin (GelMA) to the hyaluronic acid methacrylate (HAMA) is (4-6):1, which can prepare a hydrogel matrix with moderate and uniform pore size, which is beneficial to nutrient exchange and cell proliferation.

[0029] On the other hand, the present application provides a method for preparing the multifunctional hydrogel, comprising the following steps: S1, mixing the black phosphorus nanosheet suspension and the Qi Ai essential oil, and then carrying out an extrusion cycle to allow the black phosphorus nanosheet to encapsulate the Qi Ai essential oil, to prepare a solution containing composite nanoparticles; S2, mixing methacrylated gelatin, hyaluronic acid methacrylate and buffer to prepare a hydrogel solution; S3. After mixing the hydrogel solution and the solution containing the composite nanoparticles, cross-linking and curing are performed under the condition of initiation by a photoinitiator or a light source to prepare a multifunctional hydrogel loaded with Qi Ai essential oil.

[0030] In some embodiments, in step S1, the concentration of the black phosphorus nanosheet suspension is 1-5 mg / mL.

[0031] It is to be understood that the present application does not limit the source of the above-mentioned black phosphorus nanosheet suspension. The black phosphorus nanosheet suspension can be purchased from a commercial source, or the black phosphorus nanosheets purchased from a commercial source are resuspended in a solvent. The black phosphorus nanosheet suspension can also be prepared by itself according to methods known in the art.

[0032] In some embodiments, in step S1, the method for preparing the above-mentioned black phosphorus nanosheet suspension comprises the following steps: mixing black phosphorus powder and an organic solvent, and then performing liquid exfoliation by ultrasound to peel off the three-dimensional black phosphorus powder layer by layer into a two-dimensional lamellar structure to obtain black phosphorus nanosheets, and then resuspending to obtain a black phosphorus nanosheet suspension.

[0033] In some embodiments, the preparation process of the above-mentioned black phosphorus nanosheet suspension is specifically as follows: black phosphorus powder and an organic solvent are mixed, and after ultrasonic treatment in an ice water bath for 8 to 15 hours, the supernatant is collected by centrifugation at a speed of 2000 to 5000 rpm for 5 to 10 minutes, and the supernatant is collected by centrifugation at a speed of 10000 to 15000 rpm for 10 to 30 minutes. The precipitate is then resuspended and washed, and then centrifuged at a speed of 15000 to 20000 rpm for 1 hour to 2 hours to obtain black phosphorus nanosheets. Finally, the black phosphorus nanosheets are resuspended in a solvent to obtain a black phosphorus nanosheet suspension.

[0034] In some embodiments, the usage ratio of the black phosphorus powder and the organic solvent is (0.5~1) mg:1 mL.

[0035] In some embodiments, the organic solvent includes but is not limited to one or more of saturated sodium hydroxide in N-methylpyrrolidone, N-methylpyrrolidone (NMP), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0036] In some embodiments, the black phosphorus nanosheets can be resuspended in a solvent such as deionized water, N-methylpyrrolidone, N,N-dimethylformamide, dimethyl sulfoxide, etc. to obtain a black phosphorus nanosheet suspension.

[0037] In some embodiments, in step S1, the preparation method of the above-mentioned Qi Ai essential oil comprises the following steps: mixing Qi Ai powder and deionized water, and then extracting by distillation to separate and obtain Qi Ai essential oil.

[0038] In some embodiments, the amount of the Qi Ai powder is 50-200 g, the amount of deionized water is 1000-2000 mL, and the distillation time is 2-8 h.

[0039] In some embodiments, the volume ratio of the black phosphorus nanosheet suspension to the Qi Ai essential oil is (5-15):1.

[0040] In some embodiments, the number of the extrusion cycles is 5 to 15, so that the black phosphorus nanosheets encapsulate the Qi Ai essential oil. In some embodiments, the pore size of the filter membrane used in the extrusion cycle is 200 to 600 nm.

[0041] In some embodiments, in step S2, the concentration of methacryloyl gelatin in the hydrogel solution is 50-150 mg / mL.

[0042] In some embodiments, in step S2, the mass ratio of the methacrylated gelatin to the hyaluronic acid methacrylate is (4-6):1.

[0043] In some embodiments, the buffer is Dulbecco's phosphate buffered saline (DPBS) or phosphate buffered saline (PBS).

[0044] It is understood that the present application does not limit the volume ratio of the solution containing composite nanoparticles to the hydrogel solution. Those skilled in the art can adjust the ratio of the two according to actual needs to obtain multifunctional hydrogels containing composite nanoparticles of different concentrations. In some embodiments, in step S3, the volume ratio of the solution containing composite nanoparticles to the hydrogel solution is (0.5-2):1.

[0045] The present application does not limit the size, thickness, etc. of the multifunctional hydrogel. Those skilled in the art can select a suitable size and thickness according to the requirements of the actual application scenario, which are all within the protection scope of the present application.

[0046] In some embodiments, in step S3, the cross-linking and curing time is 5 to 10 minutes.

[0047] In some embodiments, in step S3, the photoinitiator is selected from one or more of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), 1,2-diketone, Irgacure 2959, and Alg-2959.

[0048] In some embodiments, the amount of the photoinitiator is 0.1% to 1% of the total mass of the methacrylated gelatin and the hyaluronic acid methacrylate.

[0049] The present application also provides the use of the multifunctional hydrogel in the preparation of anti-inflammatory drugs, and the multifunctional hydrogel can polarize M1 macrophages to M2 macrophages.

[0050] The present application also provides the use of the multifunctional hydrogel in preparing a dressing that promotes wound healing.

