Spreadable graphene oxide hybrid soybean protein hydrogel and preparation method and application thereof

Abstract

The present invention discloses a spreadable graphene oxide hybrid soybean protein hydrogel and a preparation method and application thereof. The present invention extracts SPI from soybean protein isolate by means of gallic acid under alkaline conditions; simultaneously grafts SPI-GA with phenol and thiol (SH) to form SPI-GA-SH; dissolves dopamine in a graphene oxide solution to react and obtain a PGO stock solution; finally, SPI-SH-GA and PGO are self-assembled into a spreadable biogel in a glycerol / water binary solvent system. Cytological studies have shown that SPI-SH-GA@PGO is non-toxic to HaCaT cells and has antibacterial properties. It also promotes cell migration, angiogenesis and inhibits cell apoptosis. The spreadable biogel prepared by the present invention forms a protective film against radiation dermatitis during radiotherapy, and can be used as a skin radiation protector to improve radiation protection performance.

Description

Technical Field

[0001] The invention relates to the technical field of biogels, and in particular to spreadable graphene oxide hybrid soybean protein hydrogel and a preparation method and application thereof. Background Art

[0002] Radiotherapy is one of the effective interventions to inhibit tumors. However, radiotherapy usually uses strong energy beyond the tolerance of normal tissues, which will inevitably cause undesirable radiobiological damage to normal tissues during radiation. The strong energy indirectly produces excessive reactive oxygen species (ROS) by reacting with water, which further damage the epidermal basal cells, which play a key role in maintaining the normal metabolism and regeneration function of skin tissue. According to the damage mechanism of radiation dermatitis, there is an urgent need for a free radical scavenger as an effective skin radioprotector to remove the excessive ROS generated by X-rays, thereby preventing the development of radiation dermatitis.

[0003] IGF-I and hyaluronic acid mitigate the negative effects of radiation on human skin keratinocytes. MKI67 and IL6 gene expression in irradiated keratinocytes was significantly increased after H-HA / L-HA treatment. The stimulatory effects of H-HA / L-HA and IGF-I were able to be confirmed in 3D culture. Cell viability, migration and gene expression were positively affected after treatment with HL-HA and IGF-I. Trehalose protects keratinocytes from UV radiation by regulating TIMP3 and ATG9A to activate autophagy. In summary, there are various forms of nanomaterials that can be used to treat radiation-induced skin damage, especially nanocarrier-loaded gene drug therapy. Subcutaneous administration, percutaneous absorption and skin penetration in topical preparations are recommended first, which is the fastest way for drugs to reach skin tissue. In terms of social significance, radiation-induced skin damage is the most common radiation-induced disease because the skin covers the entire body and has the largest area among all organs in the body. When external irradiation is used for tumor radiotherapy, the irradiation first passes through the skin and then reaches the tumor area. Therefore, the rational design and development of nanomedicines for the treatment of radiation skin diseases has important practical significance.

[0004] Studies have found soft hydrogels with tissue-like mechanical properties and epidermal hydration effects as alternatives to wound dressings. Among them, based on the in situ gelation process, injectable hydrogels can be directly applied to completely cover the damaged surface. Chemically cross-linked hydrogels form Michael addition thiol-ene reactions or Schiff base aminoaldehyde reactions. However, a double-valve applicator must be installed to separate the reaction components before they are mixed and extruded through a syringe. Such pre-gel solutions also require a certain amount of time to fully cure, which will burden the duration of the operation and the risk of infection. In addition, some chemical hydrogels require a UV source for curing, reducing radiation safety. In contrast, physical hydrogels are converted from liquid form to gel without involving related chemical reactions or UV irradiation. Despite this, physical hydrogels still need to wait for time to cure before use. Soy protein is one of the most abundant proteins in natural plants and has good biocompatibility, biodegradability, and extremely low immunogenicity. To date, many SPI-based materials, such as gels, films, and adhesives, have been reported. However, most SPI-based adhesive materials require a hot pressing process to obtain strong adhesion, which is not suitable for fragile skin. Therefore, there is a need for a spreadable biogel that can be applied directly to the skin to form a protective film to resist radiation dermatitis during radiotherapy. Summary of the invention

[0005] In view of the above prior art, the object of the present invention is to provide a spreadable graphene oxide hybrid soy protein hydrogel and its preparation method and application. The present invention uses cost-effective and naturally derived soy protein as the main matrix and polydopamine-reduced graphene oxide (PGO) nanosheets as a radiation protection component to develop a biogel that can be directly applied to the skin and form a protective film against radiation dermatitis during radiotherapy. The hydrogel is used as a skin radiation protector to improve the radiation protection performance.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a method for preparing a spreadable graphene oxide hybrid soy protein hydrogel is provided, comprising the following steps:

[0008] (1) Preparation of gallic acid grafted thiolated soy protein isolate SPI-SH-GA: Soy protein isolate was dispersed in deionized water at room temperature, and then gallic acid was added. After mixing evenly, NaOH and urea were added and stirred to form a SPI-GA solution. EDTA was added to the SPI-GA solution, and then Traut reagent (2-iminothiolane hydrochloride) solution was added dropwise and stirred at room temperature to obtain uniform SPI-SH-GA. The solution obtained after SPI-SH-GA was dialyzed was freeze-dried to obtain SPI-SH-GA powder.

