Preparation method and application of a composite gel containing chitosan, nano-gold and sodium hyaluronate

Abstract

The invention discloses a preparation method of a composite gel containing chitosan, nano-gold and sodium hyaluronate, which comprises the following steps: First step, mix polyethylene glycol 400 and polyethylene glycol 4000, heat to 50-80 °C and keep for 10-30 min. When it is naturally cooled to 40-50 °C, maintain the temperature, add a chitosan solution to the above solution that has been completely dissolved, continue stirring for 15-30 min and then stop heating. Wait for the solution to be completely cooled to room temperature and then perform ultrasonic treatment; Second step, add a nano-gold solution and stir; Third step, add a sodium hyaluronate solution and stir to obtain the composite gel containing chitosan, nano-gold and sodium hyaluronate; The gel prepared by this method is an ointment integrating antibacterial and tissue repair functions. The preparation process is simple, and the raw materials are cheap and easily available. It not only has excellent broad-spectrum antibacterial properties, but also has a significant function of promoting tissue repair.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to a preparation method and application of a composite gel containing chitosan, nano-gold and sodium hyaluronate. Background Art

[0002] As a clinical drug, antibiotics have promoted the mobilization and horizontal transfer of a wide range of antibiotic resistance genes (ARGs) to other bacterial species, especially those causing diseases (Nat Rev Microbiol, 2015, 13, 310 - 317). This continuous evolution and accumulation have increased the difficulty of preventing and treating bacterial infections (Nat Rev Microbiol, 2021, 20, 257 - 269). At the same time, it has given rise to the emergence of more drug-resistant bacteria and multi-drug resistant bacteria, greatly increasing the morbidity and mortality of patients, which makes antibiotic treatment more restrictive. In the face of the global spread of microbial drug resistance and the increasing cost of developing new antibiotics, non-antibiotic antibacterial agents have become an attractive and promising alternative for treating infections caused by multi-drug resistant (MDR) microorganisms (Int J Antimicrob Agents, 2021, 58, 106380 - 106400).

[0003] Chitosan is a natural polymer with unique properties such as good biodegradability, biocompatibility, non-toxicity, antibacterial activity, anti-tumor, immune enhancement, and low cost (Molecules, 2021, 26(12), 3694 - 3710), and has been widely studied for many fields. By fully utilizing solvents (organic acid solutions such as formic acid and acetic acid, and inorganic acids such as dilute phosphoric acid and dilute hydrochloric acid) to protonate the amino groups of chitosan and bind to the negatively charged bacterial cell wall, resulting in cell rupture, thereby changing the membrane permeability, and then attaching to DNA, DNA replication is inhibited and subsequently leads to cell death through a broad-spectrum antibacterial mechanism (Functional Chitosan, 2020, 457 - 489), chitosan has also become a new type of non-antibiotic antibacterial agent (Polymers (Basel), 2021, 13(6), 904 - 924).

[0004] Gold nanoparticles (AuNPs) have the characteristics of controllable size, easy chemical synthesis and modification, and non-toxicity to human and animal cells. When this nanomaterial is functionalized with sugar molecules or polysaccharides, they are called glyconanoparticles and have high antibacterial activity (Materials Science and Engineering C, 2016, 69, 366 - 372); the use of nanogold avoids the generation of drug resistance and can effectively and safely combat a variety of pathogenic microorganisms. In addition, nanogold can be used as a drug carrier, effectively enhancing chemical stability and biological activity against different microorganisms (Materials Science & Engineering C, 2019, 96, 693 - 707).

[0005] The polysaccharide hyaluronic acid (HA) is a multifunctional and polymorphic glycosaminoglycan with a wide range of biological functions. HA is distributed throughout the body and is mainly present in the skin, so it plays an important role in wound healing (Int Wound J, 2014, 11(2), 159 - 163). Hyaluronic acid is also called hyaluronic acid, and the sodium salt form of hyaluronic acid is called sodium hyaluronate, which belongs to a functionalized cosmetic raw material and is widely used in various skin care products.

[0006] The patent application with the publication number CN114097788A discloses a preparation method of a Fenton-like slow-release antibacterial hydrogel, which comprises the following steps: dissolving a natural polymer material in a solvent, first adding a nano metal oxide and dispersing it evenly, then adding a peroxide and dispersing it evenly, and finally adding an ammonium salt and / or a weak acid, stirring and mixing to obtain the Fenton-like slow-release antibacterial hydrogel; wherein the ratio of the natural polymer material, the solvent, the nano metal oxide, the peroxide, and the ammonium salt and / or the weak acid is 0.1-1 g: 10-25 mL: 0.1-0.5 g: 0.01-0.5 g: 0.01 g. Preferably, the ratio of the natural polymer material, the solvent, the nano metal oxide, the peroxide, and the ammonium salt and / or the weak acid is 0.3 g: 20 mL: 0.25 g: 0.01-0.5 g: 0.01 g. Preferably, the natural polymer material is at least one of sodium carboxymethyl cellulose, carboxymethyl chitosan, sodium alginate, sodium hyaluronate, agarose, and chitosan. Preferably, the nano metal oxide is at least one of nano zinc oxide, nano calcium oxide, nano magnesium oxide, and nano aluminum oxide. Preferably, the peroxide is at least one of sodium percarbonate, urea peroxide, calcium peroxide, and hydrogen peroxide. Preferably, the ammonium salt is at least one of ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium persulfate, ammonium citrate, and ammonium tannate. Preferably, the weak acid is one of silicic acid, hydrocyanic acid, hypochlorous acid, tartaric acid, citric acid, tannic acid, malic acid, salicylic acid, acetic acid, and rosin. Preferably, the solvent is at least one of water, ethanol, and dimethyl sulfoxide; more preferably water. Preferably, the nano metal oxide is first added to the solvent and ultrasonically dispersed at 20-40 Hz for 2-3 h, and then the dispersion is added to the natural polymer material solution, wherein the ratio of the nano metal oxide to the solvent is 0.1-0.5 g: 5 mL, and the solvent in the dispersion is the same as the solvent in the polymer solution.