[0051] This application uses methacrylated gelatin and hyaluronic acid methacrylate to form a hydrogel matrix with a three-dimensional network structure, and at the same time, the black phosphorus nanosheets containing Qi Ai essential oil are uniformly loaded in the hydrogel, and a hydrogel with multiple functions is prepared through the synergistic effect between the components, including hemostasis, anti-inflammatory, antibacterial, antioxidant, and angiogenesis-promoting functions, and the hydrogel has good biocompatibility, strong water retention capacity, and excellent mechanical properties, and can be used to prepare a dressing that promotes wound healing. In addition, the multifunctional hydrogel provided in this application has the characteristics of near-infrared light response. When the above-mentioned multifunctional hydrogel is irradiated with near-infrared light, Qi Ai essential oil can be effectively released. The released Qi Ai essential oil cooperates with the efficient photothermal effect of the black phosphorus nanosheets to further enhance the function of the hydrogel.

[0052] It should be understood that materials with the same or similar types, models, qualities, properties or functions as the reagents and instruments used in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0053] The above technical solution is described in detail below in conjunction with specific embodiments.

[0054] The main reagents and materials used in the following examples are from the following sources: Black phosphorus (BP) was purchased from Aladdin Biochemical Technology Co., Ltd.; Qi Ai was purchased from Hubei Lizchen Biotechnology Co., Ltd.; methacryloyl gelatin (GelMA) and hyaluronic acid methacrylate (HAMA) were purchased from EFL Co., Ltd.; YTO9 / propidium iodide (PI) kit was purchased from Thermo Fisher Scientific; calcein-AM / PI kit, CCK-8 kit, DMEM medium, PBS, human keratinocytes (HaCaT), human monocytic THP-1 cells and human umbilical vein endothelial cells (HUVECs) were purchased from Cell Biotechnology Co., Ltd.; fetal bovine serum (FBS) was purchased from Gibco (Grand Island, USA); phorbol 12-myristate 13-acetate (PMA) was purchased from Merck (Darmstadt, Germany); Staphylococcus aureus (S. aureus, ATCC 29213) and Escherichia coli (E. coli, ATCC 25922) were purchased from Shanghai Biological Resources Center, China.

[0055] Example 1 1.1 Preparation of Artemisia argyi essential oil (WEO) Grind 100 g of Artemisia argyi leaves into powder and place it in a round-bottom flask, add 1500 mL of distilled water, then assemble the round-bottom flask, distillation head, condenser, receiving tube and recovery flask into a Clevenger-type device, heat and boil and distill for 4 h, collect the extracted Artemisia argyi essential oil (WEO), transfer it to an EP tube and store it at 4 °C for later use.

[0056] 1.2 Preparation of black phosphorus (BP) nanosheets 20 mg of black phosphorus powder was dispersed in 20 mL of saturated sodium hydroxide solution in N-methylpyrrolidone, and ultrasonically treated for 10 h in an ice bath to liquid-exfoliate the blocky black phosphorus. The exfoliated solution was then centrifuged at 3000 rpm for 5 min to remove the precipitate. The supernatant was centrifuged at 12000 rpm for 30 min, and the supernatant was collected. The precipitate was then suspended in ultrapure water, ultrasonically treated for 2 h in the dark, and centrifuged at 15000 rpm to obtain BP nanosheets, the characteristics of which are as follows: Figure 1 Then the BP nanosheets were suspended in ultrapure water to obtain a BP nanosheet suspension with a concentration of 3 mg / mL, which was stored in a dark environment at 4°C for later use.

[0057] The results show that Figure 1 Content A. Figure 1 Content B. Figure 1 Content C. Figure 1 It can be seen from content D that the present application prepares BP nanosheets with a particle size of 200-600 nm and a thickness of 2.5-7.5 nm.

[0058] 1.3 Preparation of WEO@BP nanoparticles 1 mL of the BP nanosheet suspension and 100 μL of the WEO were ultrasonically oscillated in an ice bath to obtain a mixture. The mixture was extruded through a polycarbonate membrane with a pore size of 400 nm using a LiposoFast-Basic extruder for 10 cycles to obtain a solution containing composite nanoparticles (ie, WEO@BP nanoparticles).

[0059] The results show that Figure 2 Content A. Figure 2 As can be seen from content B, WEO is encapsulated in BP nanosheets. Figure 2 It can be seen from content C that the particle size of WEO@BP nanoparticles is 200~600nm, indicating that whether WEO is encapsulated has little effect on the particle size of BP nanosheets.

[0060] 1.4 Preparation and characterization of multifunctional hydrogel (WEO@BP / GH hydrogel) Dissolve 5 mg of LAP in 20 mL of PBS to prepare LAP solution for later use. Dissolve 100 mg of methacryloyl gelatin (GelMA) and 20 mg of hyaluronic acid methacrylate (HAMA) in 1 mL of LAP solution and ultrasonicate for 5 min until the solution is clear and transparent to obtain GH solution. Mix 1 mL of the above solution containing composite nanoparticles and 1 mL of GH solution and cross-link and cure under 405 nm ultraviolet light for 7 min to prepare WEO@BP / GH hydrogel.

[0061] 1 mL of the above GH solution was cross-linked and cured under 405 nm ultraviolet light for 7 min to prepare GH hydrogel. 1 mL of the above BP nanosheet solution and 1 mL of GH solution were mixed and cross-linked and cured under 405 nm ultraviolet light for 7 min to prepare BP / GH hydrogel. The morphologies of GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were observed, and then the above hydrogels were freeze-dried to observe their microstructures.

[0062] The results show that Figure 3 As shown in Figure A, WEO@BP / GH hydrogel is gray. Figure 3 As shown in Content B, GH, BP / GH and WEO@BP / GH hydrogels all exhibited a porous three-dimensional interconnected architecture. The high-magnification (20 μm) SEM image showed that the internal surface of the GH hydrogel was smooth, while the internal surface of the WEO@BP / GH hydrogel exhibited a rough texture, which was attributed to the addition of WEO@BP nanoparticles (as shown by the red arrow).