[0009] (2) Preparation of polydopamine-reduced graphene oxide PGO: dopamine was dissolved in graphene oxide solution at room temperature, NaOH was added under stirring to react, and after the reaction was completed, centrifugation was performed to obtain a PGO stock solution;

[0010] (3) Preparation of SPI-PGO biogel: The PGO stock solution obtained in step (2) was diluted in deionized water, glycerol was added and mixed evenly to obtain a mixed solution, and then the SPI-SH-GA powder obtained in step (1) was added to the mixed solution and stirred to obtain the SPI-PGO biogel.

[0011] Preferably, in step (1), the ratio of the added amounts of the soy egg isolate, deionized water, gallic acid, NaOH, urea, EDTA, and Traut's reagent solution is 100 g: 1000 mL: 2 g: 10 g: 10 g: 1.6 g: 10 mL;

[0012] The concentration of the Traut's reagent solution is 20 mg / mL.

[0013] Preferably, in step (1), the stirring time is 30 min; EDTA is added to the SPI-GA solution by stirring, and the stirring time is 10 min.

[0014] Preferably, in step (1), the dialysis is performed in deionized water for 3 days, the molecular weight cut-off of the dialysis is 14000, and the deionized water is renewed every 12 hours.

[0015] Preferably, in step (2), the ratio of the added amounts of dopamine, graphene oxide solution and NaOH is 1 g: 250 mL: 3.75 g;

[0016] The concentration of the graphene oxide solution is 10 mg / mL.

[0017] Preferably, in step (2), the stirring time is 3 hours; and the centrifugation is performed at 10000 g for 5 minutes at room temperature.

[0018] Preferably, in step (3), the volumes of the PGO stock solution, deionized water and glycerol are 1:8:1; and the concentration of the PGO stock solution is 100 mg / mL.

[0019] Preferably, in step (3), the ratio of the added amount of the PGO stock solution to the SPI-SH-GA powder is 1 mL:2 g, and the stirring time is 12 h.

[0020] The second aspect of the present invention provides a spreadable graphene oxide hybrid soy protein hydrogel obtained by the above preparation method.

[0021] The third aspect of the present invention provides a spreadable graphene oxide hybrid soy protein hydrogel for use in at least one of the following 1) to 2):

[0022] 1) Prepared as skin radiation protectant;

[0023] 2) Prepare RISI wound repair products.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention has developed a spreadable biogel that can be directly applied on the skin and forms a protective film against radiation dermatitis during radiotherapy. The present invention uses cost-effective and naturally derived soy protein as the main matrix and polydopamine-reduced graphene oxide (PGO) nanosheets as the radiation protection component to obtain a biogel that can be directly applied on the skin.

[0026] (2) The spreadable biogel of the present invention has comprehensive advantages, including conformal adhesion, good mechanical compliance of the skin in contact with the biogel, and the radiation protection and antioxidant capacity of the PGO nanosheets, making it promising as a skin dressing for skin radiation protection. The spreadable biogel of the present invention can be used as a skin radiation protectant both in vitro and in vivo to improve radiation protection performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :Preparation process of SPI-SH-GA@PGO and its application in RISI wound healing;

[0028] Figure 2: Characterization of SPI-SH-GA@PGO biogels; (A) Photos showing fluid-state and solid-state biogels with different SPI-SH-GA concentrations, (B) Rheological characterization of biogels with different formulations under frequency sweep from 0 to 70 Hz: (1) to (3) are SPI rheological characterization, SPI-SH-GA rheological characterization, SPI-SH-GA@PGO rheological characterization, (C) SPI-SH-GA@PGO biogels are applied through a needle and fixed on any irregular surface by convenient topical application, (D) Photos of biogels directly applied on the skin after 5 minutes and the film-forming properties of the biogels on the skin, showing their ability to be applied on the skin and good adhesion to the skin, (E) Adhesion strength of SPI-SH-GA hydrogel and SPI-SH-GA@PGO hydrogel, (F) SPI-SH-GA@PGO gel coated on PVC film, (G) SEM images of SPI-GA-SH hydrogel: (1)~(2) are SEM images of SPI-SH-GA and SEM images of SPI-SH-GA@PGO, respectively. (H) AFM images of SPI-GA-SH@PGO biogel: (1)~(2) are AFM images of SPI-SH-GA and AFM images of SPI-SH-GA@PGO, respectively. (I) The photo shows the flexibility of SPI-SH-GA@PGO biogel: (1)~(2) are handheld SPI-SH-GA@PGO biogel and tweezer-held SPI-SH-GA@PGO biogel, respectively. (J) The elongation of the biogel film is about 45%. (K) The average tensile strength of the biogel is 0.25 MPa.

