Multifunctional hydrogel loaded with artemisia argyi essential oil, preparation method and application thereof

Abstract

The application belongs to the technical field of hydrogel materials, and more particularly relates to a multifunctional hydrogel loaded with wormwood oil, a preparation method and application thereof. The multifunctional hydrogel loaded with wormwood oil provided by the application comprises a hydrogel matrix and composite nanoparticles compounded in the interior thereof; wherein the composite nanoparticles are black phosphorus nanoplates loaded with wormwood oil. The hydrogel has multiple functions of hemostasis, anti-inflammation, antibiosis, antioxidation, promotion of angiogenesis, and the like, has strong water retention capacity, excellent mechanical properties, good biocompatibility, and high biological safety, and can be used for preparing a hydrogel dressing for promoting wound healing. In addition, the multifunctional hydrogel has near-infrared light response characteristics, can realize controlled release of the wormwood oil, and the released wormwood oil can cooperate with the photothermal effect of the black phosphorus nanoplates to further improve the performance of the hydrogel.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of hydrogel materials, and more particularly relates to a multifunctional hydrogel loaded with Chinese wormwood essential oil, a preparation method and application thereof. BACKGROUND

[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. Clinically, surgical debridement, application of negative pressure wound dressings and other methods are often used to treat diabetic wounds, but these strategies are often subject to certain limitations, such as surgical debridement, which is an invasive procedure that can damage the integrity of the skin and tissues, providing a new entry route for pathogens such as bacteria, and the wound after debridement can have excessive local inflammation, leading to delayed wound healing; traditional wound dressings such as gauze and cotton are prone to adhere to the wound and have the disadvantage of high loss.

[0003] The literature (International Journal of Biological Macromolecules, 263 (2024), 130386) uses graphene oxide (GO) as an encapsulation carrier to wrap essential oil to obtain nanoparticles, and uses GelMA as a hydrogel skeleton to embed the nanoparticles into the GelMA hydrogel, which can promote wound healing, but the encapsulation carrier graphene oxide used by the hydrogel has certain toxicity, which can increase the risk of biological safety when used for a long time, and is not conducive to popularization and application. SUMMARY

[0004] In view of the defects of the prior art, the purpose of the present application is to provide a multifunctional hydrogel loaded with Chinese wormwood essential oil, a preparation method and application thereof, which can polarize M1 type macrophages to M2 type, has excellent anti-inflammatory effect, and has excellent hemostatic, anti-inflammatory, antioxidant, and pro-angiogenic functions, and the hydrogel has good biocompatibility, strong water retention capacity, and excellent mechanical properties, and is suitable for preparing a hydrogel dressing for promoting wound healing, especially for diabetic wound healing.

[0005] To achieve the above purpose, in a first aspect, the present application provides a multifunctional hydrogel loaded with Chinese wormwood essential oil, which comprises a hydrogel matrix and composite nanoparticles compounded in the interior thereof; the composite nanoparticles comprise black phosphorus nanosheets and Chinese wormwood essential oil encapsulated in the interior thereof.

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

[0007] Preferably, the hydrogel matrix is obtained by crosslinking methacrylated gelatin and hyaluronic acid methacrylate.

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

[0009] In a second aspect, the application provides a preparation method of the multifunctional hydrogel, comprising the following steps:

[0010] S1, mixing the black phosphorus nanosheet suspension and the Wuhu Ai essential oil, and then loading the black phosphorus nanosheet with the Wuhu Ai essential oil through extrusion circulation to prepare a solution containing composite nanoparticles;

[0011] S2, mixing the methacrylated gelatin, the hyaluronic acid methacrylate and the buffer to prepare a hydrogel solution;

[0012] S3, mixing the hydrogel solution and the solution containing composite nanoparticles, and then cross-linking and solidifying under the condition of light initiation or light source initiation to prepare the multifunctional hydrogel.

[0013] Preferably, in step S1, the concentration of the black phosphorus nanosheet suspension is 1-5 mg / mL.

[0014] Preferably, the volume ratio of the black phosphorus nanosheet suspension and the Wuhu Ai essential oil is (5-15):1.

[0015] Preferably, in step S1, the number of extrusion circulations is 5-15 times.

[0016] Preferably, in step S2, the concentration of the methacrylated gelatin in the hydrogel solution is 50-150 mg / mL.

[0017] Preferably, in step S2, the mass ratio of the methacrylated gelatin and the hyaluronic acid methacrylate is (4-6):1.

[0018] Preferably, in step S3, the time of cross-linking and solidifying is 5-10 min.

[0019] Preferably, the light initiator is selected from one or more of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 1,2-diketone, Irgacure 2959, Alg-2959.

[0020] In a third aspect, the application provides the use of the multifunctional hydrogel in serving as or preparing an anti-inflammatory drug, which can polarize M1 type macrophages to M2 type.

[0021] In a fourth aspect, the application provides the use of the multifunctional hydrogel in serving as or preparing a hydrogel auxiliary material for promoting wound healing.

[0022] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages:

[0023] (1) The present application uniformly loads the black phosphorus nanosheets loaded with WOE in the hydrogel matrix, utilizes the synergistic effect between the components to prepare a hydrogel with multiple functions, including hemostasis, anti-inflammatory, antibacterial, antioxidant, and pro-angiogenic functions, and the hydrogel has strong water retention capacity, excellent mechanical properties, good biocompatibility, and high biological safety. In addition, the multifunctional hydrogel provided by the present application has near-infrared light response characteristics, and when the multifunctional hydrogel is irradiated with near-infrared light, the WOE can be effectively released, and the released WOE cooperates with the high-efficiency photothermal effect of the black phosphorus nanosheets to further improve the performance of the hydrogel, effectively promote wound healing, and can be used for preparing a hydrogel dressing for promoting wound healing.

[0024] (2) The preparation method of the multifunctional hydrogel loaded with WOE provided by the present application has the advantages of simple preparation steps, wide source of raw materials, and low price, and in actual application, the components constituting the hydrogel matrix and the ratio between the components, the size of the black phosphorus nanosheets, the amount of the loaded WOE, and the crosslinking time can be adjusted according to the actual needs, and different molds can be used to quickly prepare multifunctional hydrogels with different shapes and thicknesses, which have a broad application prospect in the field of wound healing, especially for diabetic wound healing.

