Polysaccharide-bacitracin conjugate, electrostatic spinning nanofiber dressing, preparation method and application of polysaccharide-bacitracin conjugate and electrostatic spinning nanofiber dressing

Abstract

The invention discloses a polysaccharide-bacitracin conjugate, an electrostatic spinning nanofiber dressing, a preparation method and application of the polysaccharide-bacitracin conjugate and the electrostatic spinning nanofiber dressing. The electrostatic spinning nanofiber dressing disclosed by the invention has good skin adhesion and moisturizing performance, and also shows good sterilization and bacteriostasis performance on common gram-positive bacteria and low cytotoxicity. In addition, the dressing also has excellent biocompatibility, and has a good repairing effect on skin deficiency injury.

Description

Technical Field

[0001] The present application belongs to the field of medical antibacterial materials, and in particular relates to a polysaccharide-bacitracin conjugate, an electrospun nanofiber dressing, a preparation method and applications thereof. Background Art

[0002] Polysaccharide is a high molecular weight compound composed of multiple monosaccharide molecules connected by glycosidic bonds, usually composed of more than 10 monosaccharides. Polysaccharide is a sugar chain connected by glycosidic bonds, with a chemical formula of (C6H 10 O5) n Polysaccharides have a complex structure, including primary structures (composition, arrangement, and connection of sugar groups) and higher-order structures (such as secondary, tertiary, and quaternary structures). Dissolving in solvents can enhance adhesion, and their inherent low cytotoxicity can be used in skin tissue engineering. Modifying polysaccharides or grafting them with active compounds can enhance the diversity of the final compound's properties.

[0003] Electrospinning is a processing technique that uses a high-voltage electrostatic field to stretch a polymer solution or melt into ultrafine fibers. It boasts simple equipment, flexible operation, and low cost, making it widely used in the preparation of nanofiber materials. Its core principle is to use a high-voltage electrostatic field to charge the polymer solution or melt. The electric field forces the polymer solution or melt to overcome surface tension and form a thin stream. When the electric field is sufficiently strong, the droplet forms a conical structure (a "Taylor cone"), and the thin stream is ejected from the cone tip. The stream is further stretched and refined in the electric field, ultimately solidifying into nanofibers. The preparation of nanofiber materials through electrospinning has been one of the most important academic and technological activities in the field of materials science and technology worldwide over the past decade. With its simple manufacturing equipment, low spinning costs, a wide variety of spinnable materials, and controllable process, electrospinning has become a major method for the effective preparation of nanofiber materials. Electrospinning technology can produce a wide variety of nanofibers. In the biomedical field, nanofibers have a diameter smaller than that of cells and can mimic the structure and biological functions of the natural extracellular matrix. Most human tissues and organs are similar to nanofibers in form and structure, which makes it possible for nanofibers to be used in tissue and organ repair. Some raw materials used for electrospinning have good biodegradability and can be used as carriers to enter the human body and be easily absorbed by the body. In addition, electrospun fibers also have high specific surface area, high porosity, and strong adsorption. Therefore, it has attracted the continuous attention of researchers in the biomedical field and has been well applied in drug controlled release, wound repair, biological tissue engineering, etc. Summary of the Invention

[0004] Purpose of the invention: In order to deal with skin wound infections caused by various factors, this study used polysaccharides and bacitracin as raw materials to synthesize a new compound through a dehydration condensation reaction, and then mixed the compound with polyvinyl alcohol and prepared it into a wound dressing through electrospinning technology. The physicochemical properties and biomedical properties of the dressing were systematically characterized. The prepared dressing has both the high adhesion of polysaccharides and the high antibacterial properties of bacitracin, and also has good morphological characteristics, mechanical properties, thermodynamic properties and hemostatic properties. It can quickly absorb wound exudate and maintain a moist steady-state microenvironment in the wound. In addition, the dressing can effectively promote rapid wound healing in a full-thickness skin injury model infected with Staphylococcus aureus. These excellent properties indicate that the electrospun nanofiber dressing developed in this study has great potential in repairing infected skin.

[0005] Technical solution: The present invention provides a polysaccharide-bacitracin conjugate (SA-BAC). The structural formula of the polysaccharide-bacitracin conjugate is as follows:

[0006]

[0007] In some embodiments, the polysaccharide-bacitracin conjugate is prepared by the following method: EDC and NHS are added to an aqueous solution of sodium alginate, followed by adding bacitracin, and after incubation, the carboxyl group in the sodium alginate is coupled with the amino group in the bacitracin, and finally the resulting reactant is purified to obtain a sugar-peptide conjugate.

[0008] The examples of the present application also provide the use of the above-mentioned polysaccharide-bacitracin conjugate in the preparation of antibacterial materials.

[0009] In some embodiments, the polysaccharide-bacitracin conjugate of the present application is used in the preparation of anti-Gram-positive bacteria materials.