[0007] At present, for the treatment of bacterial infections, various single or multi-component composite systems have been developed, generally traditional antibacterial gels such as chitosan antibacterial gels, nano silver antibacterial sprays, cationic antibacterial agents, and antibiotics. On the one hand, they are simply physically mixed, and the effect is single, which is easy to cause the generation of drug resistance and is difficult to effectively address the difficult-to-heal problems brought by bacterial infectious diseases. On the other hand, their broad-spectrum antibacterial performance is poor, they cannot effectively target clinically drug-resistant pathogenic microorganisms, and they cannot relieve the pain of elderly patients and other deficiencies.

[0008] Therefore, there is an urgent need in the industry for a non-antibiotic antibacterial healing ointment with strong broad-spectrum antibacterial performance, mildness and no irritation, and strong wound healing ability. Summary of the Invention

[0009] The first object of the present invention is to provide a preparation method of a composite gel containing chitosan, nano-gold and sodium hyaluronate. The gel prepared by this method is an ointment integrating antibacterial and tissue repair functions. The preparation process is simple, and the raw materials are cheap and easily available. It not only has excellent broad-spectrum antibacterial properties, but also has a significant function of promoting tissue repair, can accelerate the healing of wound infection wounds, has good biocompatibility and water solubility, has no skin irritation and toxicity, and is safe and effective.

[0010] The second object of the present invention is to provide an application of the composite gel prepared by the above method in the preparation of antibacterial drugs, tissue repair-promoting drugs, and wound healing-promoting drugs.

[0011] In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0012] The first aspect of the present invention provides a preparation method of a composite gel containing chitosan, nano-gold and sodium hyaluronate, comprising the following steps:

[0013] In the first step, polyethylene glycol 400 and polyethylene glycol 4000 with a mass ratio of 1:(0.1-10) are mixed, heated to 50-80°C for 10-30 min, and maintained at 40-50°C after natural cooling. When the temperature is maintained, a chitosan solution is added to the completely dissolved above solution, and stirring is continued for 15-30 min, then heating is stopped. After the solution is completely cooled to room temperature, ultrasonic treatment is carried out for 10-30 min;

[0014] In the second step, a nano-gold solution is added and stirred for 10-30 min;

[0015] In the third step, a sodium hyaluronate solution is added and stirred for 10-30 min to obtain the composite gel containing chitosan, nano-gold and sodium hyaluronate.

[0016] The deacetylation degree of the chitosan >50%, >80%, >85%, >90%, >95%; the molecular weight of the chitosan is selected from 1000 Da, >3000 Da, >10000 Da, >10 2 KDa, >10 3 KDa, >10 4 Kda; the viscosity of the chitosan is selected from 50-800 Pa·s (preferably 50-100 Pa·s, 100-200 Pa·s, 200-400 Pa·s, 400-800 Pa·s); the concentration of the chitosan solution is selected from 0.125-10 mg / mL (preferably 0.125, 0.25, 0.5, 1, 2, 2.5, 3, 5, 10 mg / mL).

[0017] The chitosan is at least one extracted from biological resources such as crab shells, shrimp shells, insects or microorganisms.

[0018] The chitosan solution is obtained by dissolving chitosan in an acidic solution with a pH of 3 - 5 (preferably 3, 4, 5).

[0019] The acidic solution is prepared by dissolving an acid in sterile water, and the acid is selected from at least one of formic acid, acetic acid, and citric acid.

[0020] The concentration of the nano - gold solution is 0.001 - 1 mg / mL (preferably 0.001, 0.01, 0.02, 0.03, 0.04, 0.1, 0.5, 1 mg / mL).

[0021] The particle size of the nano - gold is selected from 1 - 10 nm.

[0022] The concentration of the sodium hyaluronate solution (prepared using sterile water) is selected from 0.0001 - 5 mg / mL (preferably 0.0001, 0.001, 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5 mg / mL).

[0023] The mass ratio of chitosan, nano - gold, and sodium hyaluronate is (250 - 50000):(1 - 200):1 (preferably 2500:100:1).

[0024] The mass ratio of chitosan and polyethylene glycol 400 is (0.001 - 1):1 (preferably 0.0035:1, 0.007:1, 0.014:1).

[0025] The ratio of polyethylene glycol 400 and polyethylene glycol 4000 can adjust the viscosity of the system.

[0026] In the composite gel containing chitosan, nano - gold, and sodium hyaluronate, the concentrations of each component are as follows: the concentration of the chitosan solution is selected from 0.125 - 10 mg / mL (preferably 0.125, 0.25, 0.5, 1, 2, 5, 10 mg / mL), the concentration of the nano - gold solution is 0.001 - 1 mg / mL (preferably 0.001, 0.01, 0.02, 0.03, 0.04, 0.08, 0.1, 0.5, 1), and the concentration of the sodium hyaluronate solution is selected from 0.0001 - 5 mg / mL (preferably 0.0001, 0.0002, 0.0004, 0.0008, 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5 mg / mL).

