Nano-copper reinforced puffball spore composite biomacromolecule hydrogel with wound repairing function as well as preparation method and application of nano-copper reinforced puffball spore composite biomacromolecule hydrogel

Abstract

The invention belongs to the field of material engineering, and particularly relates to nano-copper reinforced puffball spore composite biomacromolecule hydrogel with a wound repairing function as well as a preparation method and application of the nano-copper reinforced puffball spore composite biomacromolecule hydrogel. Traditional Chinese medicines including puffball, polymer carbon nitride loaded nano-copper and biomass macromolecular hydrogel are combined to develop the functional hydrogel capable of resisting bacteria, stopping bleeding and promoting wound repair, so that the synergistic effect of a nano-material and the traditional Chinese medicines is exerted, and the defect that pure hydrogel lacks antibacterial and antioxidant capabilities is overcome; finally, the composite biomass-based hydrogel tissue engineering dressing which has multiple functions of quickly stopping bleeding, promoting revascularization, repairing wounds and the like and is good in biocompatibility is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of material engineering, and particularly relates to a nano-copper reinforced malleus spore composite biomacromolecule hydrogel with wound repair function and a preparation method and application thereof. BACKGROUND

[0002] Traditional Chinese medicine malleus has been used in the field of trauma treatment for a long time due to its excellent hemostatic performance. The spore structure of malleus can quickly adsorb platelets in blood and form a physical blocking layer, and the active ingredients such as sterols and polysaccharides contained in malleus can slightly regulate the inflammatory response of trauma. However, single malleus spore powder has the limitations of weak antibacterial activity and limited inflammatory regulation ability, which is difficult to cope with bacterial infection and excessive inflammatory response in the process of wound repair, resulting in its limited application in complex wound repair.

[0003] Sodium alginate-chitosan hydrogel as a mature biomedical carrier material has good biocompatibility, hydrophilicity and degradability, and can provide a stable moist repair microenvironment for the wound site, and is often used as a matrix for functional wound dressings. However, this kind of biomacromolecule hydrogel still has defects in hemostatic and antibacterial functional factors.

[0004] The rise of nanomaterials provides an effective way to strengthen the functions of traditional biomaterials. The highly dispersed nano-copper supported by polymer carbon nitride exhibits excellent antibacterial and anti-inflammatory performance due to its unique electronic structure and high catalytic activity. It can kill various pathogenic bacteria by destroying the integrity of bacterial cell membranes and producing reactive oxygen species (ROS), and can also remove excessive inflammatory factors at the wound site. However, nano-copper does not show outstanding functional characteristics in hemostasis. SUMMARY

[0005] To solve the above problems, the present application provides a preparation method of a nano-copper reinforced malleus spore composite biomacromolecule hydrogel, comprising the following steps: (1) Mix the nano-copper supported by polymer carbon nitride with a chitosan acetic acid solution, ultrasonic, centrifuge at 6000 rpm for 10 minutes, take the supernatant, and obtain a nano-copper dispersion liquid; (2) Mix the malleus spores with a sodium alginate solution, centrifuge at 4000 rpm for 10 minutes, and take the spore precipitate and re-disperse it with an acetic acid solution with a pH of 4 to obtain a malleus spore dispersion liquid; (3) Slowly drop the above nano-copper dispersion liquid into the malleus spore dispersion liquid, and observe that the particles in the suspension aggregate and become larger and precipitate. Stop adding, and freeze-dry to obtain a nano-copper / malleus spore composite powder; (4) Mix the above nano-copper / malleus spore composite powder with an oxidized sodium alginate solution uniformly, and then mix the mixed solution with a carboxymethyl chitosan solution uniformly; (5) Let stand for 3-5 minutes to obtain nano-copper reinforced puffball spore composite biomacromolecule hydrogel.

[0006] Furthermore, the pH value of the chitosan acetate solution in step (1) is 4.

