Preparation and use of gramicidin and its antibacterial hydrogel formulations

Abstract

The application provides a preparation of gramicidin and an antibacterial hydrogel preparation of the gramicidin, and discloses medical uses of the gramicidin, and provides a recombinant gramicidin, heterologous expression of the gramicidin is realized by constructing a Pichia pastoris recombinant strain, and the antibacterial activity of the gramicidin is retained, and the method has the advantages of simplicity and rapidness. Meanwhile, the hydrogel preparation of the gramicidin provided by the application shows good potential for treating wound infection of a diabetic mouse, and has the performance of rapid adhesion and hemostasis, lays a foundation for development of a new antibacterial drug, and provides a new direction for treatment of chronic inflammatory wounds and open bleeding.

Description

Technical Field

[0001] This invention provides a method for preparing genomicon and its antibacterial hydrogel formulation, and also discloses its medical uses, belonging to the field of biopharmaceutical technology. Background Technology

[0002] To promote wound healing, the medical community has developed various wound dressings, ranging from traditional materials such as medical gauze and bandages to the currently actively researched membranes, nanofibers, hydrogels, and microneedles. Research indicates that a moist environment is conducive to wound healing, and hydrogels have attracted significant attention due to their high porosity, drug delivery capabilities, and similarity to biological soft tissue. During wound healing, hydrogels can fill the wound surface, absorb wound exudate, provide a moist healing environment, and simultaneously load various bioactive substances, such as growth factors, cells, or antibacterial agents, enabling slow release. However, the large-scale preparation of hydrogels presents storage challenges. Researchers are continuously exploring new materials or chemically modifying existing materials to achieve rapid in-situ synthesis of hydrogels that eliminate the need for long-term storage and offer new functions, such as rapid closure, adhesion, hemostasis, reduced secondary damage to wounds caused by movement, and prevention of dressing detachment due to movement.

[0003] Due to the unique characteristics of diabetic wounds, the selection of hydrogel materials needs to consider many factors in addition to the requirements mentioned above. For example, they should possess the ability to accelerate cell migration, reduce inflammatory responses, promote macrophage polarization towards M2, reduce oxidative stress levels, and exhibit antibacterial activity. Materials with high biocompatibility, such as hyaluronic acid, gelatin, chitosan, and alginate, often have the ability to promote cell migration and are commonly used as hydrogel framework components, but their inherent antibacterial activity is limited. To prevent bacterial infection, hydrogels often carry various antibacterial substances, such as silver nanoparticles, titanium dioxide, quaternary ammonium salts, and antibiotics. However, some antibacterial agents exhibit cytotoxicity while maintaining antibacterial concentrations, and with the continuous emergence of drug-resistant bacteria, the available antibiotics are decreasing. Therefore, providing new, green, and natural antibacterial agents is the objective of this invention. Summary of the Invention

[0004] This invention provides a recombinant expressed gibberellin and its construction method, suitable for expression in Pichia pastoris, exhibiting broad-spectrum antibacterial activity and low susceptibility to drug resistance. It relates to the heterologous expression and production of gibberellin and the preparation of hydrogel formulations, providing new ideas for the development of novel antibacterial drugs and a new direction for the clinical treatment of diabetic comorbid infected wounds.

[0005] The recombinant expressed cytosine of the present invention is characterized in that its nucleotide sequence suitable for expression in Pichia pastoris is shown in SEQ ID NO. 1.

[0006] The Pichia pastoris strain of the present invention is characterized in that: the vector used is pPIC9K and the starting strain is Pichia pastoris GS115.

[0007] The present invention discloses a method for preparing recombinant expressed gibberellin, characterized by comprising the following steps:

[0008] 1) The nucleotide sequence of Jileicin was linked to the Pichia pastoris vector pPIC9K to construct the methanol-inducible recombinant vector pPIC9K-Jileicin. The recombinant vector was transformed into host cells, and the recombinant strain GS115-Jileicin was obtained by stepwise screening in YPD solid medium containing 0.2-1 mg / mL G418.