[0027] The second aspect of the present invention provides the application of the composite gel prepared by the above - mentioned method in the preparation of antibacterial drugs, tissue - promoting repair drugs, and wound - promoting healing drugs.

[0028] The bacteria in the antibacterial drug are selected from methicillin-resistant Staphylococcus aureus (ATCC43300), Pseudomonas aeruginosa (ATCC27853), methicillin-sensitive Staphylococcus aureus (MSSA, CMCC26003), Escherichia coli (ATCC25922), vancomycin-resistant Enterococcus faecalis VRE (ATCC51299), Klebsiella pneumoniae (ATCC13883), vancomycin-sensitive Enterococcus faecalis VSE (ATCC21922), Staphylococcus epidermidis (CMCC26069), methicillin-sensitive Staphylococcus aureus (MSSA, ATCC25923), beta-hemolytic Streptococcus group C (CMCC32206), and the clinical strains are selected from: Acinetobacter baumannii (Ab1617), Pseudomonas aeruginosa (3887).

[0029] Due to the above technical solutions, the present invention has the following advantages and beneficial effects:

[0030] The method of the present invention combines gold nanoparticles and chitosan through electrostatic interaction to produce a synergistic antibacterial effect, supplemented by hyaluronic acid for multi-level self-assembly to promote wound healing, forming a ternary composite system, and adding polyethylene glycol excipients to successfully construct a new non-antibiotic antibacterial and repair integrated material. This product cleverly utilizes the synergy of nanomaterials and natural polysaccharide molecules to achieve antibacterial and tissue repair functions. The maximum antibacterial rate exceeds 90%, and the wound gap of mice infected with Staphylococcus aureus can reach a healing rate of more than 60% within 14 days, providing a safe and effective product for the treatment of clinical bacterial infection wounds and having broad application prospects.

[0031] The composite gel containing chitosan, gold nanoparticles and sodium hyaluronate prepared by the method of the present invention has cheap and easily available raw materials, simple operation, low requirements for equipment and reaction conditions, can meet the needs of future large-scale production, has strong clinical practicability, promotes the proliferation and differentiation of wound epidermal cells, and has a better synergistic effect.

[0032] The antibacterial mechanism of the present invention lies in that the composite system formed by the electrostatic interaction of deacetylated chitosan and carboxyl-modified gold nanoparticles has a positive charge. Gold nanoparticles, as a carrier, effectively improve the contact between chitosan and the bacterial surface, promote the adsorption of the composite material on the bacterial surface to hinder the material exchange between bacteria and the outside world. At the same time, part of the composite material penetrates into the bacteria and undergoes flocculation in the cytoplasm, disrupting normal physiological activities and thus killing the bacteria.

[0033] The product of the present invention has been verified through antibacterial experiments, animal experiments, and safety evaluations. The results of the antibacterial performance evaluation indicate that the product has broad-spectrum antibacterial properties, with a maximum antibacterial rate exceeding 90%. Animal experiments show that the gel of the present invention can achieve a healing rate higher than 60% for the wound gaps of mice infected with Staphylococcus aureus within 14 days. In addition, a systematic safety evaluation of the ternary compound gel, including in vitro cytotoxicity, subcutaneous irritation, skin sensitization, histopathology, and hemotoxicity, was carried out, and the results all meet the biological safety evaluation standards for medical devices (GB / T 16886), proving that the gel constructed by the present invention is safe and effective and can be used for promoting healing and antibacterial conditions such as bacterial infectious wounds and diabetic foot. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagrams of the Zeta potential, TEM, and DLS characterizations of the composite gel containing chitosan, nano-gold, and sodium hyaluronate.

[0035] Figure 2 Schematic diagrams of the wound healing effect of the composite gel containing chitosan, nano-gold, and sodium hyaluronate.

[0036] Figure 3 Schematic diagrams of the in vitro cytotoxicity of the composite gel containing chitosan, nano-gold, and sodium hyaluronate.

[0037] Figure 4 Schematic diagrams of the skin irritation and sensitization experiments of the composite gel containing chitosan, nano-gold, and sodium hyaluronate.

[0038] Figure 5 Schematic diagrams of the antibacterial activities of chitosans with different viscosities and their antibacterial properties varying with concentration.

[0039] Figure 6 Schematic diagrams of the antibacterial properties of nano-gold with different concentrations against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

[0040] Figure 7 Schematic diagrams of the construction of the composite gel system containing chitosan, nano-gold, and sodium hyaluronate. DETAILED DESCRIPTION OF THE INVENTION

[0041] To more clearly illustrate the present invention, the following further describes the present invention in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific descriptions are illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0042] The raw materials involved in the embodiments of the present invention are all commercially purchased and are of cosmetic grade and pharmaceutical grade.

[0043] In the embodiments of the present invention, chitosan is at least one extracted from biological resources such as crab shells, shrimp shells, insects or microorganisms.