[0063] A series of WEO concentrations (0.04, 0.12, 0.20, 0.28, 0.36 mg / mL) of ethanol solutions were prepared and the absorbance at a wavelength of 203 nm was measured using a UV-visible spectrophotometer to establish a WEO standard curve. The standard curve is as follows: A = 0.0102c + 0.0266 (R 2 =0.9968), it can be seen that at a wavelength of 203nm, the absorbance of WEO has a strong linear correlation with the concentration. 1mL of the above solution containing composite nanoparticles was ultrasonically treated for 1h to destroy the nanoparticle wall, and the supernatant was collected at 10000rpm for 8min to measure the absorbance and calculate the encapsulation rate of WEO in WEO@BP / GH hydrogel. Encapsulation rate (%) = (M1 / M0) × 100%, where M0 is the total mass of WEO and M1 is the mass of WEO in the supernatant. The calculated encapsulation rate is 58.2%, indicating that BP nanosheets have excellent WEO loading capacity.

[0064] 50mg, 150mg, 100mg, 100mg, 80mg, 120mg of GelMA and 20mg, 20mg, 50mg, 100mg, 20mg, 20mg of HAMA were dissolved in 1mL of the above LAP solution, and ultrasonic treatment was performed for 5min until the solution was clear and transparent to obtain 5G2H solution, 15G2H solution, 10G5H solution, 10G10H solution, 8G2H solution, and 12G2H solution. Then, 1mL of the above different GH solutions and 1mL of the above solution containing composite nanoparticles were mixed, and cross-linked and cured under 405nm ultraviolet light for 7min to prepare hydrogels with different GelMA / HAMA ratios.

[0065] The results show that Figure 3 As shown in content C, the pore sizes of 5G2H and 15G2H hydrogels are uneven, and the three-dimensional interconnected architecture is unstable; the pore sizes of 10G5H and 10G10H hydrogels are small, which is not conducive to nutrient exchange and cell proliferation. The pore sizes and pore uniformity of 8G2H hydrogels and 12G2H hydrogels are the same as those of the above-mentioned WEO@BP / GH hydrogel, which is conducive to nutrient exchange and cell proliferation.

[0066] Figure 3 Content D is the FTIR spectrum of WEO@BP / GH hydrogel. - ) has a dissociation vibration at 1420 cm -1 , the vibration of methyl (-CH2) is at 2923cm -1 , confirming the successful modification of carboxymethyl groups in the hydrogel. In addition, + ) has a protonation-related asymmetric deformation vibration at 1595 cm -1, the stretching vibration of aromatic amine (-CN) is at 1321cm -1 There are amino groups in the hydrogel. Amide I (1645 cm -1 ) and amide II (1591 cm -1 ) showed that after the cross-linking curing reaction, amide bonds (-NHCO - ). Figure 3 Content E is the Raman spectrum. It can be seen that the characteristic peak corresponding to BP is observed at 354 cm in WEO@BP / GH hydrogel. - ¹、433cm - ¹ and 462cm - ¹, indicating successful integration of BP into GH hydrogel.

[0067] After freeze-drying the GH hydrogel, BP / GH hydrogel and WEO@BP / GH hydrogel with a diameter of 8 mm and a thickness of 2 mm, the initial weight of the hydrogel was weighed (denoted as W0), and then the hydrogel was immersed in 37°C PBS for a period of time and then taken out, the excess liquid on the surface was removed and weighed (denoted as Wh) until the hydrogel reached water absorption equilibrium, and the water absorption ratio of the hydrogel was calculated, where the water absorption ratio (g / g) = (Wh-W0) ⁄ W0. The fully expanded hydrogel (weight denoted as Wp) was then placed in a fume hood at a constant temperature of 37°C to dry until its weight stabilized (denoted as We), and the water retention rate of the hydrogel was calculated, where the water retention rate (%) = (We / Wp) × 100%. The rheological properties of the GH hydrogel with a diameter of 8 mm and a thickness of 2 mm and the WEO@BP / GH hydrogel were evaluated using a rheometer.

[0068] The results show that Figure 4 Content A. Figure 4 As shown in Content B, WEO@BP / GH hydrogel has excellent hygroscopicity and water retention, which can effectively absorb wound exudate and maintain a moist wound environment, which is beneficial to the water and function of cells.

[0069] like Figure 4 As shown in Figure C, the viscosity of WEO@BP / GH hydrogel remains relatively stable over time (shear rate: 10 rad / s, 37°C), indicating that the hydrogel has strong adhesion. Figure 4 As can be seen from content D, under UV irradiation, the storage modulus (G') of WEO@BP / GH hydrogel increased from 10Pa to 1000Pa, and the mechanical properties were significantly enhanced, which could maintain structural integrity and prevent the penetration of external bacteria due to material failure. Figure 4 As shown in Figure E, strain sweep experiments show that WEO@BP / GH hydrogel exhibits a gelation threshold at approximately 100% strain. Figure 4As shown in Figure F, the dynamic frequency scanning results show that as the frequency increases from 0.1 to 10 Hz, the storage modulus (G') of the hydrogel exceeds the loss modulus (G''), indicating that the WEO@BP / GH hydrogel has excellent elasticity. Through the dynamic amplitude test, a large strain amplitude of 200% was applied to the hydrogel to destroy the cross-linked network of the hydrogel, and then a small strain amplitude of 1% was applied to evaluate the recovery ability of the hydrogel, as shown in Figure 2. Figure 4 As shown in Figure G, at a strain amplitude of 200%, G' is significantly lower than G'', indicating that fracture occurred inside the hydrogel. When the strain amplitude dropped to 1%, G' increased significantly and was higher than G'', indicating the recovery of the hydrogel state, indicating that WEO@BP / GH hydrogel has self-healing ability.