[0007] Furthermore, the preparation method of polymer carbon nitride-supported copper nanoparticles in step (1) includes the following steps: Dissolve 5.0–15.0 g of urea and 0.15–0.35 g of copper chloride dihydrate in 20 ml of deionized water, stir magnetically for 1 h, and dry at 80 ℃ for 12 h. Grind the dried sample into powder, spread it evenly in a ceramic boat, place the ceramic boat in the middle of a tube furnace, and calcine at 550 ℃ for 2 h with a heating rate of 25 ℃ / min and a N2 flow rate of 40–50 ml / min. After naturally cooling to room temperature, the resulting brownish-yellow powder is the polymer carbon nitride-supported nano-copper.

[0008] Furthermore, the preparation method of the nano-copper / puffball spore complex powder in step (3) also includes: Add 5-20 mg of puffball spores to 10 ml of sodium alginate solution with a concentration of 2 mg / ml, mix well, centrifuge at 4000 rpm for 10 minutes, take the precipitate, and redisperse it with acetic acid solution at pH 4. Repeat 3 times to obtain puffball spore sodium alginate solution. The nano-copper dispersion was slowly added dropwise to the sodium alginate solution of puffball spores at a volume ratio of 1:20 to 1:40. After the suspended particles aggregated and precipitated, the addition was stopped, and the mixture was freeze-dried at -80 °C to obtain the nano-copper and puffball spore complex powder.

[0009] This invention also provides a nano-copper reinforced puffball spore composite biomacromolecule hydrogel, which is prepared by the above-described method.

[0010] This invention also provides the application of the above-mentioned nano-copper reinforced puffball spore composite biomacromolecule hydrogel in the preparation of hemostatic drugs.

[0011] The present invention has the following beneficial effects: This invention provides a method for preparing a nano-copper-reinforced puffball spore biomacromolecule composite hydrogel with wound-healing functions. This method combines traditional Chinese medicine, nanomaterials, and hydrogel wound-healing technology to develop a composite hydrogel that promotes wound healing. This hydrogel possesses stable mechanical properties, high water absorption, self-healing properties, and injectability, adapting to various wound environments and providing physical protection and a moist environment for the wound. Furthermore, the nano-copper / puffball spore complex provides the hydrogel with antioxidant, antibacterial, and hemostatic properties. Simultaneously, it improves the hydrogel's mechanical strength and swelling properties, and endows it with excellent cell migration and proliferation-promoting properties, while minimizing scarring. This method is simple, environmentally friendly, and suitable for the large-scale preparation of high-quality composite hydrogels. The nano-copper-reinforced puffball spore biomacromolecule composite hydrogel is a safe, reliable, and natural wound dressing with great application potential in wound repair. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is the X-ray diffraction (XRD) pattern of the polymer carbon nitride-supported copper nanoparticles prepared in Example 1.

[0014] Figure 2 This is a transmission electron microscope (TEM) image of the nano-copper / puffball spore complex prepared in Example 1.

[0015] Figure 3 The rheological properties are those of the nano-copper reinforced puffball spore composite biomacromolecule hydrogel prepared in Example 1.

[0016] Figure 4 This refers to the antibacterial properties of the nano-copper-reinforced puffball spore composite biomacromolecule hydrogel prepared in Example 1.

[0017] Figure 5 This demonstrates the biocompatibility of the nano-copper-enhanced puffball spore composite biomacromolecule hydrogel prepared in Example 1.

[0018] Figure 6 This refers to the hemolytic activity of the nano-copper-reinforced puffball spore composite biomacromolecule hydrogel prepared in Example 1.

[0019] Figure 7The in vitro hemostatic ability of the nano-copper-enhanced puffball spore composite biomacromolecule hydrogel prepared in Example 1 is demonstrated.

[0020] Figure 8 The in vivo hemostatic ability of the nano-copper-enhanced puffball spore composite biomacromolecule hydrogel prepared in Example 1 is demonstrated.