[0044] Example 1

[0045] Prepare a composite gel with a total volume of about 50 mL containing chitosan, nano-gold and sodium hyaluronate, using chitosan with a deacetylation degree > 95%, molecular weight > 10 2 KDa, and viscosity 200 - 400 Pa·s. As Figure 7 shown, Figure 7 is a schematic diagram of the construction of a composite gel system containing chitosan, nano-gold and sodium hyaluronate.

[0046] 1) Add 0.32 mL of medical-grade acetic acid to 100 mL of sterile water to obtain an acetic acid solution with pH = 3. Using this acetic acid solution as the solvent, prepare a chitosan solution with a concentration of 5 mg / mL, and stir with a magnetic stirrer overnight to fully dissolve it.

[0047] 2) Prepare 10 mL of a sodium hyaluronate solution with a concentration of 0.1 mg / mL using sterile water for later use. The purchased nano-gold (1 - 10 nm) (the solvent is a citric acid solution) has a concentration of 1 mg / mL;

[0048] 3) Add PEG400 (7 g) and PEG4000 (21 g) with a mass ratio of 1:3 (m / m) to a conical flask, stir mechanically, and heat in a water bath at 65 °C for 15 min;

[0049] 4) When it cools naturally to 50 °C and maintains the temperature, add 10 mL of the chitosan solution with a concentration of 5 mg / mL to the above-mentioned completely dissolved solution, continue to stir for 15 min, then stop heating. Wait for the solution to cool completely to room temperature and ultrasonicate for 30 min;

[0050] 5) Add 2 mL of nano-gold with a concentration of 1 mg / mL and stir mechanically for 30 min;

[0051] 6) Slowly add 0.2 mL of the sodium hyaluronate solution with a concentration of 0.1 mg / mL and stir mechanically for 30 min to obtain the composite gel containing chitosan, nano-gold and sodium hyaluronate, a red viscous liquid, where the concentrations of each component are: chitosan 1 mg / mL, nano-gold 0.04 mg / mL, sodium hyaluronate 0.0004 mg / mL. The total mass is about 40 g and the total volume is about 50 mL.

[0052] Example 2

[0053] Prepare a composite gel with a total volume of about 50 mL containing chitosan, nano-gold and sodium hyaluronate, using chitosan with a deacetylation degree > 95%, molecular weight > 10 2Chitosan with a molecular weight of >10

[0054] 1) Add 0.32 mL of medical-grade acetic acid to 100 mL of sterile water to obtain an acetic acid solution with a pH of 3. Using this acetic acid solution as the solvent, prepare a chitosan solution with a concentration of 10 mg / mL, and stir with a magnetic stirrer overnight to fully dissolve it.

[0055] 2) Prepare 10 mL of a sodium hyaluronate solution with a concentration of 0.1 mg / mL using sterile water for later use. The concentration of the purchased nano-gold (1 - 10 nm) (the solvent is a citric acid solution) is 1 mg / mL;

[0056] 3) Add PEG400 (7 g) and PEG4000 (21 g) with a mass ratio of 1:3 (m / m) to a conical flask, stir mechanically, and heat in a water bath at 65 °C for 15 min;

[0057] 4) When it cools naturally to 50 °C, maintain the temperature, add 10 mL of the chitosan solution with a concentration of 10 mg / mL to the above-mentioned completely dissolved solution, continue stirring for 15 min, then stop heating. After the solution cools completely to room temperature, sonicate for 30 min;

[0058] 5) Add 4 mL of nano-gold with a concentration of 1 mg / mL and stir mechanically for 30 min;

[0059] 6) Slowly add 0.4 mL of the sodium hyaluronate solution with a concentration of 0.1 mg / mL, stir mechanically for 30 min to obtain the composite gel containing chitosan, nano-gold, and sodium hyaluronate, a red viscous liquid, where the concentrations of each component are: chitosan 2 mg / mL, nano-gold 0.08 mg / mL, and sodium hyaluronate 0.0008 mg / mL. The total mass is about 40 g, and the total volume is about 50 mL.

[0060] Example 3

[0061] Prepare a composite gel containing chitosan, nano-gold, and sodium hyaluronate with a total volume of about 50 mL, using chitosan with a degree of deacetylation >95%, molecular weight >10 2 KDa, and a viscosity of 200 - 400 Pa·s.

[0062] 1) Add 0.32 mL of medical-grade acetic acid to 100 mL of sterile water to obtain an acetic acid solution with a pH of 3. Using this acetic acid solution as the solvent, prepare a chitosan solution with a concentration of 2.5 mg / mL, and stir with a magnetic stirrer overnight to fully dissolve it.

[0063] 2) Prepare 10 mL of a sodium hyaluronate solution with a concentration of 0.1 mg / mL using sterile water for later use. The concentration of the purchased nano-gold (1 - 10 nm) (the solvent is a citric acid solution) is 1 mg / mL;

[0064] 3) Add PEG400 (7 g) and PEG4000 (21 g) with a mass ratio of 1:3 (m / m) into a conical flask, stir mechanically, and heat in a water bath at 65 °C for 15 min;

[0065] 4) When it cools naturally to 50 °C and maintain the temperature, add 10 mL of chitosan solution with a concentration of 2.5 mg / mL into the above-mentioned completely dissolved solution, continue stirring for 15 min, then stop heating. Wait for the solution to cool completely to room temperature and sonicate for 30 min;

[0066] 5) Add 1 mL of nano-gold with a concentration of 1 mg / mL and stir mechanically for 30 min;

[0067] 6) Slowly add 0.1 mL of sodium hyaluronate solution with a concentration of 0.1 mg / mL and stir mechanically for 30 min to obtain the composite gel containing chitosan, nano-gold and sodium hyaluronate, a red viscous liquid, where the concentrations of each component are: chitosan 0.5 mg / mL, nano-gold 0.02 mg / mL, sodium hyaluronate 0.0002 mg / mL. The total mass is about 40 g and the total volume is about 50 mL.