[0025] (3) The present application provides the application of the multifunctional hydrogel loaded with WOE in use or preparation of anti-inflammatory drugs, and experiments show that the multifunctional hydrogel can effectively inhibit the expression of M1 macrophage marker CD86, promote the expression of M2 macrophage marker CD206, down-regulate the expression of CD86 mRNA and IL-1β mRNA, up-regulate the expression of CD206 mRNA and IL-10 mRNA, and polarize M1 macrophages to M2. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the feature of the black phosphorus nanosheets provided by 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;

[0027] Figure 2 is the feature of the WEO@BP nanoparticle provided by the present application, wherein content A is a SEM image of WEO, content B is a SEM image of the WEO@BP nanoparticle, and content C is a particle size distribution of the WEO@BP nanoparticle;

[0028] Figure 3Physical properties of the hydrogel provided by the embodiments of the present application, wherein content A is an optical image of the hydrogel, content B is an SEM image of the hydrogel at different magnifications, content C is an SEM image of other hydrogels, content D is an FTIR spectrum of the WEO@BP / GH hydrogel, and content E is a Raman spectrum of the WEO@BP / GH hydrogel;

[0029] Figure 4 Performance of the WEO@BP / GH hydrogel provided by the embodiments of the present application, wherein content A is moisture absorption performance, content B is water retention performance, content C is adhesion, content D is mechanical performance, content E is gelation threshold, content F is elasticity, and content G is self-healing ability;

[0030] Figure 5 Near-infrared photothermal performance of the WEO@BP / GH hydrogel provided by the embodiments of the present application, wherein content A, content B and content C are, respectively, an infrared thermal image, a temperature rise curve and photothermal stability of the WEO@BP / GH hydrogel precursor;

[0031] Figure 6 Cumulative release of WEO of the WEO@BP / GH hydrogel provided by the embodiments of the present application over time, wherein content A is 0-30 min and content B is 0-120 h;

[0032] Figure 7 Effect of the WEO@BP / GH hydrogel provided by the embodiments of the present application on polarizing M1 type macrophages into M1 type, wherein content A is a fluorescence intensity microscopic image of CD206 and CD86 in the cells, content B and content C are, respectively, relative fluorescence quantitative analysis of CD206 and CD86 in the cells, and content D, content E, content F and content G are, respectively, relative expression of CD206 mRNA, IL-10 mRNA, CD86 mRNA and IL-1β mRNA in the cells;

[0033] Figure 8 In vitro antibacterial performance of the WEO@BP / GH hydrogel provided by the embodiments of the present application, wherein content A is a plate clone map of S. aureus and E. coli, content B is a live / dead cell staining fluorescence map of the plate of S. aureus and E. coli, content C is survival rate of S. aureus, content D is survival rate of E. coli, content E is PI fluorescence value of S. aureus, and content F is PI fluorescence value of E. coli;

[0034] Figure 9Antioxidant performance of WEO@BP / GH hydrogel provided in the embodiments of the present application, wherein content A is Calcein-AM / PI staining fluorescence image of HaCaT cells after being cultured with 500 μM H2O2 and different treatment groups, content B is reactive oxygen species (ROS) staining fluorescence image, content C is percentage of dead cells, content D is activity of HaCaT cells after treatment, content E is relative DCF fluorescence intensity;

[0035] Figure 10 Hemostatic performance of WEO@BP / GH hydrogel provided in the embodiments of the present application, wherein content A is hemostatic optical image, content B is bleeding amount;

[0036] Figure 11 Pro-angiogenic performance of WEO@BP / GH hydrogel provided in the embodiments of the present application, wherein content A and content B are cell scratch healing and tubular structure formation of HaCaT cells after being cultured with different treatment groups at 0h and 24h of incubation, respectively, content C is branch point number, and content D is tubular length;

[0037] Figure 12 In vivo antibacterial effect of WEO@BP / GH hydrogel provided in the embodiments of the present application, wherein content A is plate colony graph of secretions of diabetic wounds at 2 days of treatment of different treatment groups, and content B is survival rate of cells in the secretions;

[0038] Figure 13 Optical image of diabetic wounds treated with WEO@BP / GH hydrogel provided in the embodiments of the present application for different treatment times;

[0039] Figure 14 Pathological analysis result of wound tissue treated with WEO@BP / GH hydrogel provided in the embodiments of the present application for 14 days, wherein content A is H&E staining image, content B is Masson trichrome staining image, content C is scar width, and content D is collagen deposition amount;

[0040] Figure 15 Immunohistochemical and fluorescent staining of wound tissue treated with WEO@BP / GH hydrogel provided in the embodiments of the present application for 14 days, wherein content A is immunofluorescent staining of CD86, content B is immunofluorescent staining of CD206, content C is immunohistochemical staining of VEGF, content D is fluorescent staining of ROS detected by DHE, content E is expression amount of CD86, content F is expression amount of CD206, content G is expression amount of VEGF, and content H is expression amount of DHE;

[0041] Figure 16In vitro hemolysis performance of the WEO@BP / GH hydrogel provided in the embodiments of the present application, wherein content A is an optical image of a blood sample after treatment by different treatment groups, and content B is the hemolysis rate of different treatment groups;

[0042] Figure 17 In vitro safety of the WEO@BP / GH hydrogel provided in the embodiments of the present application, wherein content A, content B and content C are Calcein-AM / PI staining fluorescence images, fluorescence expression of Calcein-AM and cell viability of HaCaT cells after treatment by different groups, respectively.

[0043] Figure 18 HE staining images of different organs of a diabetic rat wound treated with the WEO@BP / GH hydrogel provided in the embodiments of the present application for 14 days;

[0044] Figure 19 Blood biochemical analysis results of a diabetic rat wound treated with the WEO@BP / GH hydrogel provided in the embodiments 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.

[0045] In all the drawings, the same reference signs are used to represent the same degree of significance, wherein: ns represents P>0.05, represents P<0.05, represents P<0.01, represents P<0.001. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the present application more clear and explicit, the present application is further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0047] The present application provides a multifunctional hydrogel loaded with Chinese herbaceous wormwood essential oil, which comprises a hydrogel matrix and composite nanoparticles compounded in the interior thereof; the composite nanoparticles comprise black phosphorus nanosheets and Chinese herbaceous wormwood essential oil encapsulated in the interior thereof.

[0048] In some embodiments, the ratio of black phosphorus nanosheets to Chinese herbaceous wormwood essential oil in the composite nanoparticles is (20-40) mg:1 mL.

[0049] In preferred embodiments, the ratio of black phosphorus nanosheets to Chinese herbaceous wormwood essential oil in the composite nanoparticles is (25-35) mg:1 mL.

[0050] In some embodiments, the black phosphorus nanosheet has a particle size of 200-600 nm and a thickness of 2.5-7.5 nm.

[0051] In some embodiments, the hydrogel matrix is cross-linked from methacrylated gelatin and hyaluronic acid methacrylate. Further, the mass ratio of the methacrylated gelatin (GelMA) and the hyaluronic acid methacrylate (HAMA) is (4-6):1, which can prepare a hydrogel matrix with moderate pore size and uniform pore size, which is conducive to nutrient exchange and cell proliferation.

[0052] In another aspect, the application provides a preparation method of the multifunctional hydrogel, comprising the following steps:

[0053] S1, mixing the black phosphorus nanosheet suspension and the Biotae essential oil, and then loading the black phosphorus nanosheet with the Biotae essential oil through extrusion circulation to prepare a solution containing composite nanoparticles;

[0054] S2, mixing the methacrylated gelatin, the hyaluronic acid methacrylate and the buffer to prepare a hydrogel solution;

[0055] S3, mixing the hydrogel solution and the solution containing composite nanoparticles, and then cross-linking and solidifying under the condition of light initiation or light source initiation to prepare the multifunctional hydrogel loaded with Biotae essential oil.