[0009] 2) Ferment the GS115-Jileicin described in step 1) to induce the expression of recombinant jileicin. The induction conditions include: the induction medium is BMMY medium, the methanol addition is 1-2%, and the induction time is 72-96 h.

[0010] 3) The fermentation supernatant obtained in step 2) was concentrated by 5KD nanofiltration and then purified by nickel column to obtain recombinant genomiconin.

[0011] The application of the jejunin described in this invention in the preparation of hydrogel formulations for treating bacterial wound infections in diabetic patients.

[0012] The application of the genomiconin described in this invention in the preparation of hemostatic hydrogel formulations.

[0013] The hydrogel formulation made from genomiconin according to the present invention is characterized in that:

[0014] The product includes a hydrogel matrix, a styraxin loaded in the hydrogel, and a photoinitiator I2959; wherein the hydrogel matrix consists of 10%-20% GelMA and 5%-10% OHA by mass, the photoinitiator I2959 by mass is 0.2%-0.5%, and the styraxin by mass is 5-10 mg.

[0015] The method for preparing a genomicon hydrogel according to the present invention is characterized by comprising the following steps:

[0016] 1) Weigh out 0.25% I2959 and dissolve it in phosphate buffer. After stirring and dissolving, add 5mg of genomiconine and dissolve it completely. Then add 20% GelMA and stir and dissolve at 37℃ to obtain the first solution.

[0017] 2) Add 10% OHA by mass to the first solution and stir at room temperature to dissolve it to obtain the second solution;

[0018] 3) Irradiate the second solution with ultraviolet light of wavelength 274nm until it solidifies to obtain the hydrogel.

[0019] The recombinant gibberellin of this invention exhibits varying degrees of antibacterial activity against indicator strains: methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. Through recombinant expression, the gibberellin expression level in the fermentation broth supernatant reaches 35 mg / mL, making gibberellin production simple and quick.

[0020] The recombinant jejunin of the present invention exhibits low toxicity and low hemolytic activity against mouse macrophage cell lines, fibroblast cell lines and mouse erythrocytes.

[0021] The present invention provides the application of recombinant genomiconin in the preparation of antibacterial hydrogel formulations. The hydrogel has good adhesion and hemostatic properties, which is beneficial for drug delivery to the wound site. It also has antibacterial activities, such as accelerating wound cell migration and promoting macrophage polarization to the M2 phenotype. It can reduce the inflammatory response in diabetic wounds and promote the repair of diabetic wounds.

[0022] The positive effects of this invention are as follows: It provides a recombinant jejunosin, achieving heterologous expression of jejunosin by constructing a recombinant Pichia pastoris strain while retaining its antibacterial activity. This method is simple and rapid. Furthermore, the hydrogel formulation of jejunosin provided by this invention shows good potential for treating wound infections in diabetic mice and exhibits rapid adhesion and hemostatic properties, laying the foundation for the development of novel antibacterial drugs and providing a new direction for the treatment of chronic inflammatory wounds and open bleeding. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the construction of recombinant expression plasmids;

[0024] Figure 2 This is a schematic diagram of the step-by-step screening of antibiotics;

[0025] Figure 3 These are SDS-PAGE verification patterns of different cytosine-expressing strains;

[0026] Figure 4 This is the BCA kit test standard curve;

[0027] Figure 5 It is a structural characterization of the components of the hydrogel;

[0028] Figure 6 It is a characterization of the basic physicochemical properties of hydrogels;

[0029] Figure 7 This is a characterization of the basic physicochemical properties of hydrogels loaded with gibberellins;

[0030] Figure 8 The effect of hydrogels on fibroblast migration;

[0031] Figure 9 The effect of gibberellin-loaded hydrogel on macrophage polarization;

[0032] Figure 10 The therapeutic effect of a beta-lactamase-loaded hydrogel formulation on wound infection in diabetic mice;

[0033] Figure 11 It refers to the in vitro and in vivo hemostatic ability of the gel loaded with apigenin hydrogel. Detailed Implementation

[0034] The present invention will now be illustrated with examples; however, the present invention is not limited to the examples described below. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products. All raw and auxiliary materials selected in the present invention, as well as the selected bacterial culture methods, are well known in the art, and all percentages mentioned in the present invention are by weight and volume percentages.