[0029] Figure 3 : Biocompatibility, angiogenesis and cell migration activity of SPI-SH-GA@PGO; (A) FITC / DAPI staining images of SPI-SH-GA@PGO and HaCaT cells co-cultured for 7 days, (B) in vitro angiogenesis assay, (C) scratch migration of HaCaT cells after co-culture with SPI-SH-GA@PGO, (D) CCK-8 assay of SPI-SH-GA@PGO and HaCaT cells co-cultured for 7 days, (E) number of blood vessels in in vitro angiogenesis assay, (F) percentage of migration area of ​​scratch migration, (G) relative expression of ROS in each group, *P<0.05;

[0030] Figure 4: Effects of SPI-SH-GA@PGO on the expression, apoptosis and antibacterial activity of cell NLRP3; (A) WB expression of NLRP3, (B) semi-quantitative analysis, (C) flow cytometry, (D) antibacterial activity of SPI-SH-GA@PGO against G+ (MRSA) and G- (Escherichia coli) bacteria, (E) total apoptosis rate results by flow cytometry, (F) bacterial counts of Escherichia coli, (G) bacterial counts of Staphylococcus aureus, *P<0.05;

[0031] Figure 5 : SPI-SH-GA@PGO in RISI mouse model, (A) Wound images on day 1 and day 14, HE and NLRP3 staining on day 14, red arrows indicate positive expression, (B): (1) Western blot of Wnt3α and β-catenin, (2) relative WB expression of Wnt3α and β-catenin on day 14, (C) wound area on day 14, (D) overall recovery time, (E) relative number of hair follicles on day 14, (F) relative expression of NLRP3 IHC on day 14 *P<0.05. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] As introduced in the background technology section, many SPI-based materials, such as gels, films, and adhesives, have been reported so far. However, most SPI-based adhesive materials require a hot press process to obtain strong adhesion, which is not suitable for fragile skin.

[0034] Based on this, the purpose of the present invention is to provide a spreadable graphene oxide hybrid soy protein hydrogel and its preparation method and application. Radiation induced skin injury (RISI) is a common complication of radiotherapy. The main influencing factors of RISI wound healing include ROS and NLRP3 inflammasome activation, wound infection, etc. The present invention uses SPI-based biogel as a RISI healing material for the first time. The present invention prepares a biomaterial with multiple radioresistance strategies. Figure 1As shown in the figure, the present invention synthesizes SPI-SH-GA in two steps: extracting SPI from soy protein isolate with the help of gallic acid under alkaline conditions; and simultaneously grafting SPI-GA with phenol and thiol (SH) to form SPI-GA-SH. In this process, NaOH / urea destroys the intermolecular and intramolecular hydrogen bonds between the entangled soy protein chains to form soluble soy protein chains. At the same time, gallic acid with catechol groups interacts with the functional groups on the soluble soy protein chains and is grafted on the SPI chains, which prevents the entanglement of SPI acid chains. This is crucial for the subsequent formation of biogels. Then SPI-GA is modified by Traut's reagent, which can react with the groups contained in SPI to simultaneously obtain phenol and thiol group-grafted SPI (SPI-GA-SH). Finally, SPI-SH-GA@PGO self-assembles the biogel into a sticky biogel in a binary solvent system (glycerol / water). The self-assembly of SPI-GA-SH is driven by hydrogen bonds between SPI-GA-SH chains and glycerol, as well as SH-SH and -SH-phenol coordination cross-linking mechanisms. At the same time, glycerol forms more stable hydrogen bonds with water molecules, providing a phase-locking effect in the biogel, thereby ensuring the long-term water retention of the biogel. The incorporation of PGO nanosheets introduces more hydrogen bonds to stabilize the biogel. Cytological studies have shown that SPI-SH-GA@PGO is nontoxic to HaCaT cells and has antibacterial properties. It also promotes cell migration, angiogenesis and inhibits cell apoptosis.

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0036] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0037] Among them: soy protein isolate (SPI) was purchased from Sigma-Aldrich, USA;

[0038] Urea and NaOH were purchased from Chengdu Kelong Reagent Co., Ltd.;

[0039] GO was purchased from RuiXi Materials Tech Co. (Xi’an, China);

[0040] Dopamine and gallic acid (GA) were purchased from Sigma-Aldrich Co. (Shanghai, China);

[0041] The percentages in the test examples are by mass unless otherwise specified. The dilution ratios in the test examples are by volume unless otherwise specified.