[0056] In some embodiments, in step S1, the concentration of the black phosphorus nanosheet suspension is 1-5 mg / mL.

[0057] It can be understood that the application does not limit the source of the black phosphorus nanosheet suspension, which can be purchased from commercially available black phosphorus nanosheet suspensions, resuspended in a solvent from commercially available black phosphorus nanosheets, or prepared by a method known in the art.

[0058] In some embodiments, in step S1, the preparation method of the black phosphorus nanosheet suspension comprises the following steps: mixing black phosphorus powder and an organic solvent, and then performing liquid exfoliation by ultrasonic to exfoliate three-dimensional black phosphorus powder into two-dimensional sheet structure to obtain black phosphorus nanosheet, and then resuspending to obtain black phosphorus nanosheet suspension.

[0059] In some embodiments, the preparation process of the black phosphorus nanosheet suspension is as follows: black phosphorus powder and an organic solvent are mixed, ultrasonic treatment is performed in an ice water bath for 8-15 hours, the supernatant is collected by centrifugation at a speed of 2000-5000 rpm for 5-10 minutes, the supernatant is centrifuged at a speed of 10000-15000 rpm for 10-30 minutes to collect the precipitate, the precipitate is resuspended and washed, and then centrifuged at a speed of 15000-20000 rpm for 1-2 hours to obtain black phosphorus nanosheets, and finally the black phosphorus nanosheets are resuspended in a solvent to obtain a black phosphorus nanosheet suspension.

[0060] In some embodiments, the amount of the black phosphorus powder and the organic solvent is (0.5-1) mg: 1 mL.

[0061] In some embodiments, the organic solvent includes, but is not limited to, one or more of saturated sodium hydroxide N-methyl pyrrolidone, N-methyl pyrrolidone (NMP), N,N-dimethyl formamide (DMF), and dimethyl sulfoxide (DMSO).

[0062] In some embodiments, the black phosphorus nanosheets can be resuspended in deionized water, N-methyl pyrrolidone, N,N-dimethyl formamide, dimethyl sulfoxide, or the like to obtain a black phosphorus nanosheet suspension.

[0063] In some embodiments, in step S1, the preparation method of the Wuzhuyu essential oil includes the following steps: mixing Wuzhuyu powder and deionized water, and then extracting by distillation to obtain Wuzhuyu essential oil.

[0064] In some embodiments, the amount of the Wuzhuyu powder is 50-200 g, the amount of deionized water is 1000-2000 mL, and the distillation time is 2-8 hours.

[0065] In some embodiments, the volume ratio of the black phosphorus nanosheet suspension and the Wuzhuyu essential oil is (5-15): 1.

[0066] In some embodiments, the number of extrusion cycles is 5-15, so that the black phosphorus nanosheets encapsulate the Wuzhuyu essential oil. In some embodiments, the pore size of the filter used in the extrusion cycle is 200-600 nm.

[0067] In some embodiments, in step S2, the concentration of the methacrylated gelatin in the hydrogel solution is 50-150 mg / mL.

[0068] In some embodiments, in step S2, the mass ratio of the methacrylated gelatin and the hyaluronic acid methacrylate is (4-6): 1.

[0069] In some embodiments, the above-mentioned buffer is Dulbecco's phosphate-buffered saline (DPBS) or phosphate-buffered saline (PBS).

[0070] It can be understood that the present application does not limit the volume ratio of the above-mentioned solution containing composite nanoparticles and the above-mentioned hydrogel solution, and those skilled in the art can adjust the ratio of the two according to actual needs to obtain a multifunctional hydrogel containing composite nanoparticles with different concentrations. In some embodiments, in step S3, the volume ratio of the above-mentioned solution containing composite nanoparticles and the above-mentioned hydrogel solution is (0.5-2):1.

[0071] The present application does not limit the size, thickness, etc. of the above-mentioned multifunctional hydrogel, and those skilled in the art can select a suitable size, thickness according to the needs of the actual application scene, which is within the scope of protection of the present application.

[0072] In some embodiments, in step S3, the above-mentioned crosslinking and curing time is 5-10 min.

[0073] In some embodiments, in step S3, the above-mentioned photoinitiator is selected from one or more of lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), 1,2-diketone, Irgacure 2959, Alg-2959.

[0074] In some embodiments, the amount of the above-mentioned photoinitiator is 0.1%-1% of the total mass of the above-mentioned methacrylated gelatin and the above-mentioned hyaluronic acid methacrylate.

[0075] The present application also provides the use of the above-mentioned multifunctional hydrogel in the preparation of an anti-inflammatory drug, and the above-mentioned multifunctional hydrogel can polarize M1 type macrophages to M2 type.

[0076] The present application also provides the use of the above-mentioned multifunctional hydrogel in the preparation of a wound healing-promoting dressing.

[0077] The present application uses methacrylated gelatin and hyaluronic acid methacrylate to form a hydrogel matrix with a three-dimensional network structure, and uniformly loads black phosphorus nanosheets loaded with Ai Oila oil in the hydrogel, thereby preparing a multifunctional hydrogel through the synergistic effect between components, which has functions such as hemostasis, anti-inflammatory, antibacterial, antioxidant, and pro-angiogenic functions, and has good biocompatibility, strong water retention capacity, and excellent mechanical properties, and can be used to prepare a wound healing-promoting dressing. In addition, the multifunctional hydrogel provided by the present application has near-infrared light response characteristics, and when the above-mentioned multifunctional hydrogel is irradiated with near-infrared light, Ai Oila oil can be effectively released, and the released Ai Oila oil cooperates with the high-efficiency photothermal effect of the black phosphorus nanosheets to further improve the function of the hydrogel.

[0078] It should be understood that materials identical or similar in type, model, quality, nature or function to 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 commercially unless otherwise specified.

[0079] The above technical solutions are described in detail in combination with specific examples.

[0080] The main reagents and materials used in the following examples are as follows:

[0081] Black phosphorus (BP) was purchased from Aladdin Biochem Technology Co., Ltd.; Wuzhuyu was purchased from Hubei Lizichen Biological Technology Co., Ltd.; Methacrylated gelatin (GelMA) and hyaluronic acid methacrylate (HAMA) were both purchased from EFL Limited; YTO9 / propidium iodide (PI) kit was purchased from Thermo Fisher Scientific; Calcein-AM / PI kit, CCK-8 kit, DMEM medium, PBS, human keratinocyte (HaCaT), human monocyte THP-1 cell and human umbilical vein endothelial cell (HUVECs) were all purchased from Selleck Biological Technology Co., Ltd.; Fetal bovine serum (FBS) was purchased from Gibco (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 both purchased from Shanghai Biological Resources Center, China.