[0068] Example 4

[0069] Before adjusting the ratio of polyethylene glycol 400 and polyethylene glycol 4000, prepare a ternary composite system with a concentration 4 times lower than each active ingredient in the product (0.25 mg / mL chitosan, 0.01 mg / mL nano-gold, 0.0001 mg / mL sodium hyaluronate), and perform Zeta potential, TEM and DLS characterizations, as Figure 1 shown. Figure 1 Schematic diagrams of Zeta potential, TEM, and DLS characterizations of the composite gel containing chitosan, nano-gold and sodium hyaluronate.

[0070] From Figure 1It can be seen that: a. zeta potential, b. TEM morphological changes, c. particle size measurement by dynamic light scattering; the results of the zeta potential in Figure a show that the carboxyl-modified gold nanoparticles are negatively charged, while the deacetylated chitosan is positively charged. After the assembly of gold nanoparticles and chitosan, a part of the negative charge will be offset, and finally it shows a positive charge, which is more conducive to its adsorption on the bacterial surface. The morphological characterization by transmission electron microscopy (TEM) in Figure b shows that there is an obvious adsorption relationship between gold nanoparticles and chitosan, because gold nanoparticles and chitosan always appear overlapped in the field of view at the same time. Try to measure the particle size changes before and after assembly by dynamic light scattering (DLS). From the results of particle size measurement by dynamic light scattering in Figure c, it can be known that the diameter of the purchased gold nanoparticles is 1-10 nm, and after multi-stage assembly, the particle size distribution of the composite is not uniform, ranging from 0-1000 nm, but the particle size is generally greater than 500 nm, indicating that the ternary system has been successfully assembled, and at the same time it provides a theoretical basis for the later explanation of the antibacterial mechanism.

[0071] Example 5

[0072] Perform a tissue repair experiment on MRSA-infected wounds using the composite gel containing chitosan, gold nanoparticles and sodium hyaluronate prepared in Example 1. The specific method is as follows: After acclimating the experimental mice to the environment for one week, fast the experimental mice on the day of the experiment, shave the hair on the back of the mice, and weigh them. Anesthetize the mice by intraperitoneal injection of 10% chloral hydrate at a ratio of 300 mg / kg. After 10 minutes, the mice enter the anesthetized state. Apply the depilatory cream evenly on the back of the mice. After 5 minutes, wipe it clean with an alcohol cotton ball and disinfect it. Under sterile conditions, use a marker pen to mark a square with a side length of 2 cm on both sides of the spine on the back of the mice, and subtract the full-thickness skin along the mark to prepare a wound surface of full-thickness skin injury. Subsequently, wrap a layer of gauze equivalent to the wound surface area on the window surface, wrap it with tape, and groupwise drop MRSA bacterial solution (10 8 CFU / mL, 100 μL) on the surface of the gauze. After 1 hour, the bacteria are in sufficient contact with the wound. Re-anesthetize the mice, groupwise add the materials, and conduct subsequent evaluation or treatment.

[0073] Divide 25-27 g of BALB-C male mice into 10 groups, one cage for each group, and 3 mice in each group. Generally, anesthetize the mice by injecting 5% chloral hydrate with a mass fraction of 0.5% according to the body weight. Inject 180 μL of chloral hydrate into the abdominal cavity of each mouse. After about 10 minutes, the mice enter the anesthetized state. Use a hair clipper to shave the back of the mice, and then evenly apply a layer of depilatory cream. Wait for a while, and then wipe the back alternately with dry and wet paper towels to remove the depilatory cream and the remaining hair. Use a circular mold with an area of 1 cm 2 to make marks on the back of the mice, and then use a scalpel and forceps to cut off the skin in the marked area to obtain skin defect wound surfaces with as similar area as possible.

[0074] On the second day, re-anesthetize the mice. For the mice in Groups 1-10, apply Staphylococcus aureus ATCC43300 at a concentration of 10 8 CFU / mL to the wound surfaces, allowing the bacteria to come into full contact with the wounds. On the third day, collect samples. Use a cotton swab dipped in PBS to evenly contact the wound surfaces of the mice, break off part of the cotton swab and put it together into a PC tube containing 1 mL of PBS, shake well, after diluting 50 times, take 100 μL and plate it on a culture plate in a sterile laminar flow hood. After plating, let it dry and invert it, incubate at 37 °C for 12 h and observe the number of colonies. After collecting samples, except for the blank control in Group 1, apply 9 different samples (which are acetic acid solvent, positive control antibiotic, hyaluronic acid, nano-gold, chitosan, composite system of sodium hyaluronate and nano-gold, composite system of nano-gold and chitosan, composite system of chitosan and sodium hyaluronate, and the composite gel containing chitosan, nano-gold and sodium hyaluronate prepared in Example 1) to the wound surfaces of the remaining 9 groups of mice respectively, collect samples and apply the samples for treatment on the 3rd, 5th, 8th, 11th, and 14th days respectively, record the changes in the number of bacteria at the wound sites and the changes in the area of the defective wound surfaces, and observe the treatment effects.