[0035] Example 1

[0036] like Figure 1 As shown, the constructed Jileicin expression cassette consists of three parts: the original Jileicin signal peptide gene and a 6×His tag. This gene has been optimized according to the codon preferences of Pichia pastoris, and its nucleotide sequence is shown in SEQ ID NO.1. The plasmid vector used is the commercially available pPIC9K, whose promoter is P... AOX1 It also possesses an α-factor signal peptide. The Jilicin expression cassette was then ligated into a plasmid vector to obtain the recombinant plasmid pPIC9K-Jileicin.

[0037] Pichia pastoris strain GS115 was selected as the expression host for the expression of jileicin. The recombinant plasmid pPIC9K-Jileicin was transformed into strain GS115. Figure 2 As shown, the recombinant strain GS115-Jileicin was obtained after antibiotic screening.

[0038] Example 2

[0039] The strain obtained in Example 1 was inoculated into round-bottom test tubes containing 2 mL of YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured overnight. 1 mL of the overnight culture was then inoculated into 50 mL of fermentation medium for shake-flask fermentation at 30°C and 220 rpm until OD... 600Cells were centrifuged at 3500 g for 10 minutes at 6.4℃, the supernatant was discarded, and the cells were washed twice with sterile water. After each wash, the cells were centrifuged under the same conditions, and the supernatant was collected. Cells were resuspended in BMMY induction medium and induced to express expression at 30℃ and 180 rpm, with methanol added every 24 hours to a final concentration of 1%. After 3 days of methanol induction culture, the cells were centrifuged and the supernatant was collected. The supernatant was concentrated through a 5KD nanofiltration membrane and purified using a nickel column. SDS-PAGE was used to verify the expression yield, and the results are shown below. Figure 3 As shown, the concentration of the single-band elution buffer of apigenin was determined using the BCA kit. Standards were serially diluted to concentrations of 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL, and measured together with the samples. The final absorbance values ​​are shown in Table 1, and regression curves were plotted as follows. Figure 4 The fitting equation was y = 7.427x - 0.0543, where y is the concentration of the antimicrobial peptide (mg / mL) and x is the absorbance value. The active clones and their expression levels are shown in Table 1.

[0040] Table 1

[0041]

[0042] Example 3

[0043] The preparation method of GelMA / OHA hydrogel in this embodiment includes the following steps:

[0044] 1) Preparation of GelMA

[0045] First, dissolve 5g of gelatin in PBS buffer at 50℃ and stir thoroughly until completely dissolved. Add 2.5mL of methacrylic anhydride dropwise to the solution and maintain vigorous stirring at 50℃ for 3 hours. Then, add 200mL of distilled water to terminate the reaction. Dialyze the reaction solution through a 3.5KD dialysis bag in distilled water for 5 days. Freeze-dry the product collected from the dialysis bag to obtain GelMA. The obtained GelMA is then processed... 1 Identification was performed using ¹H NMR and FTIR; see attached table for details. Figure 5 AB. FTIR results show that GelMA at 1720 cm⁻¹ -1 The weak absorption peaks at 973 and 939 cm⁻¹ may represent the stretching vibration of the ester C=O, while the peaks at 973 and 939 cm⁻¹... -1 The absorption peak at that point is due to the out-of-plane bending vibration (OHA) of the terminal olefin CH=CH2. This is related to the preparation of grafted GelMA. 1 1H NMR spectroscopy showed that the amino signal essentially disappeared at 2.9 ppm, and the unsaturated =CH of the methacrylic acid structural unit appeared between 5.5 and 5.3 ppm, indicating that the gelatin successfully grafted rate-polymerizable methacrylic acid groups.