[0082] Example 1

[0083] 1.1 Preparation of Wuzhuyu essential oil (WEO)

[0084] 100 g of Wuzhuyu leaves were ground into powder and placed in a round-bottom flask, and 1500 mL of distilled water was added. Then the round-bottom flask, distillation head, condenser tube, receiving tube and recovery flask were assembled into a Clevenger-type device. After heating and boiling, the device was distilled for 4 h. The extracted Wuzhuyu essential oil (WEO) was collected and transferred to an EP tube for storage at 4°C for standby use.

[0085] 1.2 Preparation of black phosphorus (BP) nanosheets

[0086] 20 mg black phosphorus powder was dispersed in 20 mL saturated sodium hydroxide solution in N-methyl pyrrolidone, and liquid exfoliation was performed by ultrasonic treatment for 10 h in an ice bath. The bulk black phosphorus was exfoliated, and then the exfoliated solution was centrifuged at 3000 rpm for 5 min to remove the precipitate. The supernatant was further centrifuged at 12000 rpm for 30 min, and the precipitate was collected. Then the precipitate was suspended in ultrapure water, and after ultrasonic treatment for 2 h in the dark, it was centrifuged at 15000 rpm to obtain BP nanosheets, the characterization of which is shown in 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 at 4°C in the dark for later use.

[0087] The results show that the particle size of the BP nanosheets is 200-600 nm, and the thickness is 2.5-7.5 nm. Figure 1 Content A, Figure 1 Content B, Figure 1 Content C, Figure 1 Content D can be seen, the particle size of the BP nanosheet prepared by the present application is 200-600 nm, and the thickness is 2.5-7.5 nm.

[0088] 1.3 Preparation of WEO@BP nanoparticles

[0089] 1 mL of the above-mentioned BP nanosheet suspension and 100 μL of the above-mentioned WEO were ultrasonically oscillated in an ice bath to obtain a mixture. The above-mentioned mixture was subjected to 10 cycles of extrusion through a polycarbonate membrane with a pore size of 400 nm using a LiposoFast-Basic extruder to obtain a solution containing composite nanoparticles (i.e., WEO@BP nanoparticles).

[0090] The results show that the particle size of the BP nanosheets is 200-600 nm, and the thickness is 2.5-7.5 nm. Figure 2 Content A, Figure 2 Content B can be seen, the WEO is encapsulated in the BP nanosheet. From Figure 2 Content C can be seen, the particle size of the WEO@BP nanoparticles is 200-600 nm, indicating that the encapsulation of WEO has little effect on the particle size of the BP nanosheet.

[0091] 1.4 Preparation and characterization of multifunctional hydrogel (WEO@BP / GH hydrogel)

[0092] 5 mg of LAP was dissolved in 20 mL of PBS to prepare a LAP solution. 100 mg of methacrylated gelatin (GelMA) and 20 mg of hyaluronic acid methacrylate (HAMA) were dissolved in 1 mL of the LAP solution, and ultrasonic treatment was performed for 5 min until the solution was clear and transparent to obtain a GH solution. 1 mL of the above-mentioned solution containing composite nanoparticles and 1 mL of the GH solution were mixed and cross-linked and cured under 405 nm ultraviolet light for 7 min to prepare a WEO@BP / GH hydrogel.

[0093] 1 mL of the GH solution was cross-linked and cured under 405 nm UV light for 7 minutes to produce a GH hydrogel. 1 mL of the BP nanosheet solution was mixed with 1 mL of the GH solution and cross-linked and cured under 405 nm UV light for 7 minutes to produce a BP / GH hydrogel. The morphologies of the GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were observed, and the hydrogels were then freeze-dried to observe their microstructures.

[0094] The results show that Figure 3 As shown in Content A, WEO@BP / GH hydrogel is gray. Figure 3 As shown in Content B, GH, BP / GH and WEO@BP / GH hydrogels all exhibit porous three-dimensional interconnected architectures. The high-magnification (20 μm) SEM image shows that the internal surface of the GH hydrogel is smooth, while the internal surface of the WEO@BP / GH hydrogel exhibits a rough texture, which is attributed to the addition of WEO@BP nanoparticles (as indicated by the red arrow).

[0095] A series of WEO concentrations (0.04, 0.12, 0.20, 0.28, and 0.36 mg / mL) in ethanol were prepared and the absorbance at 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), indicating a strong linear correlation between the absorbance of WEO and its concentration at a wavelength of 203 nm. 1 mL of the above-mentioned solution containing composite nanoparticles was sonicated for 1 hour to destroy the nanoparticle walls. The supernatant was collected for absorbance measurement and the encapsulation efficiency of WEO in the WEO@BP / GH hydrogel was calculated. Encapsulation efficiency (%) = (M1 / M0) × 100%, where M0 is the total mass of WEO and M1 is the mass of WEO in the supernatant. The calculated encapsulation efficiency was 58.2%, demonstrating the excellent WEO loading capacity of the BP nanosheets.

[0096] 50 mg, 150 mg, 100 mg, 100 mg, 80 mg, and 120 mg of GelMA and 20 mg, 20 mg, 50 mg, 100 mg, 20 mg, and 20 mg of HAMA were dissolved in 1 mL of the above LAP solution and sonicated for 5 minutes until the solution became clear and transparent, resulting in 5G2H solutions, 15G2H solutions, 10G5H solutions, 10G10H solutions, 8G2H solutions, and 12G2H solutions. Subsequently, 1 mL of each of the above GH solutions was mixed with 1 mL of the above solution containing composite nanoparticles and cross-linked and cured under 405 nm UV light for 7 minutes to prepare hydrogels with different GelMA / HAMA ratios.

[0097] Results show that, as Figure 3 Content C shows that the pore size of 5G2H, 15G2H hydrogel is not uniform, and the three-dimensional interconnected architecture is unstable; the pore size of 10G5H, 10G10H hydrogel is small, which is not conducive to nutrient exchange and cell proliferation. The pore size and uniformity of 8G2H hydrogel and 12G2H hydrogel are the same as the above WEO@BP / GH hydrogel, which is conducive to nutrient exchange and cell proliferation.

[0098] Figure 3 Content D is the FTIR spectrum of WEO@BP / GH hydrogel, the dissociation vibration of carboxyl group (-COO - ) is at 1420 cm -1 , the vibration of methyl group (-CH2) is at 2923 cm -1 , which confirms the successful modification of carboxymethyl group in the hydrogel. In addition, the asymmetric deformation vibration related to the protonation of amine group (-NH3 + ) is at 1595 cm -1 , the stretching vibration of aromatic amine (-CN) is at 1321 cm -1 , which indicates that there are amino groups in the hydrogel. The stretching vibration of amide I (1645 cm -1 ) and amide II (1591 cm -1 ) indicates that amide bond (-NHCO - ) is formed inside the hydrogel after cross-linking and curing reaction. Figure 3 Content E is the Raman spectrum, it can be seen that the characteristic peaks of BP observed in WEO@BP / GH hydrogel are 354 cm - ¹, 433 cm - ¹ and 462 cm - ¹, which indicates that BP is successfully integrated into GH hydrogel.