[0075] The results are as Figure 2 shown, Figure 2 It is a schematic diagram of the wound surface healing effect of the composite gel containing chitosan, nano-gold and sodium hyaluronate; the upper figure is a schematic diagram of the healing effect of mice at different times and the anti-Methicillin-resistant Staphylococcus aureus effect of epidermal colonies, and the lower figure is a schematic diagram of the wound healing rate. Figure 2 In the upper figure above, it is the general view of the mouse wounds after being treated with different components from 1 to 14 days, as well as the number of colonies on the corresponding wound surfaces. The experimental results prove that the composite gel containing chitosan, nano-gold and sodium hyaluronate has good synergistic healing ability and good antibacterial performance. The synergistic effect of multiple components not only improves the antibacterial ability but also increases the healing effect; the lower figure is a schematic diagram of the statistical analysis results of the wound healing rate of mouse wounds using imageJ software, which proves that the composite gel containing chitosan, nano-gold and sodium hyaluronate has a wound healing ability of 60%, which is 30% higher than that of the blank group. The composite gel containing chitosan, nano-gold and sodium hyaluronate has good healing ability. From the 1st day to the 14th day, the mouse wounds were continuously treated with the composite gel containing chitosan, nano-gold and sodium hyaluronate prepared in Example 1, and the wound healing rate exceeded 60%, far exceeding the blank control group and the other experimental groups. As a cheap, simple and easy-to-prepare material with good antibacterial and repair integration, it has broad clinical application prospects.

[0076] Example 6

[0077] Perform an in vitro cytotoxicity experiment on the composite gel containing chitosan, nano-gold and sodium hyaluronate prepared in Example 1. The results are as Figure 3 shown, Figure 3Schematic diagram of the in vitro cytotoxicity of the composite gel containing chitosan, gold nanoparticles and sodium hyaluronate.

[0078] The seeding density of mouse fibroblasts (L-929) was 1.2×10⁵ cells / mL. Different concentrations of the material were incubated with the cells for 48 h. First, measure the background before adding the Cell Counting Kit-8 (CCK-8) cell counting reagent CCK-8, and then add CCK-8, 10 μL / well. After incubating for 1.5 h, measure the absorbance at 450 nm (when processing the data, subtract the background from the final value, and the obtained OD value is proportional to the cell viability). The experimental results are as Figure 3 shown in the left figure in the middle. For the single chitosan component, the cytotoxicity increases with the increase of its concentration. When the concentration is 1.25 mg / mL, the cell survival rate is 50%. When the concentration is greater than 1.25 mg / mL, the cytotoxicity is significantly enhanced and reaches an unacceptable range. From Figure 3 the right figure, 1, 2, and 3 represent the composite system diluted 32-fold, 16-fold, and 8-fold respectively. Different concentrations of the composite material CS / AuNPs / HA (0.625, 1.25, 2.5 mg / mL; 25, 50, 100 μg / mL; 0.00025, 0.0005, 0.001 mg / mL) are used. It can be seen that when the composite system is diluted 16-fold (CS 1.25 mg / mL, AuNPs 50 μg / mL, HA 0.0005 mg / mL), the cell survival rate exceeds 50%. According to "GB / T 14233.2-2005", the cytotoxicity grade of the test article should not be greater than grade 2 to be qualified, that is, the cell survival rate is above 50%. Therefore, in the composite gel containing chitosan, gold nanoparticles and sodium hyaluronate prepared in Example 1, the concentrations of each component are CS 0.25 mg / mL, AuNPs 10 μg / mL, and HA 0.0001 mg / mL, which can not only ensure good antibacterial and healing effects, but also keep the cytotoxicity within an acceptable range.

[0079] Example 7

[0080] The composite gel containing chitosan, gold nanoparticles and sodium hyaluronate prepared in Example 1 was subjected to skin irritation and sensitization experiments. The results are as Figure 4 shown. Figure 4 Schematic diagram of the skin irritation and sensitization experiments of the composite gel containing chitosan, gold nanoparticles and sodium hyaluronate.

[0081] 190 - 210 g male SD rats were divided into 4 groups. For the subcutaneous injection of the positive control (Freund's complete adjuvant), 3 rats were set up and named No. 1, No. 2, and No. 3 respectively. For the subcutaneous injection of the stimulation experiment, 3 rats were set up and named No. 4 (low concentration: CS 0.25 mg / mL, AuNPs 10 μg / mL, HA 0.0001 mg / mL), No. 5 (medium concentration: CS 1 mg / mL, AuNPs 40 μg / mL, HA 0.0004 mg / mL), and No. 6 (high concentration: CS 2 mg / mL, AuNPs 80 μg / mL, HA 0.0008 mg / mL). The product application sensitization experiment was set for rat No. 7, and the product application positive control (2,4-dinitrofluorobenzene) was set for 2 rats and named No. 8 and No. 9 respectively.