[0021] Figure 9 The in vitro wound healing effect of the nano-copper reinforced puffball spore composite biomacromolecule hydrogel prepared in Example 1 on mice. Detailed Implementation

[0022] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1 provides a method for preparing a nano-copper reinforced puffball spore composite biomacromolecule hydrogel, the steps of which are as follows: 1) Dissolve 10.0 g of urea and 0.27 g of copper chloride dihydrate in 20 ml of deionized water, stir for 1 h, and dry at 80 °C for 12 h; grind the dried sample into powder, spread it evenly in a ceramic boat, place the ceramic boat in the middle of a tube furnace, and calcine at 550 °C for 2 h with a heating rate of 25 °C / min and a N2 flow rate of 40~50 ml / min. After naturally cooling to room temperature, brownish-yellow nano-copper powder is obtained.

[0028] 2) Dissolve 10 mg of nano copper powder in 10 ml of chitosan acetate solution with a concentration of 1 mg / ml at pH 4, disperse using a cell disruptor by sonication for 15 minutes, shake for 2 seconds, pause for 2 seconds, centrifuge at 6000 rpm for 10 minutes, and collect the pale yellow supernatant to obtain the nano copper dispersion.

[0029] 3) Add 10 mg of puffball spores to 10 ml of sodium alginate solution with a concentration of 2 mg / ml, mix well, centrifuge at 4000 rpm for 10 minutes, take the precipitate, redisperse it with 10 ml of pre-prepared pH 4 acetic acid solution, and repeat 3 times.

[0030] 4) The nano copper dispersion in (2) above was slowly added dropwise to the puffball spore dispersion. When the volume ratio of the added dropwise reached 1:30, the suspended particles were found to aggregate and become larger and then precipitated. The addition was stopped and the mixture was freeze-dried at -80 °C to obtain nano copper / puffball spore complex powder.

[0031] 5) Dissolve 0.10 g of carboxymethyl chitosan (substitution degree ≥90%) in 2.5 ml of deionized water.

[0032] 6) Dissolve 0.10 g of sodium alginate (substitution degree ≥70%) in 2.5 ml of deionized water, then add 5 mg of nano copper / puffball spore complex powder, stir for 30 minutes, and sonicate for 30 minutes.

[0033] 7) Mix the solution in 5) with the suspension in 6) evenly, let stand for 3-5 min to obtain nano-copper reinforced puffball spore composite biomacromolecule hydrogel.

[0034] Figure 1 This is the X-ray diffraction pattern of the polymer carbon nitride dispersed copper nanoparticles prepared in this embodiment. It can be found that there is only a broad carbon nitride (002) diffraction peak around 27 degrees, and no peaks of metallic copper are observed, indicating that the nano-sized copper particles exist in a highly dispersed state on the carbon nitride support.

[0035] Figure 2This is a transmission electron microscope (TEM) image of the nano-copper / puffball spore complex prepared in this embodiment. It can be observed that the nano-copper / puffball spore complex retains the spherical main structure of puffball spores, but its surface is covered by an irregular, fluffy nano-copper coating layer, exhibiting a rough composite phase interface, indicating that nano-copper was successfully loaded onto the surface of puffball spores.

[0036] Figure 3 This describes the rheological properties of the nano-copper reinforced puffball spore composite hydrogel in this embodiment. (a) and (b) are the storage modulus under varying angular frequencies. G ') and loss modulus ( G The spectrum shows that within the angular frequency range of 0.1~100 rad / s, G '> G The '' indicates that the composite hydrogel is an elastic hydrogel with a certain mechanical strength, and that as the angular frequency increases, G 'and G The change is not significant, indicating that the hydrogel has a stable elastic network structure within the test frequency range; (c) shows the change in composite viscosity with shear rate, and it can be found that the composite viscosity of the hydrogel decreases exponentially and rapidly with the increase of shear rate, indicating that the hydrogel is injectable; (d) shows the storage modulus under vibration strain. G ') and loss modulus ( G (e) The strain spectrum shows that when the strain amplitude reaches about 400%, the loss modulus is greater than the storage modulus, indicating that the hydrogel can withstand a maximum strain of 400%; (e) is the storage modulus under alternating strains of 1% to 400%. G ') and loss modulus ( G The changes in strain showed that the hydrogel network structure was destroyed at 400% strain, while the network structure of the hydrogel completed self-healing when the strain was 1%, indicating that the composite hydrogel can withstand a certain strain and can automatically heal after being destroyed.