[0010] In some embodiments, the polysaccharide-bacitracin conjugate of the present application is used in the preparation of an antibacterial agent for inhibiting Staphylococcus aureus.

[0011] In some embodiments, the MIC of the polysaccharide-bacitracin conjugate against Staphylococcus aureus is 40 μg / mL.

[0012] An embodiment of the present application also provides an electrospun nanofiber dressing, which includes polyvinyl alcohol and the above-mentioned polysaccharide-bacitracin conjugate, and the mass ratio of the polyvinyl alcohol to the polysaccharide-bacitracin conjugate is (15-20):1.

[0013] In some embodiments, the electrospun nanofiber dressing comprises polyvinyl alcohol and polysaccharide-bacitracin, and the mass ratio of polyvinyl alcohol to polysaccharide-bacitracin is 16:1.

[0014] In some embodiments, the adhesion force of the electrospun nanofiber dressing is 0.29±0.02N.

[0015] In some embodiments, the contact angle of the electrospun nanofiber dressing is 57.17±2.1°, and the penetration time of the electrospun nanofiber dressing is 10.836±0.72 s.

[0016] In some embodiments, the polyvinyl alcohol (PVA) is PVA-1788.

[0017] An embodiment of the present application also provides an electrospun nanofiber dressing, which includes polyvinyl alcohol, sodium alginate and bacitracin. The mass ratio of the polyvinyl alcohol to the sum of the masses of the sodium alginate and the bacitracin is (15-20):1, and the mass ratio of the sodium alginate to the bacitracin is 1:(2-5).

[0018] In some embodiments, the electrospun nanofiber dressing is prepared by the following method:

[0019] Polyvinyl alcohol and polysaccharide-bacitracin conjugate are mixed to obtain a spinning solution, which is then electrospun to obtain a nanofiber dressing;

[0020] In some embodiments, the electrospun nanofiber dressing is prepared by the following method: polyvinyl alcohol, sodium alginate and bacitracin are mixed to obtain a spinning solution, and then electrospinning is performed to obtain a nanofiber dressing.

[0021] In some embodiments, the electrospinning parameters are: voltage: 20 kV; flow rate: 0.001-0.0013 mm / s; receiving distance: 18 cm; receiving cylinder speed: 100 r / min; environmental parameter temperature: 25.0±2.0° C.; humidity: 27.0±2.0%.

[0022] The present invention also provides a method for preparing an electrospun nanofiber dressing, comprising the following steps:

[0023] Polyvinyl alcohol and polysaccharide-bacitracin conjugate are mixed to obtain a spinning solution, which is then electrospun to obtain a nanofiber dressing.

[0024] The present invention also provides a method for preparing an electrospun nanofiber dressing, wherein polyvinyl alcohol, sodium alginate and bacitracin are mixed to obtain a spinning solution, and then electrospinning is performed to obtain a nanofiber dressing; sodium alginate and bacitracin are mixed to obtain a spinning solution;

[0025] The embodiments of the present application also provide an electrospun nanofiber dressing or an electrospun nanofiber dressing prepared by the above-mentioned preparation method of the electrospun nanofiber dressing, and its application in the field of antibacterial materials or medical devices.

[0026] The embodiments of the present application also provide an electrospun nanofiber dressing or an electrospun nanofiber dressing prepared by the above-mentioned preparation method of the electrospun nanofiber dressing for use in tissue repair or scar repair.

[0027] The embodiments of the present application also provide an electrospun nanofiber dressing or an electrospun nanofiber dressing prepared by the above-mentioned preparation method of the electrospun nanofiber dressing, and its use in wound dressing and tissue engineering dressing.

[0028] As a specific embodiment, the sodium alginate-bacitracin conjugate of the present invention is prepared by the following method:

[0029] Among them, the polysaccharide is sodium alginate as an example. 0.1g sodium alginate (SA), 0.1g N-hydroxysuccinimide (NHS), and 0.1g 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) are weighed into a 50mL beaker, 20mL distilled water is added, and magnetic stirring is performed for 30 minutes. After thorough mixing, the mixture is transferred to a dialysis bag (MW1000) and dialyzed for 8 hours, with the water changed every hour. After dialysis is completed, the solution in the dialysis bag is collected and transferred to a beaker. 10mL of 0.3g bacitracin aqueous solution is added and stirred overnight. After complete reaction, the mixture is transferred to a dialysis bag (MW7000) and dialyzed for 72 hours, with the water changed every 6 hours. The solution in the dialysis bag is then collected and freeze-dried to obtain sodium alginate-bacitracin conjugate (SA-BAC).