[0070] Example 2 Photothermal properties of multifunctional hydrogel loaded with Qi Ai essential oil 2.1 Near-infrared photothermal properties of multifunctional hydrogel loaded with Qi Ai essential oil 1mL of the above-mentioned solution containing composite nanoparticles was mixed with 1mL of the above-mentioned GH solution, and 100μL of the mixed solution was placed in a centrifuge tube and irradiated with near-infrared (NIR) laser at a wavelength of 808nm and power densities of 0.5W / cm², 1.0W / cm² and 2.0W / cm², respectively. The temperature was monitored and infrared images were taken using a FLIR infrared camera until the peak temperature was reached to evaluate the thermal conversion efficiency of the mixed solution (i.e., WEO@BP / GH hydrogel precursor solution). The mixed solution was irradiated with an NIR laser at a wavelength of 808nm and a power density of 1.0W / cm² for 5min, then cooled for 10min and irradiated again with the laser, for a total of 4 cycles, and the photothermal stability of the mixed solution was evaluated by photothermal cycle testing.

[0071] The results show that Figure 5 Content A. Figure 5 As shown in Content B, when the mixed liquid is irradiated with NIR laser at power density of 0.5W / cm², 1.0W / cm² and 2.0W / cm² for 5 minutes, the temperature can reach 38.8°C, 55.6°C and 69.4°C. Figure 5 As shown in content C, after 4 cycles of laser irradiation and cooling, the mixed solution maintained a consistent temperature curve, which indicates that the WEO@BP / GH hydrogel precursor solution prepared in the present application has excellent photothermal stability.

[0072] 2.2 Controlled release of multifunctional hydrogel loaded with Qi Ai essential oil The WEO@BP / GH hydrogel with a diameter of 8 mm and a thickness of 2 mm was placed in a centrifuge tube and irradiated with NIR at a wavelength of 808 nm and a power density of 1 W / cm² for 5 min. It was then cooled for 5 min and irradiated again. The irradiation cycle was repeated 4 times in total. The amount of WEO released was detected by UV spectrophotometry to evaluate the controllable release of WEO in the WEO@BP / GH hydrogel.

[0073] The results show that Figure 6 As shown in Figure A, the amount of WEO released from the NIR-irradiated hydrogel is significantly higher than that from the non-NIR-irradiated hydrogel. As the treatment time increases, the amount of WEO released from the NIR-irradiated hydrogel gradually increases. When the treatment time is extended to 120h, the amount of WEO released can reach 91.4%, which is significantly higher than that from the non-NIR-irradiated hydrogel ( Figure 6 Content B), which indicates that the multifunctional hydrogel prepared in this application has NIR responsive release capability.

[0074] Example 3 Anti-inflammatory properties of multifunctional hydrogel loaded with Qi Ai essential oil Human mononuclear cells (THP-1 cells) were cultured at a rate of 1 × 10 5The cells were seeded at a concentration of 100 / well in a 6-well plate in DMEM medium containing 10% fetal bovine serum and cultured at 37°C. Then, 50 ng / mL of phorbol ester 12-myristate 13-acetate (PMA) was added for 24 h to induce THP-1 cells to polarize to the M0 phenotype. Then, 20 ng / mL of interferon γ (IFNγ) and 1 μg / mL of lipopolysaccharide (LPS) were added to polarize to the M1 phenotype. Subsequently, 1 mL of PBS, GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were added to a 6-well plate and incubated for 48 hours as the PBS group, GH group, BP / GH group, and WEO@BP / GH group; WEO@BP / GH hydrogel was added to a 6-well plate and incubated for 48 hours, and then irradiated with NIR at a wavelength of 808 nm and a power density of 1 W / cm² for 5 minutes as the WEO@BP / GH+NIR group, wherein the preparation methods of the above-mentioned various hydrogels are the same as those in Example 1, and the diameters of the above-mentioned hydrogels are all 8 mm and the heights are all 2 mm. Then, THP-1 cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% BSA at room temperature, and the treated cells were incubated with polyclonal antibodies of CD86 and CD206 at 4°C for 12 hours, with a dilution ratio of 1:150. Subsequently, the cells were incubated with a Cy3-labeled goat anti-rabbit IgG secondary antibody for 1 hour in the dark, with a dilution ratio of 1:200. Finally, the cell nuclei were stained with DAPI and observed using a Nikon C2 Plus fluorescence microscope. The mRNA expression levels of M1 macrophage-related genes (CD86 and IL-1β) and M2 macrophage-related genes (CD206 and IL-10) were detected by quantitative reverse transcription polymerase chain reaction (RT-PCR). Total RNA was isolated from macrophages using an RNA rapid extraction kit, and then reverse transcribed using a Prime Script RTMaster Mix kit and a PCR instrument. A PCR reaction mixture containing TB Green Premix Ex Taq II, forward primer, reverse primer, and cDNA was prepared, and then quantitative analysis was performed using a real-time PCR system. The primer information of the various genes mentioned above is shown in Table 1. GAPDH was used as a reference gene, and the relative mRNA expression level of the target gene was normalized relative to the control sample. The relative mRNA expression level of the target gene was quantified by measuring the fluorescence intensity in the reaction mixture.