[0042] Example:

[0043] (1) Synthesis of gallic acid grafted thiolated soy protein isolate (SPI-SH-GA)

[0044] 100 g of soy protein isolate (SPI) was dispersed in 1000 mL of deionized (DI) water at room temperature. 2 g of gallic acid (GA) was added to the SPI dispersion. After mixing for 20 min, 10 g of NaOH and 10 g of urea were added to the mixture and stirred for 30 min to form a gallic acid-grafted soy protein isolate (SPI-GA) solution. 1.6 g of EDTA was added to the SPI-GA solution by stirring for 10 min, and then 10 mL of Traut reagent solution (20 mg / mL) was added dropwise and stirred at room temperature for 30 min to obtain a uniform SPI-SH-GA solution. Then, the solution was transferred to a dialysis bag (molecular weight cutoff 14000) and dialyzed in deionized water for 3 days. The deionized water was refreshed every 12 h. Finally, the solution was lyophilized to obtain purified SPI-SH-GA.

[0045] (2) Preparation of polydopamine-reduced graphene oxide (PGO)

[0046] GO was dispersed in deionized water to obtain a dispersion with a concentration of 10 mg / mL, and 1 g of DA was dissolved in the dispersion at room temperature to obtain a mixed solution. Then, 3.75 g of NaOH was added to the mixed solution under stirring within 3 hours to react. After the reaction, the solution was centrifuged at 10000 g for 5 min at room temperature to obtain a PGO stock solution.

[0047] (3) Preparation of SPI-PGO biogel

[0048] 1 mL of PGO stock solution (100 mg / mL) was diluted in 8 mL of deionized water, and then glycerol (1 mL) was added and mixed well. Then 2 g of SPI-SH-GA powder was added and stirred for 12 h to obtain a spreadable graphene oxide hybrid soy protein hydrogel.

[0049] Comparative Example:

[0050] The SPI-GA-SH prepared in step (1) of the example was dissolved in a glycerol / water binary solvent at a concentration higher than 20 wt % to obtain a SPI-GA-SH hydrogel film.

[0051] Test Example 1: Characterization

[0052] Fourier transform infrared (FTIR) spectroscopy analysis: The SPI-GA-SH hydrogel films prepared in the comparative example and the SPI-GA-SH@PGO prepared in the example were measured using FTIR spectroscopy (Nicolet 6700 FTIR spectrometer) at room temperature. All spectra were recorded between 500 and 4000 cm -1 .

[0053] Rheological testing of biogels: The rheological properties of biogels were performed on a rheometer (MCR301, Anton Paar, Austria) equipped with cone-plate geometry. During the rheological measurements, the gap was kept at 47 μm and the temperature was kept at 25 °C. To avoid water evaporation, the sample stage was filled with dimethylsiloxane. A strain amplitude sweep (0.01–1000%) was first performed to determine the linear viscoelastic region. A dynamic frequency sweep was then performed at an angular velocity from 0.1 to 75 Hz at a strain amplitude of 1.0%.

[0054] Adhesion behavior of biogels: Adhesion strength measurements of biogels (SPI-SH-GA, SPI-SH-GA@PGO) were performed using a universal mechanical testing machine (INSTRON 5567, INSTRON, USA). Porcine skin, hydrophobic polytetrafluoroethylene (PTFE) and rectangular (5 cm × 3 cm) glass slides were used as test substrates. 200 μl of gel was placed between a pair of identical substrates with an overlap area of ​​2 cm × 2 cm. It was fixed between the two clamps of a universal testing machine and then tested under a load of 10 N. The crosshead speed was 2 mm / min. The test was completed when the biogel was separated from both sides of the substrate. Five samples in each group (n = 5) were used for the adhesion test. The adhesion strength (As) was calculated by the following formula:

[0055] AS = F / A; where F is the maximum load of bonding and A is the overlapping area of ​​the samples.

[0056] Morphology of biogel-based films: To observe the morphology of biogels (SPI-SH-GA, SPI-SH-GA@PGO), the biogels were uniformly coated on transparent polyvinyl chloride (PVC) plates (0.4 mm thick) by a film scraper (1000 μm) and allowed to air-dry at room temperature for 7 days to obtain dry films.

[0057] The surface roughness of the membranes was analyzed by atomic force microscopy (AFM Bruker Multimode 8). AFM images were acquired in tapping mode. The scan area was 5 × 5 μm 2 , the test resolution is 512×512.