[0030] As a specific embodiment, the preparation method of the electrospun nanofiber dressing described in this application includes the following steps: adding 0.64g of PVA to 10mL of H2O and magnetically stirring for 12 hours. After the solute is completely dissolved, 0.04g of SA-BAC is added (so that the mass ratio of SA-BAC to PVA is 1:16) and continuously stirred for 1 hour until completely dissolved. The SA-BAC spinning solution is filtered and centrifuged to obtain a uniform SA-BAC spinning solution. The solution is then transferred to a 10mL syringe to remove air bubbles and installed on an electrospinning machine. The spinning parameters are adjusted to: voltage, 20kV; flow rate, 0.001-0.0013mm / s; receiving distance, 18cm; receiving drum speed, 100r / min; environmental parameters: temperature, 25.0±2.0℃; humidity, 27.0±2.0%. PSA-BAC dressings were prepared using the electrospinning device. PSA, PBAC, and PSA+BAC dressings were also prepared as controls. After preparation, the prepared dressing was placed in a vacuum drying oven and vacuum dried for at least 12 hours to remove the residual organic solvent on the dressing.

[0031] Beneficial Effects: This application provides a polysaccharide-bacitracin conjugate, an electrospun nanofiber dressing, a preparation method, and applications thereof. The electrospun nanofiber dressing exhibits excellent skin adhesion and moisturizing properties, as well as excellent bactericidal and antibacterial properties against common Gram-positive bacteria and low cytotoxicity. Furthermore, the dressing exhibits excellent biocompatibility and demonstrates a favorable repair effect on skin loss injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 This is the synthesis step of sodium alginate-bacitracin in Example 1 of the present application;

[0034] Figure 2 Synthesis and characterization of sodium alginate-bacitracin prepared in Example 1 of the present application, wherein Figure a shows the synthesis steps of sodium alginate-bacitracin, Figure b shows infrared spectra of sodium alginate, bacitracin, and sodium alginate-bacitracin, Figure c shows a 1H NMR spectrum of sodium alginate, Figure d shows a 1H NMR spectrum of bacitracin, and Figure e shows a 1H NMR spectrum of sodium alginate-bacitracin;

[0035] Figure 3 The antibacterial effect and cytotoxicity results of sodium alginate-bacitracin prepared in Example 1 of the present application, wherein Figure a shows the relationship between bacterial survival rate and time in a sodium alginate-bacitracin (40 μg / mL) solution, and Figure b shows the cytotoxic effects of different concentrations of sodium alginate-bacitracin on L929 and HaCaT cells;

[0036] Figure 4 Preparation and characterization of the nanofiber dressing prepared in Example 4 of the present application, wherein Figure a is a scanning electron microscope image of the dressing, Figure b is the diameter distribution of the dressing, Figure c is a curve diagram of the dressing diameter change, Figure d is a comparative analysis result of infrared spectra of sodium alginate-bacitracin dressing and sodium alginate-bacitracin, Figure e is a TGA curve of the dressing, and Figure f is a typical stress-strain curve of the dressing;

[0037] Figure 5 Performance characterization of the nanofiber dressing prepared in Example 4 of the present application, wherein Figure a shows the blood compatibility of the dressing, Figure b shows the hemolysis rate of the dressing, Figure c shows the determination of the adhesion of the dressing, Figure d shows the adhesion statistics of the dressing, Figure e shows a representative water contact angle image of the dressing, Figure f shows the static water contact angle of the dressing, and Figure g shows the ultrapure water penetration time of the dressing;

[0038] Figure 6 The cytocompatibility and antibacterial properties of the nanofiber dressing prepared in Example 4 of the present application are shown in Figure a, wherein Figure a is a live / dead staining image of L929 cells under the influence of the dressing on day 1 and day 3, Figure b is a live / dead staining image of HaCaT cells under the influence of the dressing on day 1 and day 3, Figure c is the L929 cell proliferation rate statistics under the influence of the dressing, Figure d is the HaCaT cell proliferation rate statistics under the influence of the dressing, Figure e is a colony growth diagram under the influence of the dressing, Figure f is the bacterial survival rate statistics of Staphylococcus aureus, and Figure g is the bacterial survival rate statistics of Escherichia coli;

[0039] Figure 7 The tissue regeneration during the skin repair process of mice using the nanofiber dressing prepared in Example 4 of the present application, wherein Figure a is a representative image of H&E staining of damaged mouse skin over time, Figure b is a representative image of Masson trichrome staining of damaged mouse skin over time, Figure c is a representative image of H&E staining and Masson trichrome staining on the 14th day, Figure d is a representative image of THF-α immunohistochemistry staining on the 14th day, Figure e is a statistical image of hair follicle growth in mice, and Figure f is a statistical image of TNF-α grayscale values ​​of skin wounds in each group on the 14th day. DETAILED DESCRIPTION

[0040] The following, in conjunction with the accompanying drawings, provides a clear and complete description of the technical solutions in the embodiments of the present application. It should be noted that the described embodiments are only a portion of the embodiments of the present application, and not all of the embodiments. All other embodiments derived by persons skilled in the art based on the embodiments of the present application without inventive effort are within the scope of protection of the present application. Furthermore, in the description of the present application, the term "including" means "including but not limited to." The terms "first," "second," and "third," etc., are used merely as designations and do not impose numerical requirements or establish a sequence. Various embodiments of the present application may be presented in the form of a range. It should be understood that describing in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application. Therefore, the range description should be considered to specifically disclose all possible subranges and individual numerical values ​​within the range. For example, a range description of 1 to 6 should be considered to specifically disclose subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.