[0075] Table 1 Primer information of genes IL-1β, CD86, IL-10, CD206

[0076] The results show that Figure 7 Content A. Figure 7 Content B. Figure 7As shown in Content C, compared with other groups, the expression level of CD206 (a marker of M2 macrophages) in the WEO@BP / GH group and the WEO@BP / GH+NIR group was significantly increased, while the expression level of CD86 (a marker of M1 macrophages) was significantly decreased. In addition, the results of RT-PCR analysis showed that ( Figure 7 Content D. Figure 7 Content E. Figure 7 Content F. Figure 7 Content G), WEO@BP / GH group can significantly upregulate the expression of M2 macrophage markers CD206 mRNA and IL-10 mRNA (anti-inflammatory factor), and downregulate the expression of M1 macrophage markers pro-inflammatory factors CD86 mRNA and IL-1β mRNA, which indicates that WEO@BP / GH hydrogel can polarize macrophages from M1 to M2. In addition, the use of NIR irradiation to treat WEO@BP / GH hydrogel can further promote the polarization of M1 macrophages to M2. This may be because when WEO@BP / GH hydrogel is irradiated with near-infrared light, it can effectively release Qi Ai essential oil. The released Qi Ai essential oil cooperates with the efficient photothermal effect of black phosphorus nanosheets to further enhance the effect of hydrogel polarizing M1 macrophages to M2.

[0077] Example 5 Antibacterial properties of multifunctional hydrogel loaded with Qi Ai essential oil Staphylococcus aureus (S. aureus, ATCC 29213) and Escherichia coli (E. coli, ATCC 25922) were inoculated into LB liquid culture medium and cultured at 37°C for 24 h to obtain Staphylococcus aureus solution and Escherichia coli solution (bacterial solution concentration was 1×10 7 CFU / mL). GH hydrogel, BP / GH hydrogel, WEO@BP / GH hydrogel (the preparation methods of the above hydrogels are the same as those in Example 1, and the diameter of the hydrogels is 8 mm and the height is 2 mm) were sterilized by ultraviolet irradiation for 1 h and set aside. 1 mL of sterile PBS, sterile GH hydrogel, sterile BP / GH hydrogel, and WEO@BP / GH hydrogel were placed in test tubes, 4 mL of the above bacterial solution was added, and incubated at 37°C for 12 h, as the PBS group, GH group, BP / GH group, and WEO@BP / GH group; the sterile WEO@BP / GH hydrogel was placed in a test tube, and 4 mL of the above bacterial solution (the concentration of the bacterial solution was 1×10 7 CFU / mL) were incubated at 37°C for 12 h and then subjected to a power density of 1 W / cm 2 The bacterial culture fluids of the different treatment groups were diluted 10% with PBS. 6The bacterial suspension was obtained by PCR, and then 100 μL of the bacterial suspension was spread on the agar plate and cultured at 37 °C for 12 h to observe the colony formation. The bacterial suspensions of different treatment groups were stained for 20 min using the SYTO9 / PI kit for live / dead cell staining to evaluate cell activity, where SYTO9 represents live bacteria (green fluorescence) and PI represents dead bacteria (red fluorescence), and then the stained cells were observed using a confocal laser scanning microscope.

[0078] The results show that Figure 8 Content A. Figure 8 Content C. Figure 8 Content D shows that compared with the PBS, GH, and BP / GH groups, the number of S. aureus and E. coli in the WEO@BP / GH group was significantly reduced, and the survival rate was significantly decreased, indicating that the WEO@BP / GH hydrogel has excellent in vitro antibacterial properties. After irradiating the WEO@BP / GH hydrogel with NIR for 5 minutes, the S. aureus and E. coli on the agar plate were completely removed, indicating that NIR irradiation can further enhance the antibacterial properties of the WEO@BP / GH hydrogel.

[0079] like Figure 8 Content B. Figure 8 Content E. Figure 8 As shown in Figure F, the plates of S. aureus and E. coli in the PBS group mainly showed green fluorescence (live bacteria), with only a small amount of red fluorescence (dead bacteria). After irradiating WEO@BP / GH hydrogel with NIR for 5 minutes, the plates of S. aureus and E. coli showed the strongest red fluorescence (dead bacteria). In summary, both the plate count test and the fluorescence experiment show that WEO@BP / GH hydrogel has strong antibacterial activity.

[0080] Example 6 Antioxidant properties of multifunctional hydrogel loaded with Qi Ai essential oil Human immortalized epidermal cells (HaCaT cells) were cultured at a rate of 1×10 4The cells were seeded at a concentration of 100 μM / well in a 96-well plate containing HaCaT complete medium and incubated at 37°C for 24 h. The medium was then replaced with a fresh medium containing 500 μM hydrogen peroxide (H2O2) as the control group (Control group), a fresh medium containing 500 μM H2O2 and 200 μL PBS as the PBS group, a fresh medium containing 500 μM H2O2 and GH hydrogel as the GH group, a fresh medium containing 500 μM H2O2 and BP / GH hydrogel as the BP / GH group, and a fresh medium containing 500 μM H2O2 and WEO@BP / GH hydrogel as the WEO@BP / GH group, and cultured at 37°C in the dark for 6 h. After the medium was replaced with a fresh medium containing 500 μM H2O2 and WEO@BP / GH hydrogel and cultured at 37°C in the dark for 6 h, the cells were subjected to a power density of 1.0 W / cm 2 The NIR irradiation of the hydrogel for 5 minutes was used as the WEO@BP / GH+NIR group (the preparation method of the above hydrogel is the same as that of Example 1, and the diameter of the hydrogel is 8 mm and the thickness is 2 mm). The bacterial culture fluid of each treatment group was collected, and the bacterial culture fluid of different treatment groups was stained for 20 minutes using the Calcein-AM / PI kit for live / dead cell staining, where live cells were stained with Calcein-AM (green fluorescence) and dead cells were stained with PI (red fluorescence) to evaluate the survival rate of the cells. The bacterial culture fluid of different treatment groups was fluorescently labeled for 30 minutes using DCFH-DA dye to detect the level of reactive oxygen species (ROS) in the cells.