[0037] Example 2 provides a method for preparing a nano-copper reinforced puffball spore composite biomacromolecule hydrogel, the steps of which are as follows: 1) Dissolve 5.0 g of urea and 0.15 g of copper chloride dihydrate in 20 ml of deionized water, stir for 1 h, and dry at 80 ℃ for 12 h; grind the dried sample into powder, spread it evenly in a ceramic boat, place the ceramic boat in the middle of a tube furnace, calcine at 550 ℃ for 2 h at a heating rate of 25 ℃ / min and a N2 flow rate of 40~50 ml / min, and then allow it to cool naturally to room temperature to obtain brownish-yellow nano-copper powder.

[0038] 2) Dissolve 5 mg of nano copper powder in 10 ml of chitosan acetate solution with a concentration of 1 mg / ml at pH 4, disperse using a cell disruptor by sonication for 15 minutes, shake for 2 seconds, pause for 2 seconds, centrifuge at 6000 rpm for 10 minutes, and collect the pale yellow supernatant to obtain the nano copper dispersion.

[0039] 3) Add 5 mg of puffball spores to 10 ml of sodium alginate solution with a concentration of 2 mg / ml, mix well, centrifuge at 4000 rpm for 10 minutes, take the precipitate, redisperse it with 10 ml of pre-prepared pH 4 acetic acid solution, and repeat 3 times.

[0040] 4) The nano copper dispersion in (2) above was slowly added dropwise to the puffball spore dispersion. When the volume ratio of the added dropwise reached 1:20, the suspended particles were found to aggregate and become larger and then precipitated. The addition was stopped and the mixture was freeze-dried at -80 °C to obtain nano copper / puffball spore complex powder.

[0041] 5) Dissolve 0.05 g of carboxymethyl chitosan (substitution degree ≥90%) in 2.5 ml of deionized water.

[0042] 6) Dissolve 0.25 g of sodium alginate (substitution degree ≥70%) in 2.5 ml of deionized water, then add 1 mg of nano copper / puffball spore complex powder, stir for 30 minutes, and sonicate for 30 minutes.

[0043] 7) Mix the solution in 5) with the suspension in 6) evenly, let stand for 3-5 min to obtain nano-copper reinforced puffball spore composite biomacromolecule hydrogel.

[0044] Example 3 provides a method for preparing a nano-copper reinforced puffball spore composite biomacromolecule hydrogel, the steps of which are as follows: 1) Dissolve 15.0 g of urea and 0.35 g of copper chloride dihydrate in 20 ml of deionized water, stir for 1 h, and dry at 80 °C for 12 h; grind the dried sample into powder, spread it evenly in a ceramic boat, place the ceramic boat in the middle of a tube furnace, and calcine at 550 °C for 2 h with a heating rate of 25 °C / min and a N2 flow rate of 40 ~ 50 ml / min. After naturally cooling to room temperature, brownish-yellow nano-copper powder is obtained.

[0045] 2) Dissolve 10 mg of nano copper powder in 10 ml of chitosan acetate solution with a concentration of 1 mg / ml at pH 4, disperse using a cell disruptor by sonication for 15 minutes, shake for 2 seconds, pause for 2 seconds, centrifuge at 6000 rpm for 10 minutes, and collect the pale yellow supernatant to obtain the nano copper dispersion.

[0046] 3) Add 20 mg of puffball spores to 10 ml of sodium alginate solution with a concentration of 2 mg / ml and mix well. Then centrifuge at 4000 rpm for 10 minutes, take the precipitate, and redisperse it with 10 ml of pre-prepared pH 4 acetic acid solution. Repeat 3 times.

[0047] 4) The nano copper dispersion in (2) above was slowly added dropwise to the puffball spore dispersion. When the volume ratio of the added dropwise reached 1:40, the suspended particles were found to aggregate and become larger and then precipitated. The addition was stopped and the mixture was freeze-dried at -80 °C to obtain nano copper / puffball spore complex powder.