[0099] 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 (denoted as W0) is weighed, then immersed in 37°C PBS for a period of time, removed, the excess liquid on the surface is removed and weighed (denoted as Wh), until the hydrogel reaches water absorption equilibrium, the water absorption ratio of the hydrogel is calculated, wherein the water absorption ratio (g / g) = (Wh-W0)⁄W0. Then the completely swollen hydrogel (weight denoted as Wp) is placed in a constant temperature 37°C fume hood to dry until its weight is stable (denoted as We), the water retention rate of the hydrogel is calculated, wherein the water retention rate (%) = (We / Wp) x 100%. And the rheological properties of GH hydrogel and WEO@BP / GH hydrogel with a diameter of 8 mm and a thickness of 2 mm are evaluated using a rheometer.

[0100] The results show that, as Figure 4 Content A, Figure 4 Content B shows that the WEO@BP / GH hydrogel has excellent moisture absorption and water retention, can effectively absorb wound exudates and maintain a moist wound environment, which is conducive to the water and action of cells.

[0101] As Figure 4 Content C shows that the viscosity of the WEO@BP / GH hydrogel remains relatively stable over time (shear rate: 10 rad / s, 37°C), indicating that the hydrogel has strong adhesion. From Figure 4 Content D, it can be seen that under ultraviolet light irradiation, the storage modulus (G') of the WEO@BP / GH hydrogel increases from 10 Pa to 1000 Pa, and the mechanical properties are significantly enhanced, which can maintain the structural integrity and prevent the penetration of external bacteria due to material failure. As Figure 4 Content E shows that the strain sweep experiment shows that the WEO@BP / GH hydrogel exhibits a gelation threshold at about 100% strain. As Figure 4 Content F shows that the dynamic frequency scanning results show that as the frequency increases from 0.1 to 10 Hz, the storage modulus (G') of the hydrogel is higher than the loss modulus (G''), indicating that the WEO@BP / GH hydrogel has excellent elasticity. Through dynamic amplitude experiments, 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 Figure 4 Content G shows that at a strain amplitude of 200%, G' is significantly lower than G'', indicating that fracture has occurred inside the hydrogel, and when the strain amplitude is reduced to 1%, G' increases significantly and is higher than G'', indicating the recovery of the state of the hydrogel, indicating that the WEO@BP / GH hydrogel has self-healing ability.

[0102] Example 2 Photo-thermal properties of multifunctional hydrogel loaded with Ai Oils

[0103] 2.1 Near-infrared photo-thermal properties of multifunctional hydrogel loaded with Ai Oils

[0104] The 1 mL of the above solution containing the composite nanoparticles and 1 mL of the above GH solution were mixed, 100 μL of the mixture was placed in a centrifuge tube, and near-infrared (NIR) laser irradiation was performed at a wavelength of 808 nm and power densities of 0.5 W / cm2, 1.0 W / cm2, and 2.0 W / cm2, respectively, and a FLIR infrared camera was used to monitor the temperature and take infrared images until the peak temperature was reached, so as to evaluate the thermal conversion efficiency of the mixture (i.e., the WEO@BP / GH hydrogel precursor solution). The mixture was irradiated with NIR laser at a wavelength of 808 nm and a power density of 1.0 W / cm2for 5 min, and then the laser irradiation was performed again after cooling for 10 min, for a total of 4 cycles, and the photothermal stability of the mixture was evaluated by the photothermal cycle test.

[0105] The results show that, as shown in Figure 5 Content A, Figure 5 Content B, the temperature of the mixture can reach 38.8°C, 55.6°C, and 69.4°C when the NIR laser irradiation is performed at power densities of 0.5 W / cm2, 1.0 W / cm2, and 2.0 W / cm2for 5 min. As shown in Figure 5 Content C, after 4 cycles of laser irradiation and cooling, the mixture maintains a consistent temperature curve, which indicates that the WEO@BP / GH hydrogel precursor solution prepared in the present application has excellent photothermal stability.

[0106] 2.2 Controllable release of multifunctional hydrogel loaded with WEO

[0107] The WEO@BP / GH hydrogel with a diameter of 8 mm and a thickness of 2 mm was placed in a centrifuge tube, and NIR irradiation was performed at a wavelength of 808 nm and a power density of 1 W / cm2for 5 min, and then the irradiation was performed again after cooling for 5 min, for a total of 4 cycles, and the release amount of WEO was detected by ultraviolet spectrophotometry, so as to evaluate the controllable release of WEO in the WEO@BP / GH hydrogel.

[0108] The results show that, as shown in Figure 6 Content A, the release amount of WEO in the NIR-irradiated hydrogel is significantly higher than that in the non-NIR-irradiated hydrogel. With the extension of the treatment time, the release amount of WEO in the NIR-irradiated hydrogel gradually increases, and when the treatment time is extended to 120 h, the release amount of WEO can reach 91.4%, which is significantly higher than that in the non-NIR-irradiated hydrogel (Content B), which indicates that the multifunctional hydrogel prepared in the present application has NIR-responsive release capability. Figure 6

[0109] Example 3 Anti-inflammatory performance of multifunctional hydrogel loaded with WEO

[0110] Human mononuclear cells (THP-1 cells) were seeded in a 96-well plate at a concentration of 1 × 10​5 THP-1 cells were seeded at a concentration of 1 x 105cells / well in 6-well plates in DMEM medium containing 10% fetal bovine serum and incubated at 37°C. Then, 50 ng / mL of phorbol 12-myristate 13-acetate (PMA) was added to induce THP-1 cells to polarize into M0 phenotype for 24 h. Then, 20 ng / mL of interferon gamma (IFNy) and 1 pg / mL of lipopolysaccharide (LPS) were added to polarize into M1 phenotype. Subsequently, 1 mL of PBS, GH hydrogel, BP / GH hydrogel, WEO@BP / GH hydrogel were added to 6-well plates for incubation for 48 h as PBS group, GH group, BP / GH group, WEO@BP / GH group, respectively; WEO@BP / GH hydrogel was added to 6-well plates for incubation for 48 h, and then irradiated with NIR at a wavelength of 808 nm and a power density of 1 W / cm2for 5 min as WEO@BP / GH+NIR group, wherein the preparation methods of the above hydrogels are the same as those in Example 1, and the diameter of the above hydrogels is 8 mm and the height is 2 mm. Then, the 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 h at a dilution ratio of 1:150. Subsequently, Cy3-labeled goat anti-rabbit IgG secondary antibody was incubated for 1 h in the dark at a dilution ratio of 1:200. Finally, the cell nucleus was 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 fast extraction kit, and then reverse transcription was performed using a Prime Script RT Master Mix kit and a PCR instrument. A PCR reaction mixture containing TB Green Premix Ex Taq II, forward primers, reverse primers, and cDNA was prepared, and then quantitative analysis was performed using a real-time PCR system. The primer information of the above various genes is shown in Table 1. GAPDH was used as a reference gene, and the relative mRNA expression level of the target gene was normalized to the control sample. The relative mRNA expression level of the target gene was quantified by measuring the fluorescence intensity in the reaction mixture.