[0058] Mechanical properties of biogels: The tensile strength and elongation at break of different biogel films were tested using a universal testing machine (INSTRON 5567). The biogel films were cut into stripes (70 mm long and 15 mm wide) and fixed on the fixture of the device with a gap of 40 mm. The biogel films were then stretched to failure at a speed of 50 mm / min. Five samples were prepared for each mechanical test for repeated tests (i.e., repeated 5 times, the same below). The elastic modulus was determined by the initial slope of the stress-strain curve in the elastic region.

[0059] Water content of biogel films: The water content of the hydrogel films was determined by gravimetric analysis. Different biogel films were weighed and then dried in an oven at 105°C to constant weight. The test was repeated three times. The water content of the hydrogel film was calculated according to the following formula: Wc (%) = (M0-M1) / M0×100%; where M0 and M1 are the weights of the film before and after drying (g), respectively.

[0060] Results and analysis:

[0061] The viscoelastic properties of the biogels were investigated by rheological tests. Figure 2 B). Frequency-dependent oscillatory sweeps show that when the concentration of SPI-SH-GA is above 20%, the storage modulus (G') dominates over the loss modulus (G") over the entire angular frequency range, and the biogel combined with PGO maintains a solid-like behavior over the entire frequency range. Figure 2 As shown in Figure C, the gel was continuously injected into the culture dish through a 1 mL syringe with a 0.5 mm needle to form a filamentous strip. In addition, the form of the gel can be easily adjusted by replacing the needle and mold ( Figure 2 C). The entire preparation process of the biogel does not involve any complicated chemical reactions and toxic agents, which enables the biogel to be directly applied on the skin surface ( Figure 2 D). The biogel was also easily and evenly spread on the hand skin and spontaneously transformed into a thin film that was conformally and stably adhered to the skin even during movement, indicating the feasibility of rapid film formation and surgical application. The adhesion properties of the biogel were qualitatively evaluated by using porcine skin as a representative tissue during adhesion-tensile tests. The adhesion strength of the biogel SPI-SH-GA@PGO gel was 18 kPa, and the SPI-SH-GA gel was significantly higher than that of the SPI-SH-GA@PGO biogel ( Figure 2 E).

[0062] The SPI-SH-GA@PGO biogel also showed universal adhesion to hydrophobic, hydrophilic, and metal substrates. The interfacial adhesion of the biogel is beneficial for its good conformability to the skin in practical environments. The film-forming property of the biogel was further demonstrated by coating on a transparent polyvinyl chloride (PVC) substrate (thickness 0.4 mm) using a film scraper (1000 μm). It was air-dried at room temperature for 7 days to obtain a dry film ( Figure 2 F). The surface morphology of the films (SPI-SH-GA and SPI-SH-GA@PGO) was observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM images showed that all the films exhibited porous and interconnected structures ( Figure 2 G). The incorporation of PGO nanosheets resulted in a more compact structure in the SPI-SH-GA film. Soybean globulin, β subunit, AFM images showed that the overall colors of the two films were relatively close, indicating that the two films were fairly uniform ( Figure 2 H). However, the SPI-GA-SH@PGO gel film is thinner than the SPI-GA-SH gel film, indicating that the addition of PGO enhances the ductility of the film ( Figure 2 I). In addition, the arithmetic mean deviation Ra (roughness) of SPI-GA-SH and SPI-GA-SH@PGO gel films are 50.6 nm and 31.9 nm, respectively, which proves that the surface of SPI-GA-SH@PGO gel film is smoother than that of SPI-GA-SH.

[0063] The SPI-GA-SH@PGO gel film also has mechanical strength and high stretchability, which is beneficial for its practical application in conformal integration with soft and curved skin surfaces. Figure 2 As shown in Figure 1, the biogel film coated on the PVC substrate is flexible to withstand external bending. After peeling off from the PVC substrate, the gel film is also independent. A tensile test was used to detect the stretchability of the gel film. The elongation of the gel film was about 45%, and the average tensile strength was 0.25 MPa ( Figure 2 J and 2K), which are lower than those of soft skin tissue (0.1 to 2 MPa), thus facilitating its mechanical compatibility with soft skin. In addition, the improvement of mechanical compatibility can reduce the shear stress generated between the skin and the biogel film.

[0064] Experimental Example 2: In vitro cytological study

[0065] Biocompatibility evaluation and cell morphology observation: The cytological experiment was divided into 5 groups, group A was a normal cell group, group B was a normal cell + 3Gy radiotherapy group, group C was a normal cell + 3Gy radiotherapy + SPI group, group D was a normal cell + 3Gy radiotherapy group + SPI-SH-GA group, and group E was a normal cell + 3Gy radiotherapy + SPI-SH-GA@PGO group. CCK-8 assay was used to evaluate cytotoxicity. HaCat cells were cultured at 2×10 3 The cells were seeded at a density of 10 cells / well into groups AE and incubated at 37°C in DMEM supplemented with 5% fetal bovine serum and 1% penicillin / streptomycin.