[0048] 5) Dissolve 0.25 g of carboxymethyl chitosan (substitution degree ≥90%) in 2.5 ml of deionized water.

[0049] 6) Dissolve 0.05 g of sodium alginate (substitution degree ≥70%) in 2.5 ml of deionized water, then add 10 mg of nano copper / puffball spore complex powder, stir for 30 minutes, and sonicate for 30 minutes.

[0050] 7) Mix the solution in 5) with the suspension in 6) evenly, let stand for 3-5 min to obtain nano-copper reinforced puffball spore composite biomacromolecule hydrogel.

[0051] Example 4 provides a method for preparing a nano-copper reinforced puffball spore composite biomacromolecule hydrogel, the steps of which are as follows: 1) Dissolve 10.0 g of urea and 0.35 g of copper chloride dihydrate in 20 ml of deionized water, stir for 1 h, and dry at 80 °C for 12 h; grind the dried sample into powder, spread it evenly in a ceramic boat, place the ceramic boat in the middle of a tube furnace, and calcine at 550 °C for 2 h with a heating rate of 25 °C / min and a N2 flow rate of 40~50 ml / min. After naturally cooling to room temperature, brownish-yellow nano-copper powder is obtained.

[0052] 2) Dissolve 5 mg of nano copper powder in 10 ml of chitosan acetate solution with a concentration of 1 mg / ml at pH 4, disperse using a cell disruptor by sonication for 15 minutes, shake for 2 seconds, pause for 2 seconds, centrifuge at 6000 rpm for 10 minutes, and collect the pale yellow supernatant to obtain the nano copper dispersion.

[0053] 3) Add 15 mg of puffball spores to 10 ml of sodium alginate solution with a concentration of 2 mg / ml, mix well, centrifuge at 4000 rpm for 10 minutes, take the precipitate, redisperse it with 10 ml of pre-prepared pH 4 acetic acid solution, and repeat 3 times.

[0054] 4) The nano copper dispersion in (2) above was slowly added dropwise to the puffball spore dispersion. When the volume ratio of the added dropwise reached 1:30, the suspended particles were found to aggregate and become larger and then precipitated. The addition was stopped and the mixture was freeze-dried at -80 °C to obtain nano copper / puffball spore complex powder.

[0055] 5) Dissolve 0.15 g of carboxymethyl chitosan (substitution degree ≥90%) in 2.5 ml of deionized water.

[0056] 6) Dissolve 0.15 g of sodium alginate (substitution degree ≥70%) in 2.5 ml of deionized water, then add 5 mg of nano copper / puffball spore complex powder, stir for 30 minutes, and sonicate for 30 minutes.

[0057] 7) Mix the solution in 5) with the suspension in 6) evenly, let stand for 3-5 min to obtain nano-copper reinforced puffball spore composite biomacromolecule hydrogel.

[0058] The activity detection steps are as follows: Select Gram-negative bacteria E. coil (K12 D31) and Gram-positive bacteria S. aureus (CGMCC1.1088) was used as a model bacterium for testing the antibacterial activity of the composite hydrogel. First, the bacteria stored at -80 °C were removed. E. coil The bacterial strain was activated. A small amount of bacterial culture was inoculated into LB liquid medium using an inoculation loop and incubated at 180 rpm and 37 °C for 12 h. Then, the inoculated culture was streaked onto agar plates and incubated at 37 °C for 24 h. A single colony was then streaked onto agar slant using an inoculation loop and incubated at 37 °C for another 24 h. The incubated slant culture was stored at 4 °C as the bacterial strain for subsequent composite gel antibacterial activity testing. A small amount of the bacterial strain was then inoculated into LB liquid medium using an inoculation loop and incubated at 180 rpm and 37 °C for 10 h, yielding a concentration of approximately 3.0 × 10⁻⁶. 8CFU / mL bacterial suspension. Hydrogel was cut into 10 mm diameter, 5 mm high hydrogel columns and placed in 24-well plates. 1 mL of bacterial suspension was added. The plates were incubated at room temperature for 2 h. 100 μL of the reaction bacterial suspension was then serially diluted, and another 100 μL was plated. The plates were incubated at 37 ℃ for 24 h, and the colony count was recorded. The control group was incubated under the same conditions for 2 h. 100 μL of the reaction bacterial suspension was serially diluted, and another 100 μL was plated. The plates were incubated at 37 ℃ for 24 h, and the colony count was recorded. Each experiment was performed in triplicate. All samples and equipment were sterilized before the experiment to eliminate the influence of external bacterial contamination. Gram-positive bacteria. S. aureus The experimental procedure is the same as above.