[0046] 2) Preparation of OHA

[0047] Dissolve 1 g of hyaluronic acid in 100 mL of distilled water and stir at room temperature until fully dissolved. Add 0.5 g of potassium permanganate to the solution and continue stirring in the dark for 6 hours. Then add 1 mL of ethylene glycol and continue stirring for 1 hour to terminate the reaction. Dialyze the above reaction solution through a 3.5 KD dialysis bag in distilled water for 4 days. The product collected from the dialysis bag is freeze-dried to obtain OHA. The obtained OHA is then processed through... 1 Identification was performed using ¹H NMR and FTIR; see attached table for details. Figure 5 A, 5C. FTIR results showed that the most significant change after hyaluronic acid oxidation was observed at 1729 cm⁻¹. -1 An absorption peak for the C=O stretching vibration of aldehydes appeared at the location. 1 The H NMR results also showed that the CH and CH2 chemical shift signals of the sugar unit were significantly weakened between 3.9 and 3.0 ppm, while the isomerized O-CH-O appeared at 4.4 and 4.3 ppm. The typical characteristic signals of the aldehyde proton and hemiacetal proton appeared between 5.1 and 4.9 ppm, indicating that the oxidized hyaluronic acid was successfully prepared.

[0048] 3) Preparation of hydrogels

[0049] 2.5 mg of photoinitiator I2959 was dissolved thoroughly in 1 mL of PBS buffer. GelMA (10% and 20% by mass) was added to this solution and dissolved in a 37°C water bath; this is the first solution. OHA (5% and 10% by mass) was added to the first solution and slowly dissolved, then vortexed to mix; this is the second solution. After preparing hydrogel precursor solutions with different proportions, crosslinking was performed using a 274 nm UV light source for 30 s to prepare GelMA / OHA hydrogels, named G20OHA10. A visual representation of the prepared hydrogels is attached. Figure 5 D.

[0050] Example 4:

[0051] This embodiment involves the detection of the physicochemical properties of hydrogels prepared in different ratios as described in Example 3, including the following tests:

[0052] 1) Determination of swelling rate

[0053] The swelling properties of hydrogels are evaluated by measuring the weight change. Hydrogels of similar weight are prepared and their weight is recorded as W0. The hydrogels are immersed in PBS buffer, and the buffer is removed at fixed intervals. After blotting off the liquid from the hydrogels with filter paper, the weight W is measured. t Calculate the swelling ratio of the hydrogel using the following formula:

[0054] Swelling ratio = (Wt -W0) / W0×100%;

[0055] See appendix for results. Figure 6 A. All hydrogels exhibited rapid swelling after 2 hours and reached equilibrium after approximately 24 hours. G10 had the highest swelling rate (38.17 ± 3.20%), while G20OHA10 had the lowest swelling rate (17.30 ± 2.10%) due to its high network density and cross-linking degree.

[0056] 2) Degradation performance

[0057] Prepare a physiological concentration of collagenase in PBS (5 U / mL). Immerse the weighed hydrogel in the solution. At fixed time intervals, remove the buffer solution, blot the hydrogel with filter paper, and weigh it. Calculate the hydrogel residue using the following formula:

[0058] Residual ratio=W t / W0×100%;

[0059] See appendix for results. Figure 6 B. Each hydrogel underwent enzymatic degradation for 6-8 days, with G20 having the longest degradation time (>168h) and G20OHA5 having the shortest degradation time (144h).

[0060] 3) Shear adhesion strength of hydrogel

[0061] Two pieces of glass (7.5 cm long, 2.5 cm wide) were coated with a 20% (w / v) gelatin solution and air-dried at room temperature. Then, 100 μL of the pre-hydrogel solution was pipetted and photocrosslinked between the two glass pieces. The bonding area was 2.5 cm × 1 cm. The shear bond strength was measured using a universal tensile testing machine at a speed of 3 mm / min, with the shear bond strength measured at the separation point. See the appendix for results. Figure 6 C, among which G20OHA10 has the highest tensile strength (176.45±9.64kPa).