[0041] Sodium alginate (SA) is a linear, unbranched polysaccharide containing varying amounts of 1,4-linked β-D-mannuronic acid and α-L-guluronic acid residues. The composition and sequence of the residues can vary widely and are arranged in a block-like pattern along the chain. These homopolymer regions of β-D-mannuronic acid blocks and α-L-guluronic acid blocks are interspersed with regions of alternating structure (β-D-mannuronic acid-α-L-guluronic acid blocks). The molecular structure of algae is similar to cellulose, containing a large number of hydroxyl groups. Sodium alginate molecules have strong intra- and inter-molecular hydrogen bonds, resulting in strong interactions. Dissolving sodium alginate in a medium can increase the viscosity of the medium.

[0042] Bacitracin (BAC) is a polypeptide antibiotic produced by Bacillus licheniformis and Bacillus subtilis. Bacitracin exhibits broad antimicrobial spectrum activity, strong activity, and low drug resistance, making it widely used in medicine and veterinary medicine. It acts by binding to undecaprenyl pyrophosphate, acting as an inhibitor of cell wall biosynthesis. This damages the cell membrane, leading to an efflux of ions and amino acids and bacterial death. In clinical practice, systemic bacitracin may cause severe nephrotoxicity and is therefore generally not recommended. Bacitracin is typically used as a topical treatment, for example, for localized skin and soft tissue infections, skin wound infections, infections following extensive surgery or burns, and conjunctival infections. Because it is mildly irritating when applied topically, allergic reactions are rare, and bacteria develop resistance relatively slowly, it is often used as a topical anti-infective.

[0043] The molecular structure of sodium alginate is shown below:

[0044]

[0045] The structure of bacitracin is shown below:

[0046]

[0047] Sodium alginate (SA) is a water-soluble polysaccharide that can improve adhesion when dissolved in water. It also has good biocompatibility and low cytotoxicity, so SA is widely used in the field of wound dressings. However, due to the weak antibacterial properties of SA itself, it may not be able to effectively inhibit bacterial growth in some infected wounds or high-risk wounds. To solve this problem, this application combines SA with antimicrobial agents such as antimicrobial peptides.

[0048] The preparation steps of the sugar-peptide conjugate are to add EDC and NHS to an aqueous solution of sodium alginate, and then add bacitracin. After the reaction is incubated, the carboxyl group in the sodium alginate is coupled with the amino group in the bacitracin. Finally, the resulting reaction product is purified to obtain a sugar-peptide conjugate with the following structure:

[0049]

[0050] The polysaccharide-peptide conjugate of the present application has good skin adhesion and moisturizing capacity, and exhibits good bactericidal performance on common gram-positive bacteria. Experimental results show that after the bacteriocin with antibacterial effect is coupled with the polysaccharide, the antibacterial activity is maintained, and the biocompatibility of the polysaccharide and the polypeptide is inherited, the toxicity problem caused by the traditional material is reduced, the toxicity and immunogenicity to mammalian cells are reduced, a synergistic effect is generated between the polysaccharide and the bacteriocin, the biological activity is collectively enhanced, the water solubility of the graft product of the polysaccharide and the bacteriocin is improved, after the polysaccharide-bacteriocin graft, the graft product has better thermal stability and hydrophilicity, is more stable in a physiological environment, can prolong the half-life of the polypeptide, and thus improves the curative effect.

[0051] Further, the sugar-peptide conjugate is mixed with polyvinyl alcohol (PVA) and prepared into an antibacterial preparation, an antibacterial material, a wound dressing, and an application in a medical material by an electrospinning technology.

[0052] The inventor team of the applicant adopts the electrospinning fiber technology to prepare the sugar-peptide conjugate and polyvinyl alcohol (PVA) into an electrospinning fiber membrane, and the following advantages are achieved: (1) the electrospinning fiber membrane has an extremely high specific surface area and porosity, which makes it excellent in applications such as filtration and adsorption; (2) the electrospinning fiber membrane usually has high mechanical strength and stability, and is suitable for application scenarios that need to bear certain mechanical stress, and compared with the hydrogel commonly seen in the prior art, the problem that the hydrogel is usually soft and has relatively weak mechanical properties is overcome; (3) the electrospinning fiber membrane can maintain stable performance in extreme environments such as dryness, wetness, and high temperature, while the hydrogel may lose water and become hard in extreme environments, resulting in reduced stability; (4) the electrospinning technology is simple to operate and has low cost, and is suitable for large-scale production. In comparison, the preparation of the hydrogel may need a complex crosslinking process; (5) the electrospinning fiber membrane performs well in medical dressings and tissue engineering scaffolds, and its structure is similar to the extracellular matrix, which is conducive to the adhesion, proliferation, and differentiation of cells.