[0059] Figure 4 The antibacterial properties of the composite hydrogel in Example 1 are shown in the figures: (a) bacterial survival rate after co-culturing with the composite hydrogel; (b) images of bacterial colonies after co-culturing the composite hydrogel with Staphylococcus aureus and Escherichia coli. The figures show that the hydrogel achieved antibacterial rates of 97.13% and 96.37% against Escherichia coli and Staphylococcus aureus, respectively, demonstrating good antibacterial effects. This is attributed to the antibacterial properties of the complex of nano-copper and puffball spores. The enzyme-like catalytic activity of nano-copper and the release of copper ions can kill bacteria, while the physical adsorption of puffball spores increases the attraction to bacteria. The synergistic effect of these two components endows the composite hydrogel with high antibacterial properties.

[0060] The biocompatibility testing steps are as follows: Mouse embryonic fibroblasts (NIH3T3 cells) were selected as model cells for testing the biocompatibility of the composite gel. First, cell activation was performed: NIH3T3 cells stored at -80 ℃ were incubated in a 37 ℃ water bath for 1 min, then rapidly transferred to a clean bench in the cell culture room. The dissolved cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and 1 ml of fresh cell culture medium was added to the centrifuge tube and dispersed evenly. The dispersed cell suspension was then transferred to a T25 cell culture flask, and 4 ml of fresh cell culture medium and 0.2 ml of antibiotics were added. The flask was incubated at 37 ℃. Fresh cell culture medium was added to the T25 flask every 24 h. This process was repeated for 4–5 days until the cells successfully recovered and grew to cover more than 80% of the bottom of the T25 flask before passage.

[0061] Cell passage: Remove the grown T25 flask from the incubator, discard the culture medium, add 1 ml of trypsin, and incubate at 37 ℃ for 2-3 min. After most of the adherent NIH3T3 cells have been digested, add 2 ml of culture medium to stop digestion, transfer to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 ml of culture medium to disperse evenly, transfer to a T25 flask, add 4 ml of culture medium and 0.2 ml of antibiotics, and continue to incubate at 37 ℃ for 2-3 days, changing the culture medium every 2 days to complete the passage.

[0062] CCK-8 cytotoxicity assay: After passing the cells as described above, digest them with trypsin, centrifuge, and resuspend. Take 10 μL and place it on a hemocytometer. Count the cells under a microscope and calculate the total number of cells in the cell suspension. Dilute the suspension. Calculate the required number of wells in a 96-well plate according to the experimental design. Add 100 μL of cell suspension to each well, 10,000 cells per well. Incubate the 96-well plate at 37 ℃ for one day. Beforehand, immerse 100 mg of the composite gel sample in 1 ml of culture medium and incubate for 1, 3, and 5 days. Remove the sample and dilute the culture medium extract of the composite gel to 10 μg / ml. Add 100 μL of this extract to each well of the prepared 96-well plate, discarding the old culture medium beforehand. After incubating at 37 ℃ for 1 day, remove the plate and add 10 μL of CCK-8 reagent to each well in the dark. Wrap the 96-well plate with aluminum foil and incubate at 37 ℃ for 2-4 hours. Remove the plate and measure the absorbance at 517 nm using a microplate reader. Calculate cell viability based on the absorbance. Results are as follows: Figure 5 As shown in (a).