[0081] PBS, GH hydrogel, BP / GH hydrogel and WEO@BP / GH hydrogel were sterilized by ultraviolet light and immersed in complete DMEM medium for 3 days, which were used as PBS group, GH group, BP / GH group and WEO@BP / GH group. WEO@BP / GH hydrogel was sterilized by ultraviolet light and immersed in complete DMEM medium for 3 days, and then irradiated with NIR with power density of 1W / cm² for 5min as WEO@BP / GH+NIR group. The immersion solution of each treatment group was aspirated with a syringe. HaCaT cells were cultured at 1×10 4 Cells were inoculated at a concentration of 100 / well in a 96-well plate containing HaCaT complete medium and incubated at 37°C for 12 h. The cell attachment and fusion reached 70%. The medium was then replaced with 100 μL of the immersion solution of the above-mentioned treatment groups. After incubation at 37°C for 12 h, 1000 μL of H2O2 was added and cultured in the dark at 37°C for 6 h. Then, 10% CCK-8 reagent was added and incubated at 37°C for 4 h. The absorbance of each treatment group at a wavelength of 450 nm was measured with an enzyme reader to calculate the cell viability.

[0082] The results show that Fig. 9As can be seen from Content A, the amount of red fluorescence in the GH group and BP / GH group was significantly higher than that in the WEO@BP / GH group and WEO@BP / GH+NIR group, and the percentage of dead cells was high ( Fig. 9 Content C). Similarly, CCK-8 experiments showed that when HaCaT cells were exposed to an environment rich in H2O2, the activity of HaCaT cells in the WEO@BP / GH group and the WEO@BP / GH+NIR group was higher than that in the other groups ( Fig. 9 Content D). In addition, the fluorescence of the fluorescent probe DCFH in the WEO@BP / GH group and WEO@BP / GH+NIR group was less than that in the GH group and BP / GH group ( Fig. 9 Content B), ROS signal was significantly lower than that of GH group and BP / GH group ( Fig. 9 Content E). In summary, WEO@BP / GH hydrogel has excellent antioxidant effect under near-infrared response, and can effectively remove ROS, improve cell survival rate, and is beneficial to wound healing.

[0083] Example 7 Hemostatic properties of multifunctional hydrogel loaded with Qi Ai essential oil The lower third of the rat tail was cut using surgical scissors, and after 15 seconds, the wound was soaked with sterile PBS as the PBS group; the wound was covered with GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel as the GH group, BP / GH group, and WEO@BP / GH group; the wound was covered with WEO@BP / GH+NIR hydrogel and then irradiated with NIR at 808nm and a power density of 1W / cm² for 5min as the WEO@BP / GH+NIR group (the preparation methods of the above hydrogels were the same as in Example 1, and the diameter of the hydrogels was 8mm and the thickness was 2mm); the wound was covered with gauze as the Gauze group, and the bleeding volume of each treatment group was recorded, and the hemostatic performance of the hydrogel was evaluated by the tail hemostasis model.

[0084] The results show that Fig.10 Content A. Fig.10 It can be seen from content B that the amount of bleeding when using different hydrogels and gauze is small, indicating that the above hydrogels can stop bleeding quickly and with small amount of bleeding.

[0085] Example 8 Angiogenesis-promoting properties of multifunctional hydrogel loaded with Qi Ai essential oil Scratch test: PBS, GH hydrogel, BP / GH hydrogel and WEO@BP / GH hydrogel were sterilized by ultraviolet light and immersed in complete DMEM medium for 3 days, which were used as PBS group, GH group, BP / GH group and WEO@BP / GH group. WEO@BP / GH hydrogel was sterilized by ultraviolet light and immersed in complete DMEM medium for 3 days, and then irradiated with NIR with a power density of 1W / cm² for 5min as WEO@BP / GH+NIR group. The immersion solution of each treatment group was drawn with a syringe. HaCaT cells were cultured at 5×10 5 The cells were inoculated at a concentration of 100 cells / well in a 6-well plate containing HaCaT complete medium. After incubation at 37°C for 12 h, the medium was removed and 4 mL of the above-mentioned immersion solution was added. Then, a 200 μL pipette tip was used to mechanically disrupt the cells (marked as 0 h). The cells were incubated at 37°C and 5% CO2 for 24 h, and the cell morphology was observed under a microscope to evaluate the effect of the hydrogel extract on the migration ability of fibroblasts.

[0086] Tubular structure formation experiment: 10 μL of matrix gel was applied to a 96-well plate and incubated at 37°C for 45 min to solidify. Then human umbilical vein endothelial cells (HUVECs) were cultured at 1×10 4 The cells were inoculated at a concentration of 100 / well in a 96-well plate and incubated at 37°C for 12 h, then the culture medium was removed and 4 mL of the above immersion solution was added and incubated at 37°C. The branch points and tubular length of the newborn blood vessels were observed under an inverted fluorescence microscope at 2 h and 4 h of incubation.

[0087] The results show that Fig.11 As shown in content A, the healing effect of cell scratches in the WEO@BP / GH group was better than that in the PBS group, GH group, and BP / GH group; NIR irradiation of the WEO@BP / GH group could further improve the healing rate of cell scratches. Fig.11 Content B. Fig.11 Content C. Fig.11 As shown in Figure D, after 2 hours of incubation, a small amount of tubular structures were produced in the PBS group, GH group and BP / GH group, while the WEO@BP / GH group and WEO@BP / GH+NIR group had more mature and complete tubular structures. After 4 hours of incubation, the WEO@BP / GH+NIR group had more branch points and longer capillaries. This may be because the WEO@BP / GH hydrogel effectively released WEO under NIR irradiation, thereby reducing oxidative stress, improving the migration of fibroblasts, and enhancing the ability of vascular production, and had excellent biocompatibility.