[0062] Example 5

[0063] Based on the characterization results of the hydrogel in Example 4, the low-swelling, high-adhesion hydrogel is more suitable for wound adhesion and hemostasis. A GelMA mass ratio of 20% and an OHA mass ratio of 10% were selected for the subsequent preparation of the hydrogel formulation. Following the hydrogel preparation method in Example 3, 5 mg of gibberellin was added to the first solution, shaken to dissolve, and then a second solution was prepared. A gibberellin-loaded hydrogel was prepared by irradiation with 274 nm ultraviolet light and named G20OHA10-JC. Its characterization methods mainly included the following tests:

[0064] 1) Rheological testing

[0065] In the frequency sweep test, G' and G'' were recorded at frequencies ranging from 0.1 to 100 rad / s, with a shear strain of 1%. In the shear strain sweep, the applied average frequency was constant at 10 rad / s, and the strain amplitude was set to 0-1000%. Results are attached. Figure 7 As the angular frequency gradually increases, each sample exhibits gel-like behavior, i.e., G'>G''. At a fixed frequency, the critical strain crossover points are 135.4% and 798.2%, respectively.

[0066] 2) Scanning electron micrograph (SEM) of the hydrogel

[0067] The freeze-dried hydrogels G20OHA10 and G20OHA10-JC were cut into blocks, sputter-coated with gold, and their microstructure was observed using scanning electron microscopy under accelerating voltage conditions. The results are shown in the appendix. Figure 7 C.

[0068] 3) In vitro release of gibberellins

[0069] Under light-protected conditions at room temperature, fluorescein isothiocyanate (FITC) was used to label jiileicin in 0.1 M carbonate buffer (pH=9) for two hours, with a FITC:protein ratio of 4:1. The FITC-jiileicin solution was dialyzed in 0.01 M PBS for 24 hours at 37°C using a dialysis bag with a molecular weight cutoff of 3.5 kDa, followed by dialyzing with deionized water for 2 hours. The dialysate was freeze-dried and concentrated, then serially diluted twofold with PBS. A standard curve was established from 2 mg / mL to 0.625 mg / mL by measuring the fluorescence intensity of the solution, with an excitation wavelength of 490 nm and an emission wavelength of 525 nm. 5 mg of FITC-jiileicin was encapsulated in a hydrogel, immersed in 2 mL of PBS, and incubated at 37°C in the dark. 100 μL of the solution was taken at intervals to measure the fluorescence intensity, and the released jiileicin was calculated using the standard curve. Results are attached. Figure 7 D. Due to the participation of cross-linking and the dense pore size of the hydrogel, the release time of genomicon is prolonged, reaching the maximum release amount (71.75±1.35%) after 96 hours.

[0070] 4) Antibacterial activity of G20OHA10-JC hydrogel

[0071] Inoculate with methicillin-resistant Staphylococcus aureus until the logarithmic growth phase, at a dose of 1×10⁻⁶. 5 / hole Inoculate into 96-well plates, add hydrogel, gibberellin, and hydrogel containing gibberellin, incubate at 37°C for 6 hours, then dilute and spread onto TSB plates for overnight incubation, and count colonies. Results are attached. Figure 7E. Hydrogels loaded with apigenin exhibit good antibacterial activity.

[0072] Example 6

[0073] 1) Cytotoxicity detection

[0074] The hydrogel was sterilized by UV irradiation and immersion in 75% alcohol, and then incubated in DMEM medium for 5 days to obtain the extract. A density of 5 × 10⁻⁶ was used. 4 100 μL of mouse fibroblast L929 cells / mL were seeded per well in a 96-well cell culture plate and incubated for 12 h in a 5% CO2 incubator at 37°C. Then, gilisin and its hydrogel extract were added, and the plate was incubated for another 24 h in a 5% CO2 incubator at 37°C. Finally, 10 μL of CCK-8 reagent was added to each well, mixed, and incubated for 1 h in the dark. Cell viability was calculated by measuring the absorbance at 450 nm in each well using a microplate reader. See attached figure for detailed results. Figure 8 Hydrogels have good cell compatibility.

[0075] 2) The effect of hydrogels on cell migration

[0076] The effect of hydrogels on L929 cell migration was determined using the cell scratch assay. L929 cells were diluted with DMEM medium at a concentration of 5 × 10⁻⁶. 5 The cells were seeded into each well of a 6-well plate and cultured for 12 hours. The culture medium was then removed, and scratches of equal width were made on the cell surface. Cell debris was washed away with PBS, and then hydrogel extract was added for further culture. The migration rate was calculated based on the migration area. See attached table for detailed results. Figure 8 Hydrogels have the ability to accelerate cell migration.