[0063] Cell viability staining assay: Discard the old culture medium from the 96-well plate containing the cultured cells as described above, add cell viability staining reagent, and incubate at 37°C for 30 min. Then, photograph the cells using a fluorescence microscope. Results are as follows: Figure 5 As shown in (b).

[0064] Figure 5 The biocompatibility of the composite hydrogel in Example 1 is shown in the figures: (a) NIH3T3 cell viability determined by CCK-8 assay at 1, 3, and 5 days; (b) live / dead staining images of NIH3T3 cells co-cultured with the hydrogel exudate at 1, 3, and 5 days. As can be seen from the figures, the cell viability in the experimental group reached over 95% compared to the control group. The live / dead staining images also indicate that the number and density of surviving cells in the experimental group were not less than those in the control group, demonstrating that this composite gel is non-toxic to humans and will not affect cell growth or wound repair.

[0065] The steps for detecting hemolysis are as follows: Whole blood from ICR mice was collected and added to a pre-extracted anticoagulated blood centrifuge tube. The tubes were centrifuged at 3000 rpm for 10 min. The lower layer of red blood cells was collected using a pipette and dissolved in sterile saline to prepare a 4% red blood cell suspension. The suspension was then stored at 4 ℃ for later use. In each sterile centrifuge tube, 0.9 ml of PBS solution and 0.1 ml of 4% red blood cell suspension were added, along with approximately 100 mg of gel. 0.9 ml of PBS solution and 0.1 ml of 4% red blood cell suspension served as a negative control, while 0.9 ml of deionized water and 0.1 ml of 4% red blood cell suspension served as a positive control. The centrifuge tubes were co-cultured at 37 ℃ for 3 h, then centrifuged at 3000 rpm for 10 min. The supernatant was collected, and its absorbance at 540 nm was measured. The hemolytic properties of the composite gel were calculated using the formula. Hemolysis rate = % Figure 6 This study investigated the hemolytic activity of the composite hydrogel in Example 1. Results showed that red precipitate formed at the bottom of all test tubes in the gel group. In contrast, all blood cells in the positive group lysed. The hemolysis rate of the gel group was below the 5% international permeability standard for biomaterials, indicating that the composite hydrogel exhibits good blood compatibility as a wound dressing.

[0066] The steps for the in vitro hemostasis experiment are as follows: ICR mice were anesthetized by intraperitoneal injection of tribromoethanol. The tails were then severed from the upper middle portion using sterile surgical scissors. Blood was collected from the tail using filter paper, and pressure was applied to the tail. The amount of bleeding was measured, and the time to cessation of bleeding at the tail was monitored and recorded. A blank control group and a gel experimental group were set up, with each group repeated three times.

[0067] Figure 7 The in vitro hemostatic ability of the composite hydrogel in Example 1 is shown in the images: (a) Changes in bleeding volume during the hemostasis process of a mouse tail amputation model within 60 seconds; (b) Blood loss during the tail amputation hemostasis process; and (c) Hemostasis time during tail amputation. The results indicate that the experimental group, treated with the hydrogel, experienced only about one-third of the blood loss and hemostasis time compared to the control group, demonstrating that the composite hydrogel possesses excellent in vitro hemostatic ability.

[0068] The in vivo hemostasis experiment steps are as follows: After creating a puncture wound in the mouse liver using a sterile needle, a composite hydrogel was applied to the bleeding site until the bleeding stopped. The amount of bleeding in the mouse liver was determined by calculating the change in filter paper weight, and the hemostasis time in the mouse liver was obtained through real-time monitoring.

[0069] Figure 8 The in vivo hemostatic ability of the composite hydrogel in Example 1 is shown in the images: (a) Changes in bleeding volume during the hemostasis process of the mouse liver model within 60 seconds; (b) Blood loss during the hemostasis process of the liver; and (c) Hemostasis time of the liver. The results show that the bleeding time of the liver model was reduced by about 50% compared with the control group, and the bleeding volume was reduced to about 25 mg, indicating that the composite hydrogel has a good in vivo hemostatic effect.