[0053] In some embodiments, the present application provides a preparation method of a polysaccharide-peptide conjugate (SA-BAC), which dissolves SA in water, then adds catalysts EDC and NHS, waits for EDC to react with the carboxyl group of SA to form an O-acylisourea intermediate, which is then mixed with NHS to generate a stable amino reactive ester, and then adds an aqueous BAC solution to synthesize SA-BAC through a dehydration condensation reaction.

[0054] Weigh 0.1g sodium alginate (SA), 0.1g N-hydroxysuccinimide (NHS), and 0.1g 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) into a 50mL beaker, add 20mL of distilled water, and stir with a magnetic stirrer for 30 minutes. After thorough mixing, transfer the solution to a dialysis bag (MW1000) and dialyze for 8 hours, changing the water every hour. After dialysis is complete, collect the solution in the dialysis bag and transfer it to a beaker. Add 10mL of 0.3g bacitracin aqueous solution and stir overnight. After complete reaction, transfer the solution to a dialysis bag (MW7000) and dialyze for 72 hours, changing the water every 6 hours. Then collect the solution in the dialysis bag, freeze-dry it, and store it.

[0055] In some embodiments, the present application provides a method for preparing an electrospun nanofiber dressing () containing a polysaccharide-peptide conjugate (SA-BAC).

[0056] 0.64 g of PVA was added to 10 mL of H₂O and magnetically stirred for 12 hours. After complete dissolution, 0.04 g of SA-BAC (making the mass ratio of SA-BAC to PVA 1:16) was added and stirred continuously for 1 hour until complete dissolution. The solution was then filtered and centrifuged to obtain a uniform SA-BAC spinning solution. The solution was then transferred to a 10 mL syringe to remove air bubbles and installed on an electrospinning machine. Spinning parameters were adjusted to: voltage, 21 kV; flow rate, 0.001-0.0013 mm / s; receiving distance, 18 cm; receiving drum speed, 100 rpm; ambient temperature, 25.0 ± 2.0°C; and humidity, 27.0 ± 2.0%. PSA-BAC dressings were prepared using the electrospinning apparatus. PSA, PBAC, and PSA+BAC dressings were also prepared as controls. After preparation, the dressings were placed in a vacuum drying oven and dried for at least 12 hours to remove any residual organic solvent.

[0057] In some embodiments, the present application provides another method for preparing an electrospun nanofiber dressing (PSA+BAC):

[0058] 0.64 g of PVA was added to 10 mL of H₂O and magnetically stirred for 12 h. After complete dissolution, 0.04 g of sodium alginate and bacitracin (mass ratio of sodium alginate to bacitracin:1:3) were added and stirred continuously for 1 h until complete dissolution. The solution was filtered and centrifuged to obtain a uniform SA-BAC spinning solution. The solution was then transferred to a 10 mL syringe to remove air bubbles and installed on an electrospinning machine. The spinning parameters were adjusted to: voltage, 21 kV; flow rate, 0.001-0.0013 mm / s; receiving distance, 18 cm; receiving drum speed, 100 rpm; ambient temperature, 25.0 ± 2.0°C; and humidity, 27.0 ± 2.0%. PSA-BAC dressings were prepared using the electrospinning device. PSA, PBAC, and PSA+BAC dressings were also prepared as controls. After preparation, the prepared dressing was placed in a vacuum drying oven and vacuum dried for at least 12 hours to remove the residual organic solvent on the dressing.

[0059] The test results of the two electrospun nanofiber dressings prepared in this application compared with the control group showed the following advantages:

[0060] (1) Morphological analysis: The fiber diameter of PSA+BAC is 211 nm, and the fiber diameter of PSA-BAC is 221 nm. The results show that the viscosity of the electrospinning solution of PSA-BAC increases, which weakens the stretching effect of the electric field force, increases the surface tension, reduces the jet stability and stretching ability, and ultimately leads to a thicker diameter of the spun fiber.

[0061] (2) Thermodynamic performance analysis: The thermal decomposition temperatures of PSA+BAC and PSA-BAC were both maintained above 90.0°C, indicating that the dressings could maintain the durability and consistency of their performance during storage and use even under temperature fluctuations.

[0062] (3) Blood compatibility analysis: The hemolysis rate of PSA+BAC was 1.46±0.06%, and the hemolysis rate of PSA-BAC was 0.15±0.04%. The results showed that the hemolysis rates of both dressings were lower than the national standard (5%), but the lower hemolysis rate of PSA-BAC indicated that adverse reactions to blood were less likely to occur.