[0088] Example 9 Effect of multifunctional hydrogel loaded with Qi Ai essential oil on diabetic wound healing Male rats weighing 200-250g were used to construct a rat model of type 1 diabetes. The specific operation was as follows: after fasting the rats for 12 hours, 1% streptozotocin (STZ, injection dose of 60mg / kg, dosage unit is drug protein content / rat body weight) was intraperitoneally injected. After 7 days, the blood glucose level was measured from the rat tail vein. When the blood glucose level exceeded 16.7mmol / L, the rat model of type 1 diabetes was successfully constructed. After anesthetizing the diabetic rats, the hair on the back was shaved, and an 8mm incision was made on each side of the center line of the spine to create a skin wound. 200 μL sterile PBS was soaked on each incised wound as the PBS group; GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were placed as the GH group, BP / GH group, and WEO@BP / GH group; WEO@BP / GH+NIR hydrogel was placed and then irradiated with NIR at 808 nm and a power density of 1 W / cm² for 5 min as the WEO@BP / GH+NIR group (the preparation method of each hydrogel was the same as in Example 1, and the diameter of the hydrogel was 8 mm and the thickness was 2 mm). The wound area of ​​the different treatment groups was measured on the 0th, 3rd, 7th, 10th, and 14th day, and the wound area ratio was evaluated. Wound area ratio = (An / A0) × 100%, An is the wound area of ​​the different treatment groups when they were placed for n days, and A0 is the wound area of ​​the different treatment groups when they were placed for 0 days. On the second day of placement, the wound secretions of the different treatment groups were cultured on agar plates, incubated at 37°C for 24 hours, and the growth of the colonies was observed. The rats were killed on the 14th day of placement, and the wound tissues were collected and fixed in 4% formaldehyde for histological and immunofluorescence analysis. Specifically, hematoxylin-eosin (H&E) staining was used for histological evaluation of wound healing; Masson trichrome staining was used to evaluate collagen deposition; vascular endothelial growth factor (VEGF) immunohistochemical staining was performed; and fluorescent in situ staining of CD86 and CD206 was performed to evaluate inflammation, angiogenesis, and macrophage differentiation at the wound site. In addition, the dihydroethidium (DHE) ROS detection kit was used to detect the generation of ROS in vivo.

[0089] The results show that Fig.12 Content A. Fig.12 As can be seen from content B, the number of bacterial colonies in the wound secretions of the WEO@BP / GH+NIR group is the smallest, the bacterial survival rate is the lowest, and it can significantly inhibit the growth of bacteria in the wound.

[0090] Depend on Fig.13 It can be seen that under the same placement time, the wound area of ​​the WEO@BP / GH and WEO@BP / GH+NIR groups was significantly smaller than that of the other groups; on the 14th day of placement, the wound area of ​​the WEO@BP / GH+NIR group was only 5%, showing the best wound healing effect.

[0091] like Fig.14 As shown in Figure A (HE staining), 14 days after surgery, the wounds in the WEO@BP / GH+NIR group were almost completely healed, with dense granulation tissue and complete epidermal regeneration, and the scar width was only 0.58±0.09mm ( Fig.14 Content C). Fig.14 As shown in content B (Masson staining), 14 days after surgery, collagen fibers in the WEO@BP / GH+NIR group were arranged in parallel around the hair follicles, and the amount of collagen deposition was the highest ( Fig.14 Content D) was significantly higher than that of other groups.

[0092] like Fig.15 Content A. Fig.15 Content B. Fig.15 Content E. Fig.15 As shown in content F, 14 days after surgery, the red fluorescence coverage area of ​​CD206 in the WEO@BP / GH group and WEO@BP / GH+NIR group was significantly higher than that in the other groups, and the green fluorescence coverage area of ​​CD86 was significantly less than that in the other groups, indicating that WEO@BP / GH hydrogel can promote the polarization of M1 macrophages to M2 type, and NIR irradiation of WEO@BP / GH hydrogel can further enhance the polarization effect.

[0093] like Fig.15 Content C. Fig.15 As shown in Content G, 14 days after surgery, the expression levels of vascular endothelial growth factor (VEGF) in the WEO@BP / GH group and the WEO@BP / GH+NIR group were significantly higher than those in the other groups, indicating that the WEO@BP / GH hydrogel has an angiogenesis-promoting effect on new skin tissue. Treatment with WEO@BP / GH+NIR can further enhance the angiogenesis-promoting effect and effectively promote the formation of new blood vessels.

[0094] like Fig.15 Content D. Fig.15 As shown in Figure H, 14 days after surgery, the ROS levels in the WEO@BP / GH group and the WEO@BP / GH+NIR group were significantly lower than those in the other groups, indicating that WEO@BP / GH hydrogel can effectively remove reactive oxygen species in the body, and WEO@BP / GH+NIR treatment can further enhance the removal effect of reactive oxygen species in the body.

[0095] Example 9 Biosafety of Multifunctional Hydrogel Loaded with Qi Ai Essential Oil 9.1 In vitro hemolysis Blood from diabetic rats was collected and centrifuged to obtain red blood cells, which were then washed three times with PBS to obtain eluted red blood cells. The red blood cells were then placed in a test tube, and 2 mL of 10% Triton X-100 was added and incubated at 37°C for 4 h as the Triton group (positive control group); 200 μL of PBS was added and incubated at 37°C for 4 h as the PBS group (negative control group); GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were added and incubated at 37°C for 4 h as the GH group, BP / GH group, and WEO@BP / GH group; WEO@BP / GH hydrogel was added and incubated at 37°C for 4 h and then subjected to a power density of 1.0 W / cm 2 The NIR irradiation of 5 min was used as the WEO@BP / GH+NIR group. The supernatants of the different treatment groups were collected by centrifugation at 2500 rpm for 5 min, and the absorbance of the supernatant was measured using an ELISA instrument to calculate the hemolysis rate, where the hemolysis rate (%) = (Ah-Ap) / (At-Ap)×100%, Ah is the absorbance of different hydrogel groups, Ap is the absorbance of the PBS group, and At is the absorbance of the Triton group.