[0082] According to the "Skin Reaction Scoring System" and "Intradermal Reaction Scoring System" in "GB / T16886.10 - 2017", when there is no erythema, the irritation score is 0; for very slight erythema (barely visible), the score is 1; for distinct erythema, the score is 2; for moderate erythema, the score is 3; for severe erythema (purple-red) to eschar formation where erythema grading cannot be performed, the score is 4. In the positive control group of rats No. 1, No. 2, and No. 3, moderate or severe erythema (greater than 2 points) appeared on both sides of the spinal column on the back of the rats. This may be because after injecting Freund's complete adjuvant into 3 stimulation areas on the right side of the spinal column, the stimulant entered the internal environment of the rats and participated in the blood circulation of the back, resulting in the spread of back irritation and causing a whole piece of erythema. For all rats injected subcutaneously, small bumps appeared in the area about 2 mm around the injection point. However, they disappeared with metabolism on the second day, which is a normal phenomenon. On the first day, due to hair removal of the rats, it had a slight impact on the back skin. Therefore, the skin may show redness on the first day, which is not considered and scored as erythema. 2 In the experimental group, no erythema occurred in the skin of rats injected subcutaneously with 3 different concentrations of the product for 4 - 5 days. Similarly, no erythema occurred in the skin of rats in the application experimental group of No. 7. The skin conditions on both the left and right sides of the spinal column were the same, and the score was not more than 1 point, meeting the requirements. From the positive control group with skin application of No. 8 and No. 9, it can be seen that distinct erythema, moderate erythema, and severe erythema occurred in the area on the right side of their spinal columns, and obvious sensitization reactions occurred, while the area on the left side of the spinal column was normal. The data of rats No. 4, No. 5, and No. 9 on the first day were missing because when first injecting 10% chloral hydrate, the anesthetic dose was too large, resulting in the death of the rats. On the second day, other healthy rats were used for synchronous experiments, and the time, process, and results of this experiment all met the national standards for the biological evaluation of medical devices.

[0083] In the experimental group, no erythema occurred in the skin of rats injected subcutaneously with 3 different concentrations of the product for 4 - 5 days. Similarly, no erythema occurred in the skin of rats in the application experimental group of No. 7. The skin conditions on both the left and right sides of the spinal column were the same, and the score was not more than 1 point, meeting the requirements. From the positive control group with skin application of No. 8 and No. 9, it can be seen that distinct erythema, moderate erythema, and severe erythema occurred in the area on the right side of their spinal columns, and obvious sensitization reactions occurred, while the area on the left side of the spinal column was normal. The data of rats No. 4, No. 5, and No. 9 on the first day were missing because when first injecting 10% chloral hydrate, the anesthetic dose was too large, resulting in the death of the rats. On the second day, other healthy rats were used for synchronous experiments, and the time, process, and results of this experiment all met the national standards for the biological evaluation of medical devices.

[0084] Example 8

[0085] Literature reports indicate that the higher the degree of acetylation of chitosan, the higher the level of exposed amino groups. In an acidic environment, the amino groups are protonated, resulting in high antibacterial properties. When purchasing commercially available chitosan with a relatively high degree of deacetylation, the molecular weight of commercial chitosan is generally not measured, and the viscosity is used as the unit. The higher the viscosity, the larger the molecular weight. However, if the viscosity is too high, the solubility decreases. When selecting chitosan with different viscosities for antibacterial properties, the higher the chitosan concentration, the better the antibacterial performance, and synergistic optimization can be carried out.

[0086] Figure 5 It is a schematic diagram of the antibacterial activity of chitosan with different viscosities and the antibacterial properties varying with concentration. Figure 5 In it, the upper two figures are schematic diagrams of the antibacterial effects of chitosan with different viscosities against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, and the lower two figures are schematic diagrams of the antibacterial properties of CS3 against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa varying with concentration. It can be seen from the upper two figures that different chitosans are CS1 with a viscosity of 50 - 100 Pa·s, CS2 with a viscosity of 100 - 200 Pa·s, CS3 with a viscosity of 200 - 400 Pa·s, CS4 with a viscosity of 400 - 800 Pa·s, and CS5 with a molecular weight less than 3000 Da. Experiments have proved that the performance of CS3 is better, which may be due to its high viscosity and large molecular weight, more exposed amino groups, and high antibacterial properties. However, when the viscosity increases further, the dissolved content is limited, resulting in weakened activity. It can be seen from the lower two figures that the antibacterial properties increase continuously with the concentration of CS3, and CS3 can be used as a preferred system for active antibacterial.

[0087] In the present invention, different solvents are used for dissolution, and the antibacterial properties are different. Specifically, citric acid has a weak acidity and poor dissolution ability, and a higher pH needs to be adjusted to dissolve chitosan. If the acidity is too high, it will affect the biological application of chitosan. Formic acid has a strong acidity and can be used as a solvent in the application of chitosan, but formic acid is not as mild as acetic acid and is not conducive to the pre-clinical biological application.

[0088] In the present invention, nano-gold is used as the only nano-carrier of the system, which has a new antibacterial mechanism, such as Figure 6 shown Figure 6 It is a schematic diagram of the antibacterial properties of nano-gold with different concentrations against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. However, nano-gold itself has weak antibacterial properties. Experiments have proved that with the increase in the concentration of nano-gold, there is no concentration-dependent antibacterial property. Based on this, the antibacterial concentration at a low concentration is selected as the assembly system of the composite material to verify the performance of synergistic enhancement.

[0089] In the present invention, if the concentration of sodium hyaluronate is too high, it will affect the state of the overall system, and the two polyelectrolytes form a gel in the solution. The concentration used in the present invention can make the system uniform, and the other concentrations can be better applied, and it will also increase the ability of wound healing.