[0063] (4) Adhesion performance analysis: The adhesion of PSA+BAC was 0.2±0.01 N, and the adhesion of PSA-BAC was 0.29±0.02 N. The results showed that PSA-BAC had stronger adhesion and could increase the adhesion on the skin.

[0064] (5) Analysis of hydrophilic and moisturizing properties: The water contact angle of PSA+BAC was 56.43±1.85°, and the water drop penetration time was 10.95±1.11s. The water contact angle of PSA-BAC was 57.17±2.1°, and the water drop penetration time was 10.836±0.72s. The results showed that the contact angles of both dressings were less than 90°, indicating that the dressings were hydrophilic, keeping the wound moist and avoiding dryness and adhesion. This property helps maintain a moist environment in the wound, thereby promoting cell proliferation and tissue repair. The water drop penetration time of the two dressings was basically the same and was in a relatively ideal state, showing good permeability, ensuring rapid absorption of wound exudate in a moist microenvironment and accelerating wound healing.

[0065] (6) Cytotoxicity analysis: The proliferation rates of L929 cells treated with PSA+BAC on the first and third days were 156.67±1.63% and 120.01±2.88%, respectively. The proliferation rates of HaCaT cells treated with PSA-BAC on the first and third days were 130.37±0.61% and 108.16±2.81%. The results showed that the cell growth rate of both cell types on the third day was lower than that on the first day, but all dressings had a growth-promoting effect on cells within three days. Therefore, the cell proliferation rate indicates that the dressing has good cytocompatibility and a positive effect on cell proliferation.

[0066] (7) Antibacterial performance analysis: The inhibition rates of PSA+BAC against Staphylococcus aureus and Escherichia coli were 85.71% and 58.71%, respectively. The inhibition rates of PSA-BAC against Staphylococcus aureus and Escherichia coli were 99% and 85.48%, respectively. The results showed that when the dressing dosage was the same, the inhibition rates of PSA+BAC and PSA-BAC against Escherichia coli were lower than those against Staphylococcus aureus. The antibacterial performance of PSA-BAC against Staphylococcus aureus and Escherichia coli was superior to that of PSA+BAC.

[0067] The applicant's inventor team further compared the two electrospun nanofiber dressings with the electrospun nanofiber dressing PSA (PVA+SA) and the electrospun nanofiber dressing PBAC (PVA+BAC). The performance of the above two electrospun nanofiber dressings (PSA-BAC, PSA+BAC) was improved.

[0068] The technical solution of the present invention is further described below with reference to the embodiments:

[0069] Example 1: Synthesis and structural characterization of sodium alginate-bacitracin (SA-BAC) conjugate

[0070] like Figure 1As shown, sodium alginate-bacitracin was synthesized by the following method: 0.1g sodium alginate (SA), 0.1g N-hydroxysuccinimide (NHS), and 0.1g 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were weighed into a 50mL beaker, 20mL of distilled water was added, and magnetic stirring was performed for 30 minutes. After thorough mixing, the mixture was transferred to a dialysis bag (MW1000) and dialyzed for 8 hours, with the water changed every hour. After dialysis, the solution in the dialysis bag was collected and transferred to a beaker. 10mL of 0.3g bacitracin aqueous solution was added and stirred overnight. After complete reaction, the mixture was transferred to a dialysis bag (MW7000) and dialyzed for 72 hours, with the water changed every 6 hours. The solution in the dialysis bag was then collected, freeze-dried, and stored. The structure of the product was confirmed by infrared spectroscopy and H-NMR spectroscopy.

[0071] SA, BAC, and SA-BAC samples were freeze-dried to remove excess moisture that could affect the analysis results. The treated samples were placed on the FT-IR (Nicolet IS50) detection table. The detection light spots were completely covered and the experimental parameters were set: the detection wavelength was 4000-500 cm -1 , the detection frequency is 4cm -1 The number of scans was 32. SA, BAC and SA-BAC samples were freeze-dried to remove the influence of excess water on the analysis results, and then the samples were dissolved in 99.9% D2O. 1 H NMR spectra were recorded at 300 K using a Bruker Avance III HD 600 NMR spectrometer equipped with a Bruker CP TCI helium cryoprobe (Bruker, Rheinstetten, Germany) at a frequency of 600 MHz using sodium 3-trimethylsilylpropionate as the internal standard.

[0072] The experimental results are as follows Figure 2 As shown in Figure b, in the SA infrared curve, the -COOH and -OH stretching vibration absorption peaks are located at 1592 cm -1 and 1022cm -1 The stretching vibration peak of the -CO- bond is located at 1024 cm -1 and 1083cm -1 The expression of these IR characteristic peaks is basically consistent with the structure of polysaccharides. In the infrared spectrum of BAC, 1645cm -1 、1515cm -1 、1386cm -1 and 1227cm -1 They are the stretching vibration absorption peaks of -C=O, NH in amide, -CN in lactam, and -CN in nitrogen-containing heterocycle. From the SA-BAC spectrum, at 1100cm -1The characteristic peak of SA was observed near 3300 cm -1 A peak belonging to BAC but not present in the SA pattern was observed at 3314 cm -1 and -C=O at 1599cm -1 Stretching vibration absorption peak. At 1725cm -1 The small peak at is considered to be the -C=O peak of the newly generated amide formed by the grafting of BAC and SA.