[0070] The steps for testing in vitro wound healing efficacy are as follows: ICR mice were selected as experimental subjects, with 5 ICR mice in each of the control and experimental groups to minimize experimental error. First, the mice were cultured in a mouse house for one week to acclimatize to the new environment. After one week, the mice were anesthetized with tribromoethanol, the hair on their backs was removed, and the backs were disinfected with povidone-iodine. A wound was created in a selected area, aiming for a circular shape. The control group received no wound treatment, relying solely on the mice's own wound healing process. In the experimental group, a composite gel was fixed to the wound on the mice's backs using gauze and bandages. The mice were then returned to the mouse house for culture, and wound healing was observed and recorded. Images of the wounds on days 0, 3, 7, 10, and 14 post-traumatic induction were compared to study the effect of the composite gel on wound healing.

[0071] Figure 9 The following is an example of the repair and regeneration of infected wounds in Example 1: (a) wound healing rate within 14 days; (b) representative images of wound healing within 14 days; (c) schematic diagram of wound boundary changes at different time points during the wound healing process. The results show that the wounds treated with hydrogel have a better healing effect, with a healing rate of over 95% within 14 days, which is nearly 20% higher than the blank control group.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a nano-copper-reinforced puffball spore composite biomacromolecule hydrogel, characterized in that, Includes the following steps: (1) Mix the polymer carbon nitride-loaded copper nanoparticles with chitosan acetate solution, sonicate, centrifuge at 6000 rpm for 10 minutes, and take the supernatant to obtain a copper nanoparticle dispersion. (2) Mix the puffball spores with sodium alginate solution, centrifuge at 4000 rpm for 10 minutes, take the spore precipitate and redisperse it with acetic acid solution at pH 4 to obtain puffball spore dispersion. (3) The above-mentioned nano copper dispersion was slowly added dropwise to the puffball spore dispersion. It was observed that the particles in the suspension aggregated, became larger and precipitated. The addition was stopped and the nano copper / puffball spore complex powder was obtained after freeze-drying. (4) Mix the above-mentioned nano copper / puffball spore complex powder with sodium oxidized alginate solution evenly, and then add the mixed solution to carboxymethyl chitosan solution and mix evenly. (5) Let stand for 3-5 minutes to obtain nano-copper reinforced puffball spore composite biomacromolecule hydrogel.

2. The preparation method according to claim 1, characterized in that, The pH value of the chitosan acetate solution in step (1) is 4.

3. The preparation method according to claim 1, characterized in that, The preparation method of polymer carbon nitride-supported copper nanoparticles in step (1) includes the following steps: Dissolve 5.0~15.0 g of urea and 0.15~0.35 g of copper chloride dihydrate in 20 ml of deionized water, stir magnetically for 1 h, and dry at 80 ℃ for 12 h. Grind the dried sample into powder, spread it evenly in a ceramic boat, place the ceramic boat in the middle of a tube furnace, and calcine at 550 ℃ for 2 h with a heating rate of 25 ℃ / min and a N2 flow rate of 40~50 ml / min. After naturally cooling to room temperature, the resulting brownish-yellow powder is the polymer carbon nitride-supported nano-copper.

4. The preparation method according to claim 1, characterized in that, The preparation method of the nano-copper / puffball spore complex powder in step (3) further includes: Add 5-20 mg of puffball spores to 10 ml of sodium alginate solution with a concentration of 2 mg / ml and mix well. Centrifuge at 4000 rpm for 10 minutes, collect the precipitate, and redisperse it with acetic acid solution at pH 4. Repeat 3 times to obtain puffball spore sodium alginate solution. The nano-copper dispersion was slowly added dropwise to the sodium alginate solution of puffball spores at a volume ratio of 1:20 to 1:

40. After the suspended particles aggregated and precipitated, the addition was stopped, and the mixture was freeze-dried at -80 °C to obtain the nano-copper and puffball spore complex powder.

5. A nano-copper-reinforced puffball spore composite biomacromolecule hydrogel, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.

6. The application of the nano-copper reinforced puffball spore composite biomacromolecule hydrogel as described in claim 5 in the preparation of hemostatic drugs.

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