[0090] In the present invention, the mass ratio of chitosan, nano-gold, and sodium hyaluronate is optimized one by one according to the antibacterial performance. Finally, it is proved that they have a synergistic effect. Experiments have shown that the concentration ratio of chitosan, nano-gold, and sodium hyaluronate can promote their synergistic antibacterial performance. The concentration of sodium hyaluronate is the optimal concentration selected to reduce the gelation of the system. The gel prepared in the present invention is a gel with antibacterial and healing properties. The composite components with an in vitro concentration of 0.25 mg / mL chitosan, 0.01 mg / mL nano-gold, and 0.0001 mg / mL sodium hyaluronate are used. The study of epidermal wounds will expand the four-fold system because there are differences between in vitro and in vivo studies.

[0091] In the present invention, different ratios of polyethylene glycol 400 and polyethylene glycol 4000 will affect the viscosity of the system. The ratio of polyethylene glycol 400 and polyethylene glycol 4000 selected in the present invention can enable the product to be better adsorbed on the wound. When the proportion of polyethylene glycol 4000 decreases and the concentration is too dilute, it is not conducive to the application of the gel product; when the proportion of polyethylene glycol 4000 increases, the viscosity of the system will increase after cooling.

[0092] Comparative Example 1

[0093] The patent application with the application number 202111249587.6 and the invention title of "A Mupirocin Ointment and Its Preparation Method" takes Example 1 of this invention as Comparative Example 1 of the present invention, specifically as follows:

[0094] Dissolve 1 g of citric acid in 391 g of polyethylene glycol 400, then add 98 g of polyethylene glycol 4000, heat it to melt in a water bath at 60 °C, then add 10 g of mupirocin, stir in a water bath at 60 °C to dissolve mupirocin, mix evenly, and cool to room temperature to obtain mupirocin ointment.

[0095] The prepared product has the advantages of good antibacterial effect and convenient application. However, it involves the use of antibiotics, and the overuse of antibiotics will induce bacterial drug resistance, especially the generation of multi-drug resistant bacteria.

[0096] Comparative Example 2

[0097] Take Example 3 in the patent application with the publication number CN114097788A as Comparative Example 2 of the present invention. The specific method steps are as follows:

[0098] (1) Measure 10 ml of dimethyl sulfoxide and dissolve 0.5 g of chitosan in it.

[0099] (2) Ultrasonically disperse 0.3 g of nano-aluminum oxide in 5 ml of water, and then add the nano-aluminum oxide dispersion to solution (1).

[0100] (3) Ultrasonically disperse 0.01 g of carbamide peroxide in solution (2).

[0101] (4) Dissolve 0.01 g of ammonium nitrate in the solution of (3).

[0102] (5) Stir the above solution at a speed of 500 r / min to obtain a kind of Fenton-like slow-release hydrogel.

[0103] The present invention prepares a novel antibacterial gel. This composite system has a high synergistic antibacterial effect. It not only tests the antibacterial properties against a variety of different Gram-positive and Gram-negative bacteria, showing broad-spectrum antibacterial ability, but also the biocompatibility and effectiveness are verified. Through the treatment of mouse epidermis and the tests of product sensitivity and irritation, it is verified that this composite gel has high biocompatibility and effectiveness and can be effectively applied to clinical research.

[0104] The above are only the preferred embodiments of the present invention, and there is no any form of limitation to the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to equivalent embodiments by using the technical content prompted above. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the present invention.

Claims

1. A preparation method of a composite gel containing chitosan, nano-gold and sodium hyaluronate, characterized in that, it comprises the following steps: First step, mix polyethylene glycol 400 and polyethylene glycol 4000 with a mass ratio of 1:(0.1 - 10), heat to 50 - 80 °C for 10 - 30 min, maintain the temperature when it cools naturally to 40 - 50 °C, add a chitosan solution to the completely dissolved solution, continue stirring for 15 - 30 min and then stop heating, wait for the solution to cool completely to room temperature, and ultrasonicate for 10 - 30 min; Second step, add a nano-gold solution and stir for 10 - 30 min; Third step, add a sodium hyaluronate solution and stir for 10 - 30 min to obtain the composite gel containing chitosan, nano-gold and sodium hyaluronate; the mass ratio of chitosan, nano-gold, and sodium hyaluronate is (250 - 50000):(1 - 200):1; the deacetylation degree of the chitosan > 95%; The molecular weight of the chitosan is selected from >10 2 Kda; the viscosity of the chitosan is selected from 200 - 400 Pa·s; the concentration of the chitosan solution is selected from 0.5 - 2 mg / mL, the concentration of the nano-gold solution is 0.02 - 0.08 mg / mL, and the concentration of the sodium hyaluronate solution is selected from 0.0002 - 0.0008 mg / mL; the mass ratio of chitosan to polyethylene glycol 400 is (0.001 - 1):1; the chitosan solution is obtained by dissolving chitosan in an acidic solution with pH = 3 - 5; the acidic solution is prepared by dissolving an acid in sterile water, and the acid is selected from acetic acid.

2. The preparation method of the composite gel containing chitosan, nano-gold and sodium hyaluronate according to claim 1, characterized in that, the particle size of the nano-gold is selected from 1 - 10 nm.

3. Application of the composite gel prepared by the method according to claim 1 or 2 in the preparation of antibacterial drugs, tissue repair-promoting drugs, and wound healing-promoting drugs.

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