[0073] The NMR spectrum of SA-BAC is as follows Figure 2 As shown in Figures c, d, and e, 1 The synthesis of SA and BAC was confirmed by H NMR. During the synthesis, -COOH on SA combined with -NH2 on BAC to form -CONH. The newly formed amide proton peak appeared at 8.64 ppm. 1 The benzene ring protons (δ 7.2) appeared in the HNMR spectrum. These results indicate that the two substances are chemically coupled, indicating that the coupling of the two substances is completed by a dehydration-condensation reaction between the amino and carboxyl groups.

[0074] Example 2: Antibacterial experiment of sodium alginate-bacitracin (SA-BAC) conjugate

[0075] The MIC of SA-BAC was investigated using the serial dilution method. SA-BAC was dissolved in PBS at concentrations of 320 μg / mL, 160 μg / mL, 80 μg / mL, 40 μg / mL, and 20 μg / mL, respectively. The test bacterial suspension was resuspended in fresh LB culture medium and then diluted with PBS until the absorbance at 600 nm was between 0.08 and 0.1 as measured by the Ludwig-Beer law on a UV spectrophotometer. At this point, the concentration of the bacterial suspension was 1×10 8 CFU / mL, after preparing the bacterial solution, add 100μL per well to a 96-well plate, set up 4 replicates for each concentration, and set up a blank control group. Add the diluted SA-BAC solution to make the final concentrations of 160μg / mL, 80μg / mL, 40μg / mL, 20μg / mL, and 10μg / mL. Then place it in a biochemical incubator and incubate at 37°C. The absorbance values ​​at a wavelength of 600nm are measured at 8h, 16h, 24h, 32h, 40h, and 48h. The absorbance values ​​of the experimental group are compared with those of the blank control to obtain the bacterial survival rate.

[0076] The experimental results are as follows Figure 3As shown in Figure a, experimental data indicates that at a concentration of 40 μg / mL, SA-BAC exhibits an inhibition rate against S. aureus exceeding 80% within 24 hours, while inhibition against E. coli is not evident until 32 hours. Therefore, the MIC (minimum inhibitory concentration) of SA-BAC against S. aureus is 40 μg / mL, but its inhibitory effect on E. coli is not significant. Overall, SA-BAC exhibits a strong inhibitory effect against S. aureus and exhibits excellent antibacterial properties against infected wounds caused by Gram-positive bacteria.

[0077] Example 3: Cytocompatibility test of sodium alginate-bacitracin (SA-BAC) conjugate

[0078] The cytotoxicity of SA-BAC was investigated by serial dilution method. SA-BAC was diluted with high-glucose DMEM cell culture medium to concentrations of (2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, and 125 μg / mL) for later use. Cells were cultured to the 3rd to 5th generation, at which time the cells had the best activity. The cells (3rd to 5th generation) were digested with 1 mL of trypsin-EDTA digestion solution, pipetted and transferred to a centrifuge tube for centrifugation (1000 rpm, 5 min), the supernatant was discarded, and the cells were resuspended with 1 mL of high-glucose DMEM cell culture medium to obtain a cell mother solution. The cell mother solution was diluted to a concentration of 8×10 4 When the cells reach 500 μg / mL, inoculate 100 μL per well into a 96-well plate and incubate overnight in a 37°C, 5% CO2 incubator. When the cells have grown to the bottom of the plate, discard the mother solution. Add the corresponding SA-BAC dilution to the experimental group and set up 5 replicates. Set up a blank control group and continue incubation for 24 hours. After incubation, discard the liquid in the plate and add CCK-8 detection solution. Set up a CCK-8 detection solution group. After incubation for 1 hour in the dark, measure the absorbance at a wavelength of 450 nm. Compare the absorbance of the experimental group with that of the blank group to express the cytotoxicity of SA-BAC.

[0079] The experimental results of SA-BAC cytotoxicity are as follows Figure 3 As shown in Figure 2(b), at the maximum SA-BAC concentration, the viability of L929 and HaCaT cells was 100.57±4.06% and 108.48±2.87%, respectively. At the lowest SA-BAC concentration, the viability of L929 and HaCaT cells was 119.86±2.68% and 124.95±3.91%, respectively. Overall, the viability of cells co-cultured with SA-BAC was higher than that of the blank control group. This indicates that SA-BAC is not cytotoxic to either cell type.