[0066] After 1, 4, and 7 days of incubation, cell viability was quantified using the CCK-8 assay. To investigate the effects of cytoskeletal morphology, HaCat cells were seeded on the AE group culture medium for 24 h, and then the cells were washed three times. They were washed with preheated phosphate-buffered saline and fixed with preheated 4% paraformaldehyde at room temperature. The fixed cells were rinsed with phosphate buffer and soaked. The cells were then incubated with phalloidin at 37 °C without light and DAPI (2-(4-amidinophenyl)-6-indolecarboxamidine dihydrochloride) for 30 min and 5 min. Fluorescence images of the stained cells were obtained using a confocal laser scanning microscope (780, Zeiss, Germany). Then, HaCat cells were stained with DAPI and phalloidin, followed by fluorescence observation.

[0067] Scratch migration: HaCaT cells were grown to confluence in complete medium and seeded in 24-well plates (2 × 10 4 Monolayers were crossed (0 h) with a pipette tip and a Zeiss video microscope for examination at 24 h. Individual experiments were repeated five times per group, and specific measurements were performed using ImageJ 1.48v software (NIH, USA), with experiments repeated three times.

[0068] In vitro formation test: Mix 2×10 4 HaCaT cells were seeded on Matrigel-coated 96-well plates and then added to the AE group. After 24 h of stimulation, the newly formed tubes were photographed using an inverted phase microscope (Olympus, USA). Each group was repeated five times. Each experiment was repeated three times.

[0069] Flow cytometry: Apoptosis assay was performed using flow cytometry assay and Annexin V-FITC / PI apoptosis detection kit (Dojindo Molecular Technologies, Japan). Cells were collected and suspended in 200 μl binding buffer containing 5 μl Annexin V-FITC and 5 μl PI. Cells were analyzed using a fluorescence activated cell sorter (FACS) (Beckman Coulter, USA) using Cell Quest software.

[0070] Cell colony formation assay: The method was the same as biocompatibility assessment and cell morphology observation. Each well was stained with 1 ml of 0.005% crystal violet for 1 hour, and the number of colonies was calculated using Image-Pro Plus 6.0 after imaging.

[0071] Bacterial co-culture: MRSA (methicillin-resistant Staphylococcus aureus) and Escherichia coli (E. coli) were obtained from the Army Medical University. The bacteria were amplified (overnight growth) to 1 × 10 9 Colony forming units (CFU) / mL, and then diluted to 1×10 4 CFU / mL. The same amount of bacterial liquid was inoculated into the coated plate, and after incubation at 37°C for 24 hours, the change in bacterial count was measured.

[0072] Statistical analysis: All tables and statistical data were drawn using GraphPad Prism 9.0 software. Each experiment was repeated three times, and all data are expressed as mean ± standard error. The two groups were compared using Student's t test, and differences were considered statistically significant when P < 0.05.

[0073] Results and Analysis

[0074] Biocompatibility, angiogenesis and cell migration activity of SPI-SH-GA@PGO:

[0075] like Figure 3 As shown in A, HaCaT cells and SPI-SH-GA@PGO were co-cultured for 7 days. FITC / DAPI staining showed no significant difference in cell morphology; the nucleus and cytoplasm were intact. CCK-8 assay showed that on the 4th day, the OD value of HaCaT cells in groups B and E decreased after 3Gy irradiation, indicating that radiation significantly inhibited cell growth ( Figure 3 D). On the 7th day, the OD value of group E recovered significantly and was significantly higher than that of group B (P < 0.05), suggesting that SPI-SH-GA@PGO may not affect the growth of irradiated cells in the short term (1–4 days), while the growth curve of irradiated cells returned to normal in the long term (>5 days).

[0076] like Figure 3 As shown in B+3E, after co-culturing endothelial cells with different groups of materials, the matrix gel ascending angiogenesis experiment was performed for about 24 hours. Group A had the largest number of normal cells and the longest blood vessels. Group B had significantly restricted blood vessel formation after 3Gy irradiation, and the SPI-SH-GA precursor material in group D partially restored blood vessel formation (P<0.05). The best angiogenesis was in group E (P<0.05).

[0077] like Figure 3As shown in C+3F, HaCaT cells were co-cultured with each group of materials for about 24 hours, and then the cell scratch test was performed. The migration rate of group B was significantly reduced at 24 hours (P<0.05), which may be related to the effect of radiation on cell migration. The migration inhibition effect of group E was significantly improved, indicating that the substance can increase the migration rate of irradiated cells (P<0.05). The cell migration rate of group E was the same as that of group D (P>0.05), indicating that SPI-SH-GA expression promoted the migration of HaCaT cells.