[0077] Example 7

[0078] To assess the effect of hydrogels on macrophage polarization, 3×10 5 RAW264.7 cells were seeded into each well of a 6-well plate and cultured for 2 hours after hydrogel extraction. Then, 100 ng LPS was added to each well, and the plates were incubated at 37°C for 24 hours. Total RNA was extracted from macrophages using Trizol, and quantitative real-time PCR analysis was performed using the Step One Plus Real-Time PCR system. β-actin was used as an internal control gene to detect the transcriptional levels of macrophage polarization markers M1 (CD86, IL-6, iNOS) and M2 (Arg-1, IL-10, CD206) phenotypic markers. Detailed results can be found in [link to relevant documentation]. Figure 9The hydrogel G20OHA10 treatment group was able to reverse the effects of LPS. Compared with the LPS control group, G20OHA10 significantly increased the mRNA levels of Arg-1, CD206 and IL-10, and decreased the mRNA levels of IL-6 and CD86. This indicates that the hydrogel can promote macrophage polarization to M2 type and accelerate the transition from inflammation to anti-inflammatory and healing.

[0079] Example 8

[0080] Sixty male C57BL / 6 mice (5 weeks old) were purchased and fed a high-sugar, high-fat diet for 30 days before the experiment. After fasting for 12 hours, the mice were intraperitoneally injected with streptozotocin (STZ) (50 mg / kg), dissolved in sterile citrate buffer (0.05 mol / L sodium citrate, pH 4.5, 50 mg / kg) for 5 consecutive days. Blood glucose was monitored after 2 weeks, and mice with blood glucose levels exceeding 16.7 mM were considered diabetic. After anesthetizing the diabetic mice with sodium pentobarbital, the back hair of the mice was completely removed with depilatory cream, and then a full-thickness wound with a diameter of 8 mm was created using a punch. Drug-resistant Staphylococcus aureus (1×10⁻⁶) was dripped into the wound. 8 Mice were randomly divided into 5 groups (n=12) after 24 hours (CFU / mL, 10 μL): wounds were treated with equal volumes of PBS, betulin, and betulin hydrogel (treatment group 200 μL: betulin content 1 mg / mL), hydrogel alone (200 μL), and baicalein dressing. Wound healing was documented by photographs on days 3, 7, and 15. The appearance of all wounds was photographed (see attached image). Figure 10 A. Assess the degree of wound closure based on the size of the remaining wound on the skin; see attached document for detailed results. Figure 10 B. From the appendix Figure 10 Results showed that a large number of bacteria were still present in the mouse wounds on the third day, and treatment with the genomicon hydrogel formulation could effectively inhibit the proliferation of bacteria in the wound infection.

[0081] Example 9

[0082] 1) In vitro hemolytic activity

[0083] Fresh blood was collected from mice using anticoagulant tubes and centrifuged at 1000g for 10 min. Red blood cells were collected as anticoagulated whole blood. 20 μL of red blood cells were collected from each of the PBS group, hydrogel group, gibberellin group, and 2% Triton 100X group. The red blood cells were resuspended in 980 μL of the corresponding solution, incubated at 37℃ for 1 h, and then centrifuged. The supernatant was then measured for absorbance at 542 nm. (See attached image). Figure 11 A. The hydrogel and genomiconin have good biocompatibility.

[0084] 2) External hemostatic properties

[0085] Fresh mouse blood was collected using anticoagulant tubes and centrifuged at 1000g for 10 min to collect red blood cells as anticoagulated whole blood. The hydrogel was spread evenly in a 24-well plate, photocrosslinked to form a 2 mm sheet, and preheated at 37°C for 10 min. 10 μL of anticoagulated whole blood was evenly dropped onto the hydrogel surface, and 1 μL of CaCl2 solution (0.2 M) was added. The plate was then incubated at 37°C for 5 min. Samples were taken every 1 min, and 2 mL of deionized water was added and gently mixed. The absorbance was measured at 550 nm. The absorbance of 10 μL of calcified whole blood in 2 mL of deionized water was used as a reference value, and the experiment was repeated three times.