[0111] Table 1 Primer information of genes IL-1β, CD86, IL-10, CD206

[0112]

[0113] The results show that, as Figure 7 Content A, Figure 7 Content B,Figure 7 As shown in Content C, compared with the 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), the WEO@BP / GH group was able to 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 CD86 mRNA and IL-1β mRNA, pro-inflammatory factors. This indicates that the WEO@BP / GH hydrogel can polarize macrophages from M1 to M2. In addition, NIR irradiation treatment of the WEO@BP / GH hydrogel further promoted the polarization of M1 macrophages to M2. This may be because when the 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, further enhancing the effectiveness of the hydrogel in polarizing M1 macrophages to M2.

[0114] Example 5 Antibacterial properties of multifunctional hydrogel loaded with Qi Ai essential oil

[0115] Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) were inoculated into LB liquid medium and cultured at 37°C for 24 h to obtain Staphylococcus aureus solution and Escherichia coli solution (bacterial concentration was 1×10 7 CFU / mL). GH hydrogel, BP / GH hydrogel, and 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 are all 8 mm and the height is all 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 the cells were 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 bacterial solution concentration 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 6The bacterial suspension was obtained, and then 100 μL of the bacterial suspension was coated on an agar plate, respectively, and cultured at 37°C for 12 h, and the colony formation was observed. The bacterial suspensions of different treatment groups were stained with a SYTO9 / PI kit for 20 min for live / dead cell staining to evaluate cell activity, wherein 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.

[0116] The results show 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 antibacterial performance in vitro. After the WEO@BP / GH hydrogel was irradiated with NIR for 5 min, the S. aureus and E. coli on the agar plate were completely removed, which indicates that NIR irradiation can further improve the antibacterial performance of the WEO@BP / GH hydrogel. Figure 8 Content A, Figure 8 Content C, Figure 8 Content D, it can be seen 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 antibacterial performance in vitro. After the WEO@BP / GH hydrogel was irradiated with NIR for 5 min, the S. aureus and E. coli on the agar plate were completely removed, which indicates that NIR irradiation can further improve the antibacterial performance of the WEO@BP / GH hydrogel.

[0117] As shown in Figure 8 Content B, Figure 8 Content E, Figure 8 Content F, the S. aureus and E. coli in the PBS group mainly presented green fluorescence (live bacteria) on the plate, and only a small amount of red fluorescence (dead bacteria) was present. After the WEO@BP / GH hydrogel was irradiated with NIR for 5 min, the S. aureus and E. coli on the plate presented the strongest red fluorescence (dead bacteria). In summary, both the plate counting test and the fluorescence experiment indicate that the WEO@BP / GH hydrogel has strong antibacterial activity.

[0118] Example 6 Antioxidant performance of multifunctional hydrogel loaded with Chinese celery essential oil

[0119] Human immortalized epidermal cells (HaCaT cells) were seeded at a density of 1×10 4The HaCaT cells were inoculated in the 96-well plate containing the HaCaT complete culture medium at a concentration of 1 x 105cells / well and incubated at 37°C for 24 h. Then, the culture medium was replaced with fresh culture medium containing 500 μM hydrogen peroxide (H2O2) as a control group, fresh culture medium containing 500 μM H2O2 and 200 μL PBS as a PBS group, fresh culture medium containing 500 μM H2O2 and GH hydrogel as a GH group, fresh culture medium containing 500 μM H2O2 and BP / GH hydrogel as a BP / GH group, and fresh culture medium containing 500 μM H2O2 and WEO@BP / GH hydrogel as a WEO@BP / GH group, and incubated at 37°C in the dark for 6 h. After the culture medium was replaced with fresh culture medium containing 500 μM H2O2 and WEO@BP / GH hydrogel and incubated at 37°C in the dark for 6 h, the NIR irradiation was performed at a power density of 1.0 W / cm2for 5 min as a WEO@BP / GH+NIR group (the preparation method of the above hydrogels is the same as in Example 1, and the diameter of the hydrogels was 8 mm and the thickness was 2 mm). The bacterial culture solution of each of the above treatment groups was collected, and Calcein-AM / PI kit was used to stain the bacterial culture solution of each of the different treatment groups for 20 min for live / dead cell staining, wherein the live cells were stained with Calcein-AM (green fluorescence) and the dead cells were stained with PI (red fluorescence) to evaluate the survival rate of the cells. DCFH-DA dye was used to fluorescently label the bacterial culture solution of each of the different treatment groups for 30 min to detect the level of reactive oxygen species (ROS) in the cells. 2 The results show that, compared with the control group, the PBS group, the GH group, and the BP / GH group, the WEO@BP / GH group and the WEO@BP / GH+NIR group have a higher cell survival rate and a lower ROS level.

[0120] The PBS, GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were sterilized by ultraviolet light and then soaked in complete DMEM culture medium for 3 days as a PBS group, a GH group, a BP / GH group, and a WEO@BP / GH group. The WEO@BP / GH hydrogel was sterilized by ultraviolet light and then soaked in complete DMEM culture medium for 3 days, and then was irradiated by NIR at a power density of 1 W / cm2for 5 min as a WEO@BP / GH+NIR group. The soaking solution of each of the treatment groups was sucked using a syringe. The HaCaT cells were inoculated in the 96-well plate containing the HaCaT complete culture medium at a concentration of 1 x 105cells / well and incubated at 37°C for 12 h until the cells were 70% confluent, and then the culture medium was replaced with 100 μL of the soaking solution of each of the above treatment groups. After incubation at 37°C for 12 h, 1000 μL of H2O2 was added and incubated at 37°C in the dark for 6 h, and then 10% CCK-8 reagent was added and incubated at 37°C for 4 h. The absorbance of each of the treatment groups at 450 nm was measured using a microplate reader to calculate the cell viability. 4 The results show that, compared with the control group, the PBS group, the GH group, and the BP / GH group, the WEO@BP / GH group and the WEO@BP / GH+NIR group have a higher cell survival rate and a lower ROS level.

[0121] The results show that, compared with the control group, the PBS group, the GH group, and the BP / GH group, the WEO@BP / GH group and the WEO@BP / GH+NIR group have a higher cell survival rate and a lower ROS level. Figure 9Content A can be seen that the amount of red fluorescence in the GH group and the BP / GH group is significantly higher than that in the WEO@BP / GH group and the WEO@BP / GH+NIR group, and the percentage of dead cells is high Figure 9 Content C). Similarly, the CCK-8 experiment 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 other groups Figure 9 Content D). In addition, the amount of fluorescence of the fluorescent probe DCFH in the WEO@BP / GH group and the WEO@BP / GH+NIR group was less than that in the GH group and the BP / GH group Figure 9 Content B), and the ROS signal was significantly lower than that in the GH group and the BP / GH group Figure 9 Content E). In summary, the WEO@BP / GH hydrogel has excellent antioxidant effect under near-infrared response, can effectively scavenge ROS, improve cell survival rate, and is beneficial to wound healing.