[0096] The results show that Fig.16 As shown in Content A, the red blood cell supernatant of the positive group was bright red, while the supernatant of the other treatment groups was colorless and transparent, with red blood cells precipitated at the bottom; except for the Triton group, the hemolysis rates of the other groups were less than 5% ( Fig.16 Content B), which indicates that the above hydrogel has little effect on red blood cell hemolysis and will not cause hemolysis.

[0097] 9.2 In vitro safety PBS, GH hydrogel, BP / GH hydrogel and WEO@BP / GH hydrogel were sterilized by UV and then immersed in complete DMEM culture medium for 24 hours, which were used as PBS group, GH group, BP / GH group and WEO@BP / GH group. WEO@BP / GH hydrogel was sterilized by UV and then immersed in complete culture medium for 24 hours, and then subjected to 1.0 W / cm 2 The hydrogels were irradiated with NIR for 5 min as the WEO@BP / GH+NIR group (the preparation methods of the above hydrogels were the same as in Example 1, and the diameters and thicknesses of the hydrogels were all 8 mm and 2 mm), and the immersion liquid of each treatment group was drawn with a syringe.

[0098] Leaching test: HaCaT cells were inoculated in 24-well plates and incubated for 12 hours, then 500 μL of the immersion solution of each treatment group was added and incubated for 1 day, 3 days, and 5 days. The live cells and dead cells were stained using the calcein-AM / PI kit. Live cells were stained with calcein-AM (green fluorescence), dead cells were stained with PI (red fluorescence), and fluorescence was observed using a Nikon C1 confocal microscope.

[0099] CCK-8 experiment: HaCaT cells were inoculated in DMEM medium containing 10% FBS and 1% penicillin-streptomycin and incubated at 37°C, 5% CO2 for 24 h. The medium was replaced with 1 mL of the immersion solution of each treatment group and incubated for 1, 3, and 5 days. 10 µL of CCK-8 was added and incubated in the dark for 1 h. The optical density at 450 nm was detected using a microplate reader to evaluate cell viability.

[0100] The results show that Fig.17 Content A. Fig.17 As shown in Content B, within the same incubation time, the green fluorescence intensity of the hydrogel group was similar to that of the PBS group, indicating that the hydrogel group did not affect cell proliferation. Fig.17 As shown in Content C, within the same incubation time, there was no significant difference in the cell viability of each treatment group, which indicated that the different hydrogel groups had no significant toxicity to the cells.

[0101] 9.3 In vivo safety The heart, liver, spleen, lung and kidney of the diabetic rats in Example 8 were collected 14 days after surgery and stained with HE, and blood was collected for blood biochemical analysis.

[0102] The results show that Fig.18 As shown in the figure, no significant pathological abnormalities or damage were found in the organs of each treatment group. Blood biochemical analysis showed that there was no significant difference in the content of HGB, ALT, AST, BUN, CRE, WBC, PLT, and RBC between the different hydrogel groups and the PBS group ( Fig.19 Contents A~H).

[0103] In summary, the multifunctional hydrogel provided in the present application has high biosafety and good clinical application prospects.

[0104] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A multifunctional hydrogel loaded with Qi Ai essential oil, characterized in that: The invention comprises a hydrogel matrix and composite nanoparticles composited therein; the composite nanoparticles comprise black phosphorus nanosheets and Qi Ai essential oil contained therein.

2. The multifunctional hydrogel according to claim 1, characterized in that In the composite nanoparticles, the ratio of black phosphorus nanosheets to Qi Ai essential oil is (20-40) mg:1 mL.

3. The multifunctional hydrogel according to claim 1, characterized in that The hydrogel matrix is ​​obtained by cross-linking methacryloyl gelatin and hyaluronic acid methacrylate; Preferably, the mass ratio of the methacrylated gelatin to the hyaluronic acid methacrylate is (4-6):

1.

4. A method for preparing the multifunctional hydrogel according to any one of claims 1 to 3, characterized in that: The steps include: S1, mixing the black phosphorus nanosheet suspension and the Qi Ai essential oil, and then loading the black phosphorus nanosheet with the Qi Ai essential oil through an extrusion cycle to prepare a solution containing composite nanoparticles; S2, mixing methacrylated gelatin, hyaluronic acid methacrylate and buffer to prepare a hydrogel solution; S3, after mixing the hydrogel solution and the solution containing composite nanoparticles, cross-linking and curing them under the condition of initiation by a photoinitiator or a light source to prepare a multifunctional hydrogel.

5. The preparation method according to claim 4, characterized in that: In step S1, the concentration of the black phosphorus nanosheet suspension is 1-5 mg / mL; Preferably, the volume ratio of the black phosphorus nanosheet suspension to the Artemisia argyi essential oil is (5-15):

1.

6. The preparation method according to claim 4, characterized in that: In step S1, the number of extrusion cycles is 5 to 15 times.

7. The preparation method according to claim 4, characterized in that: In step S2, in the hydrogel solution, the concentration of the methacryloyl gelatin is 50-150 mg / mL; The mass ratio of the methacrylated gelatin to the hyaluronic acid methacrylate is (4-6):

1.

8. The preparation method according to claim 4, characterized in that: In step S3, the cross-linking and curing time is 5 to 10 minutes; and / or, The photoinitiator is selected from one or more of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, 1,2-diketone, Irgacure2959, and Alg-2959.

9. Use of the multifunctional hydrogel according to any one of claims 1 to 3 in the preparation of an anti-inflammatory drug, characterized in that: The multifunctional hydrogel is able to polarize M1 macrophages to M2 macrophages.

10. Use of the multifunctional hydrogel according to any one of claims 1 to 3 as or in the preparation of a hydrogel auxiliary material for promoting wound healing.

Images