[0078] exist Figure 3 G, it can be seen that irradiation can significantly increase ROS expression in Hacat cells, while SPI-SH-GA can effectively eliminate ROS in group D. The results of free radical scavenging activity and reducing power assays showed that GA significantly improved the antioxidant activity of SPI. Specifically, compared with unmodified SPI, the DPPH radical scavenging rate and ABTS radical scavenging ability increased by nearly 5 times and 1.5 times, respectively. In addition, the reducing power was more than 3 times compared with the SPI control.

[0079] Cellular NLRP3 expression, apoptosis and antibacterial activity of SPI-SH-GA@PGO:

[0080] NLRP3 expression in western blot analysis Figure 4 A+4B. The expression of NLRP3 in group E was significantly lower than that in group B (P<0.05) and group D (P<0.05), indicating that the substance can enter the cytoplasm of HaCaT cells through endocytosis, thereby playing a relevant role. Radiation significantly increased the apoptosis rate ( Figure 4 C+4E). Compared with group B, the apoptosis rate of group E was lower (P<0.05), and the effect of group E was higher than that of group D. This indicates that SPI-SH-GA@PGO significantly reduces the apoptosis rate of cells, and the increase of PGO in group E plays an important role in regulating the apoptosis rate of cells. Cytological studies have shown that SPI-SH-GA@PGO is nontoxic to HaCaT cells and has antibacterial properties. It promotes migration and angiogenesis and inhibits apoptosis.

[0081] Antibacterial effect such as Figure 4 As shown in D, F and G, based on the growth of MRSA (G+) and E. coli (G-), SPI-SH-GA could not effectively inhibit the growth of bacteria (p>0.05), but SPI-SH-GA@PGO showed antibacterial activity (p<0.05).

[0082] Test Example 3: In vivo cytological test

[0083] RISI mouse model: About 0.2 ml of pentobarbital was injected intraperitoneally to anesthetize each mouse. Then a linear accelerator emitting 6 Mev electron rays was used (irradiation field 1 cm × 1 cm; one irradiation; 30 Gy; 10 min; dose 300 cGy / min). In this case, the source skin was kept at a distance of 1 meter, and the lead plate was used to shield the remaining skin. After irradiation of each group, the irradiated area was still coated with biogel at 48-hour intervals, with a total duration of 7-14 days, and divided into five groups, each containing five mice. The five groups were AE group: normal mouse group (Group A), normal mouse + 30gy irradiation (Group B), normal mouse + 30gy irradiation + SPI (Group C), normal mouse + 30gy irradiation + SPI-SH-GA (Group D), normal mouse + 30gy irradiation + SPI-SH-GA@PGO (Group E).

[0084] To determine recovery activity, a comparative assessment of wound area before / after recovery was performed using the IPP6.0® as follows:

[0085] Wound recovery = (initial wound area - wound area after fixed recovery time) / initial wound area × 100%. The Douglas and Fowler method was then used to calculate the RISI score.

[0086] Hematoxylin-eosin (HE) staining and histology-based analysis: Wound samples from each mouse were collected 14 days after trauma to prepare paraffin sections, which were then stained with HE before obtaining high-quality images for dataset results.

[0087] Western blotting and immunohistochemical staining for NLRP3 expression: Two weeks after injury, intact defect samples (10 mm × 10 mm) were collected to combine the neoepidermis with the granulation tissue before rapid freezing using liquid nitrogen according to the kit protocol (VarioskanFlash®; Thermo Scientific). Then diluted primary antibodies (1:100) of wnt3+ / β-catenin (Biuss), anti-β-actin (Fineasy, China), and anti-NLRP3 (Biuss, China) were used, and diluted (1:2000) goat anti-rabbit conjugated with goat horseradish peroxidase (Zhongshan Biotechnology™, China) were used as secondary antibodies. The polyvinylidene difluoride membrane was finally collected and retained.

[0088] Effects of SPI-SH-GA@PGO on RISI mouse model:

[0089] like Figure 5As shown in A, all mice were shaved, photographed, and treated on day 1. On day 14, the mice were photographed again, and local skin tissues were finally collected for HE staining. Compared with group B, wound recovery in groups D and E was enhanced (p<0.05), and compared with group D, group E was significantly improved (p<0.05, Figure 5 C and 5D). The results showed that SPI-SH-GA@PGO enhanced the migration and proliferation activities of epidermal cells to induce wound recovery.

[0090] During wound repair, the level and standard of angiogenesis have a great impact on the level of wound recovery. H&E staining results showed that the hair follicles of mice in each group were almost the same ( Figure 5 A and 5E), since radiation reduces hair follicle density, this reduction can lead to wound formation. SPI-SH-GA@PGO greatly enhanced hair follicle density and wound recovery. Group E has SPI and PGO, indicating a strong enhancement effect (p<0.05). SPI-SH-GA@PGO gel achieved wound recovery without scars, regenerating thickened dermis and several embedded hair follicles within two weeks, showing enhanced full-thickness wound recovery and scar surround properties. This shows that graphene oxide has the effect of promoting the repair of damaged hair follicles.