[0122] Example 7 Hemostatic performance of multifunctional hydrogel loaded with Chinese cypress essential oil

[0123] The lower third of the rat tail was cut off using a surgical scissors, and 15s later, the wound was soaked with sterile PBS as the PBS group; the GH hydrogel, the BP / GH hydrogel, and the WEO@BP / GH hydrogel were respectively covered on the wound as the GH group, the BP / GH group, and the WEO@BP / GH group; the WEO@BP / GH+NIR hydrogel was covered on the wound 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 method of each hydrogel was the same as that in Example 1, and the diameter of each hydrogel was 8mm and the thickness was 2mm); gauze was covered as the Gauze group, and the amount of bleeding of each treatment group was recorded to evaluate the hemostatic performance of the hydrogel through the tail hemostasis model.

[0124] The results showed that the amount of bleeding was small when different hydrogels and gauze were used, indicating that the above hydrogels could quickly stop bleeding and had small amount of bleeding. Figure 10 Content A, Figure 10 Content B can be seen that the amount of bleeding was small when different hydrogels and gauze were used, indicating that the above hydrogels could quickly stop bleeding and had small amount of bleeding.

[0125] Example 8 Pro-angiogenic performance of multifunctional hydrogel loaded with Chinese cypress essential oil

[0126] Scratch test: PBS, GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were sterilized by UV and then immersed in complete DMEM medium for 3 days, as the 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 DMEM medium for 3 days, and then irradiated with NIR at a power density of 1 W / cm² for 5 minutes as the WEO@BP / GH+NIR group. The immersion solution of each treatment group was aspirated with a syringe. HaCaT cells were cultured at a rate of 5×10 5 The cells were seeded at a concentration of 100 μg / 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. The cells were then mechanically disrupted using a 200 μL pipette tip (marked as 0 h). The cells were incubated at 37°C and 5% CO2 for 24 h. The cell morphology was observed under a microscope to evaluate the effect of the hydrogel extract on the migration ability of fibroblasts.

[0127] Tube formation assay: 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 a rate of 1×10 4 The cells were seeded at a concentration of 100 cells / well in a 96-well plate and incubated at 37°C for 12 h, after which the culture medium was removed and 4 mL of the above-mentioned soaking solution was added. The cells were incubated at 37°C and the branch points and tubular length of the newborn blood vessels were observed using an inverted fluorescence microscope after 2 h and 4 h of incubation.

[0128] The results show that Figure 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. Figure 11 Content B. Figure 11 Content C. Figure 11 As shown in Content D, after 2 h 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 h 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 disorders, improving fibroblast migration, and enhancing blood vessel production capacity, and had excellent biocompatibility.

[0129] Example 9 Effect of multifunctional hydrogel loaded with Qi Ai essential oil on diabetic wound healing

[0130] Male rats weighing 200-250 g were used to construct a type I diabetic rat model. Specifically, after fasting for 12 h, the rats were intraperitoneally injected with 1% streptozotocin (STZ, injection dose of 60 mg / kg, dose unit of drug protein content / rat weight), and 7 days later, the blood glucose level was measured from the tail vein of the rats. When the blood glucose level exceeded 16.7 mmol / L, a type I diabetic rat model was successfully constructed. After the diabetic rats were anesthetized, the dorsal hair was shaved, and an 8-mm incision was made on each side of the center line of the spine to create a skin wound. 200 μL of sterile PBS was soaked on each incised wound as the PBS group; GH hydrogel, BP / GH hydrogel, and WEO@BP / GH hydrogel were placed on the wounds as the GH group, the BP / GH group, and the WEO@BP / GH group, respectively; and WEO@BP / GH+NIR hydrogel was placed on the wound 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 methods of the hydrogels were the same as in Example 1, and the diameter and thickness of each hydrogel were 8 mm and 2 mm, respectively). The wound area was measured on days 0, 3, 7, 10, and 14 after placement in the different treatment groups, and the wound area ratio was evaluated. The wound area ratio = (An / A0) x 100%, where An is the wound area on day n after placement in the different treatment groups, and A0 is the wound area on day 0 after placement in the different treatment groups. On day 2 after placement, the wound exudates in the different treatment groups were cultured on agar plates, which were incubated at 37°C for 24 h, and the growth of bacterial colonies was observed. On day 14 after placement, the rats were sacrificed, 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's 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 generation of ROS in vivo was detected using a dihydroethidium (DHE) ROS detection kit.

[0131] The results showed that the number of bacterial colonies in the wound exudates of the WEO@BP / GH+NIR group was the least, and the bacterial survival rate was the lowest, which could significantly inhibit the growth of bacteria at the wound site. Figure 12 Content A, Figure 12 Content B It can be seen that the number of bacterial colonies in the wound exudates of the WEO@BP / GH+NIR group was the least, and the bacterial survival rate was the lowest, which could significantly inhibit the growth of bacteria at the wound site.

[0132] The results showed that the number of bacterial colonies in the wound exudates of the WEO@BP / GH+NIR group was the least, and the bacterial survival rate was the lowest, which could significantly inhibit the growth of bacteria at the wound site. Figure 13 It can be seen that the wound area of the WEO@BP / GH and WEO@BP / GH+NIR groups was significantly smaller than that of the other groups at the same placement time; on day 14 after placement, the wound area of the WEO@BP / GH+NIR group was only 5%, showing the best wound healing effect.

[0133] AsFigure 14 As shown in Content A (HE staining), at 14 days after operation, the wound of the WEO@BP / GH+NIR group was almost completely recovered, dense granulation tissue appeared, and the epidermis was completely regenerated, and the scar width was only 0.58±0.09 mm (P<0.01) (Content B (Masson staining) shows that at 14 days after operation, the collagen fibers in the WEO@BP / GH+NIR group were arranged in parallel around the hair follicles, and the collagen deposition was the highest (Content D), which was significantly higher than that in other groups. Figure 14 Figure 14 Figure 14

[0134] As shown in Content A, Figure 15 Content B, Figure 15 Content C, Figure 15 Content D, Figure 15 Content E, Content F, at 14 days after operation, the CD206 red fluorescence coverage area in the WEO@BP / GH group and the WEO@BP / GH+NIR group was significantly higher than that in other groups, and the CD86 green fluorescence coverage area was significantly less than that in other groups, which indicated that the WEO@BP / GH hydrogel could promote the polarization of M1 type macrophages to M2 type, and the NIR irradiation of the WEO@BP / GH hydrogel could further improve the polarization effect.