[0086] See appendix for results. Figure 11 B. The BCI of both the hydrogel group and the fibrin glue group was significantly lower than that of the untreated group. After 2 minutes of incubation, the BCI of the G20OHA10-JC group was significantly lower than that of the fibrin glue group, which indicates that the G20OHA10-JC hydrogel has a faster hemostatic ability.

[0087] 3) In vitro platelet adhesion properties

[0088] Platelet-rich plasma (PRP) was obtained by centrifuging anticoagulated whole blood at 1000g for 10 min. Platelets were diluted with PBS and counted under a microscope. Platelets serially diluted twofold were lysed using 1% Triton X-100. LDH release was detected using an LDH detection kit at 450 nm absorbance. A standard curve of platelet count versus absorbance was plotted. Hydrogels and fibrin glue were fixed onto 48-well plates, and 50 µL of PRP was added to the surface and incubated at 37°C. After 30 min and 60 min, the hydrogels were removed, and unadhered platelets were carefully washed away with PBS. Platelet lysis and LDH release were detected as described above, with empty culture dishes containing PRP serving as controls. Results are shown in Figure 11C. After 30 min of incubation, the number of platelets attached to the G20OHA10-JC hydrogel was 2.38 × 10⁻⁶. 5 ±1.1×10 4 The value was significantly higher than that of fibrin glue (1.68×10). 5 ±8×10 3 After 60 minutes of incubation, both G20OHA10-JC hydrogel and fibrin glue were able to adhere to a large number of platelets, with negligible differences. This indicates that G20OHA10-JC hydrogel can accelerate hemostasis by promoting platelet adhesion and erythrocyte aggregation.

[0089] 4) Hemostatic properties of G20OHA10-JC hydrogel in a mouse liver hemorrhage model

[0090] Mice were anesthetized with sodium pentobarbital. The liver was exposed through an abdominal incision, and the surrounding serous fluid was removed. The liver was carefully pulled out and placed on weighing filter paper. The liver was then punctured with a 20G needle, and 100 μL of hydrogel was immediately applied and cured under UV light for 5 seconds. Fibrin glue was used as a positive control, and the weight of the blood absorbed on the filter paper was measured. Results are attached. Figure 11 D. Although there was no significant difference, the experimental results using fibrin glue as a hemostasis control group showed greater bleeding than the G20OHA10-JC group. Because G20OHA10-JC hydrogel has strong tissue adhesion and coagulation-promoting properties, coupled with its adjustable curing ability, it can rapidly coagulate blood on the liver surface and seal bleeding points within 5 seconds.

[0091] In summary, the above embodiments are merely descriptions of preferred implementations of this experiment and are not intended to limit the scope of this experiment. Without departing from the spirit of this experiment design, all modifications and improvements made by those skilled in the art to the technical solutions of this experiment should fall within the protection scope determined by this experiment.

Claims

1. A hydrogel formulation made from bezoar, characterized in that: It consists of a hydrogel matrix, a styraxin loaded in the hydrogel, and a photoinitiator I2959; wherein the hydrogel matrix is ​​composed of 10%-20% GelMA and 5%-10% OHA by mass, the photoinitiator I2959 by mass is 0.2%-0.5%, and the styraxin by mass is 5-10 mg. The optimized nucleotide sequence of the above-mentioned cytokinin codon is shown in SEQ ID NO.

1.

2. The method for preparing a hydrogel formulation made from genomiconin according to claim 1, characterized in that, Includes the following steps: 1) Weigh out 0.25% I2959 and dissolve it in phosphate buffer. After stirring and dissolving, add 5mg of genomiconine and dissolve it completely. Then add 20% GelMA and stir and dissolve at 37℃ to obtain the first solution. 2) Add 10% OHA by mass to the first solution and stir at room temperature to dissolve it to obtain the second solution; 3) Irradiate the second solution with ultraviolet light with a wavelength of 274nm and wait for it to solidify to obtain the hydrogel.

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