[0091] The upregulation of NLRP3 inflammasome and its initiation play a key role in RISI. SPI-SH-GA@PGO downregulated ROS, and thus IFI6 protein significantly downregulated NLRP3 inflammasome. Therefore, by regulating the targets associated with NLRP3 inflammasome, new prevention and treatment options can be provided for combating RISI, as well as the study of the mechanism of biogel promoting wound healing.

[0092] Mechanisms of biogel promoting wound recovery Figure 5 As shown in A and 5F, NLRP3 was significantly upregulated after 30gy radiation. Nevertheless, SPI-SH-GA@PGO (group E) downregulated this gene. From the perspective of the classical pathway, Wnt3α / β-catenin protein significantly downregulated NLRP3 inflammasome. Qualitative and semi-quantitative analysis of the proteomic expression of the Wnt3α / β-catenin junction pathway is shown in Figure 5 B. In addition, stress after irradiation activates the Wnt3α / β-catenin repair mechanism, and Wnt3a enhances cell migration activity. The mouse model shows that Wnt3a can promote wound recovery. SPI-SH-GA@PGO (group E) can further enhance the expression of the pathway in the wound tissue (p<0.05), improve and promote the Wnt3α / β-catenin repair mechanism. This indicates that SPI-SH-GA@PGO may promote RISI wound recovery through the Wnt3α / β-catenin-related pathway.

[0093] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a spreadable graphene oxide hybrid soybean protein hydrogel, characterized in that: The following steps are involved: (1) Preparation of gallic acid grafted thiolated soy protein isolate SPI-SH-GA: Soy protein isolate was dispersed in deionized water at room temperature, and then gallic acid was added. After mixing evenly, NaOH and urea were added and stirred to form a SPI-GA solution. EDTA was added to the SPI-GA solution, and then Traut's reagent solution was added dropwise and stirred at room temperature to obtain uniform SPI-SH-GA. The solution obtained after SPI-SH-GA was dialyzed was freeze-dried to obtain SPI-SH-GA powder. (2) Preparation of polydopamine-reduced graphene oxide PGO: dopamine was dissolved in graphene oxide solution at room temperature, NaOH was added under stirring to react, and after the reaction was completed, centrifugation was performed to obtain a PGO stock solution; (3) Preparation of SPI-PGO biogel: The PGO stock solution obtained in step (2) was diluted in deionized water, glycerol was added and mixed evenly to obtain a mixed solution, and then the SPI-SH-GA powder obtained in step (1) was added to the mixed solution and stirred to obtain the SPI-PGO biogel.

2. The preparation method according to claim 1, characterized in that: In step (1), the ratio of the added amounts of the soy protein isolate, deionized water, gallic acid, NaOH, urea, EDTA, and Traut's reagent solution is 100 g: 1000 mL: 2 g: 10 g: 10 g: 1.6 g: 10 mL; The concentration of the Traut's reagent solution is 20 mg / mL.

3. The preparation method according to claim 1, characterized in that: In step (1), the stirring time is 30 minutes; 1.6 g EDTA is added to the SPI-GA solution by stirring for 10 minutes.

4. The preparation method according to claim 1, characterized in that: In step (1), the dialysis is performed in deionized water for 3 days, the molecular weight cut-off is 14000, and the deionized water is renewed every 12 hours.

5. The preparation method according to claim 1, characterized in that: In step (2), the ratio of the added amounts of dopamine, graphene oxide solution and NaOH is 1 g: 250 mL: 3.75 g; The concentration of the graphene oxide solution is 10 mg / mL.

6. The preparation method according to claim 1, characterized in that: In step (2), the stirring time is 3 hours; and the centrifugation is performed at 10000 g for 5 minutes at room temperature.

7. The preparation method according to claim 1, characterized in that: In step (3), the volumes of the PGO stock solution, deionized water and glycerol are 1:8:1; and the concentration of the PGO stock solution is 100 mg / mL.

8. The preparation method according to claim 1, characterized in that: In step (3), the ratio of the added amount of the PGO stock solution to the SPI-SH-GA powder is 1 mL: 2 g; and the stirring time is 12 h.

9. Spreadable graphene oxide hybrid soybean protein hydrogel obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the spreadable graphene oxide hybrid soy protein hydrogel according to claim 9 in at least one of the following 1) to 2): 1) Prepared as skin radiation protectant; 2) Prepare drugs for repairing radiation-induced skin injury (RISI) wounds.

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