[0135] Figure 15 Content C, Figure 15 Content D, Content E,

[0136] Content F, at 14 days after operation, the expression amount of vascular endothelial growth factor (VEGF) in the WEO@BP / GH group and the WEO@BP / GH+NIR group was significantly higher than that in other groups, which indicated that the WEO@BP / GH hydrogel had the effect of promoting angiogenesis of new skin tissue, and the WEO@BP / GH+NIR treatment could further improve the effect of promoting angiogenesis and effectively promote neovascularization. Figure 15 Content G, Figure 16 Content H, at 14 days after operation, the ROS level in the WEO@BP / GH group and the WEO@BP / GH+NIR group was significantly lower than that in other groups, which indicated that the WEO@BP / GH hydrogel could effectively remove the active oxygen in the body, and the WEO@BP / GH+NIR treatment could further improve the removal effect of the active oxygen in the body.

[0137] Example 9 Biological safety of multifunctional hydrogel loaded with Chinese herb oil

[0138] 9.1 In vitro hemolysis

[0139] ​​The blood of diabetic rats was collected and centrifuged to obtain red blood cells, which were then washed with PBS three times to obtain eluted red blood cells. Then the red blood cells were placed in a test tube, 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, WEO@BP / GH hydrogel were added and incubated at 37°C for 4 h as the GH group, BP / GH group, WEO@BP / GH group; WEO@BP / GH hydrogel was added and incubated at 37°C for 4 h, and then irradiated with NIR radiation with a power density of 1.0 W / cm 2 for 5 min as the WEO@BP / GH+NIR group. The supernatant was collected by centrifugation at 2500 rpm for 5 min, and the absorbance of the supernatant was measured using a microplate reader to calculate the hemolysis rate, where the hemolysis rate (%) = (Ah-Ap) / (At-Ap) x 100%, Ah is the absorbance of the different hydrogel groups, Ap is the absorbance of the PBS group, and At is the absorbance of the Triton group.

[0140] The results show that, as shown in Content A, the supernatant of red blood cells in the positive group is bright red, and the supernatant of other treatment groups is colorless and transparent, with red blood cell sediment at the bottom; the hemolysis rate of other groups is less than 5% (Content B) except for the Triton group, which indicates that the above hydrogels have little effect on red blood cell hemolysis and do not cause hemolysis. Figure 16 Figure 17

[0141] 9.2 In vitro safety

[0142] PBS, GH hydrogel, BP / GH hydrogel and WEO@BP / GH hydrogel were sterilized by ultraviolet and then soaked in complete DMEM medium for 24 h as the PBS group, GH group, BP / GH group, WEO@BP / GH group. WEO@BP / GH hydrogel was sterilized by ultraviolet and then soaked in complete medium for 24 h, and then irradiated with NIR radiation with a power density of 1.0 W / cm 2 for 5 min as the WEO@BP / GH+NIR group (the preparation method of the above hydrogels is the same as in Example 1, and the diameter of the hydrogels is 8 mm and the thickness is 2 mm), and the soaking liquid of each treatment group was taken up using a syringe.

[0143] ​​Leaching test: HaCaT cells were seeded in 24-well plates for 12 h, then 500 μL of soaking solution of each treatment group was added, and incubated for 1 day, 3 days, 5 days. Live and dead cells were stained using calcein-AM / PI kit. Live cells were stained with calcein-AM (green fluorescence), and dead cells were stained with PI (red fluorescence), and observed using a Nikon C1 confocal microscope.

[0144] CCK-8 experiment: HaCaT cells were seeded 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 soaking solution of each treatment group, and incubated for 1 day, 3 days, 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 assess cell viability.

[0145] Results show that, as shown in Figure 17 Content A, Figure 17 Content B, the green fluorescence intensity of the hydrogel group is similar to that of the PBS group at the same incubation time, indicating that the hydrogel group does not affect cell proliferation. Figure 18 Content C, the cell viability of each treatment group is not significantly different at the same incubation time, indicating that different hydrogel groups have no significant toxicity to cells.

[0146] 9.3 In vivo safety

[0147] The heart, liver, spleen, lung and kidney of the diabetic rats at 14 days after operation in Example 8 were collected and subjected to HE staining, and blood was collected for blood biochemical analysis.

[0148] Results show that, as shown in Figure 19 each treatment group did not show significant pathological abnormalities or damage. Blood biochemical analysis showed that there was no significant difference in the content of HGB, ALT, AST, BUN, CRE, WBC, PLT, RBC between different hydrogel groups and the PBS group (p>0.05) (Content A~H). ​

[0149] In summary, the multifunctional hydrogel provided by the present application has high biological safety and good clinical application prospect.

[0150] Those skilled in the art will readily understand 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 replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. A multifunctional hydrogel loaded with Bupleurum falcatum essential oil, characterized by, It comprises a hydrogel matrix and composite nanoparticles compounded in the interior thereof; the composite nanoparticles comprise black phosphorus nanosheets and elecampane essential oil encapsulated in the interior thereof; In the composite nanoparticles, the ratio of black phosphorus nanosheets to elecampane essential oil is (20-40) mg:1 mL; The hydrogel matrix is crosslinked by methacrylated gelatin and hyaluronic acid methacrylate at a mass ratio of (4-6):

1.

2. A method for preparing the multifunctional hydrogel of claim 1, characterized by, It comprises the following steps: S1, mixing the black phosphorus nanosheet suspension and the elecampane essential oil, and then loading the black phosphorus nanosheets with the elecampane essential oil through extrusion circulation to prepare a solution containing composite nanoparticles; S2, mixing methacrylated gelatin, hyaluronic acid methacrylate and buffer to prepare a hydrogel solution; S3, mixing the hydrogel solution and the solution containing composite nanoparticles, and then crosslinking and curing under the condition of initiation by a photoinitiator or a light source to prepare a multifunctional hydrogel.

3. The preparation method according to claim 2, characterized in that In step S1, the concentration of the black phosphorus nanosheet suspension is 1-5 mg / mL; The volume ratio of the black phosphorus nanosheet suspension to the elecampane essential oil is (5-15):

1.

4. The production method according to claim 2, characterized by, In step S1, the number of extrusion circulations is 5-15.

5. The preparation method according to claim 2, characterized in that In step S2, in the hydrogel solution, the concentration of the methacrylated gelatin is 50-150 mg / mL; The mass ratio of the methacrylated gelatin to the hyaluronic acid methacrylate is (4-6):

1.

6. The preparation method according to claim 2, characterized in that In step S3, the crosslinking and curing time is 5-10 min; and / or, The photoinitiator is selected from one or more of phenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 1,2-diketone, Irgacure 2959, Alg-2959.

7. Use of the multifunctional hydrogel according to claim 1 in the preparation of an anti-inflammatory drug, characterized in that, The multifunctional hydrogel can polarize M1 type macrophages to M2 type.

8. Use of the multifunctional hydrogel of claim 1 in the preparation of a hydrogel adjuvant for promoting wound healing.

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