[0080] Example 4: Preparation of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate and testing of its morphology and structure

[0081] 0.64 g of PVA was added to 10 mL of H₂O and magnetically stirred for 12 h. After complete dissolution, 0.04 g of SA-BAC (making the mass ratio of SA-BAC to PVA 1:16) was added and stirred continuously for 1 h until complete dissolution. The solution was filtered and centrifuged to obtain a uniform SA-BAC spinning solution. The solution was then transferred to a 10 mL syringe to remove air bubbles and installed on an electrospinning machine. The spinning parameters were adjusted to: voltage, 21 kV; flow rate, 0.001-0.0013 mm / s; receiving distance, 18 cm; receiving drum speed, 100 rpm; ambient temperature, 25.0 ± 2.0°C; and humidity, 27.0 ± 2.0%. PSA-BAC (PVA + SA-BAC) electrospun nanofiber dressings were prepared using the electrospinning apparatus. PSA, PBAC, and PSA + BAC dressings were also prepared as controls. The preparation method is as follows:

[0082] PSA: The preparation method is the same as that of PSA-BAC, except that the composition of PSA is SA and PVA with a mass ratio of 1:16 to obtain electrospun nanofiber dressing PSA (PVA+SA).

[0083] PBAC: The preparation method is the same as that of PSA-BAC, except that the composition of PBAC is BAC and PVA with a mass ratio of 1:16 to obtain electrospun nanofiber dressing PBAC (PVA+BAC).

[0084] PSA+BAC: The preparation method is the same as that of PSA-BAC, except that the composition of PSA+BAC is (BAC+SA) and PVA with a mass ratio of 1:16, wherein the mass ratio of SA to BAC is 1:3, to obtain electrospun nanofiber dressing PSA+BAC electrospun nanofiber dressing (PVA+SA+BAC).

[0085] After preparation, the prepared dressing was placed in a vacuum drying oven and vacuum dried for at least 12 hours to remove the residual organic solvent on the dressing. The dressing was then placed under a scanning electron microscope to observe its morphological characteristics. The microstructure of PSA, PBAC, PSA+BAC, and PSA-BAC electrospun nanofiber dressings was characterized using a scanning electron microscope. The sample dressing was cut and stuck on a gold spraying table for gold spraying. The fiber diameter size and distribution of the dressing were then observed to see if they were continuous. Finally, ImageJ software was used to measure the diameter of the nanofibers in the dressing. To ensure the accuracy of the statistical analysis, more than 80 fibers were randomly selected from each group and their diameters were measured to ensure that the average fiber diameter of each dressing was reliable and representative.

[0086] The experimental results are as follows Figure 4 As shown, Figure 4 Figure a is a scanning electron microscope image of the dressing. Figure 4 Figure b shows the diameter distribution of the dressing. Figure 4 Figure c shows the dressing diameter change curve. As can be seen from the figure, the average fiber diameter of PSA-BAC increased from 181 nm to 221 nm with the grafting of SA and BAC. The blending of SA and BAC increased the fiber diameter of PSA+BAC from an average of 181 nm to 211 nm. This result shows that the grafting of SA increased the viscosity of the electrospinning solution, weakened the tensile effect of the electric field, increased surface tension, reduced jet stability and tensile strength, and ultimately led to a thicker spun fiber diameter.

[0087] Example 5: Chemical structure of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0088] The chemical structures of SA-BAC and PSA-BAC dressings were confirmed using Fourier transform infrared spectroscopy. The dressings were folded in half and placed on a test bench to completely block the infrared detection light. The wavelength range of the spectral analysis was 4000 cm -1 Up to 500cm -1 , with a resolution of 4cm -1 To obtain detailed and accurate spectral data, a total of 16 scans were performed. The infrared spectrum of PSA-BAC was then compared with the previously measured infrared spectrum of SA-BAC to determine the positions of key functional group peaks.

[0089] The experimental results are as follows Figure 4 As shown in Figure d, the structures of SA-BAC and PSA-BAC dressings were characterized by Fourier transform infrared spectroscopy. The infrared spectra of sodium alginate-bacitracin dressing and sodium alginate-bacitracin dressing were compared and analyzed. It can be seen that the FTIR spectrum of SA-BAC has 3332.1 cm-1 at the OH and CH stretching vibrations, respectively.-1 and 2915.9cm -1 The absorption peak at 1251.6cm -1 and 1029.3cm -1 The absorption peaks at 1606.3 cm-1 are CH bending vibration and CO stretching vibration, respectively, while the absorption peak of C=O stretching vibration is located at 1606.3 cm-1. -1 The FTIR spectrum of PSA-BAC dressing showed that the absorption peaks of OH and CH stretching vibration were located at 3330.6 cm -1 and 2915.7cm -1 . At 1250.9cm -1 and 1087.1cm -1 The absorption peaks at 1729.3 cm-1 are CH bending vibration and CO stretching vibration, while the absorption peak of C=O stretching vibration is located at 1729.3 cm-1. -1 This indicates that the chemical structure of SA-BAC does not change when it is prepared into a dressing.

[0090] Example 6: Thermodynamic properties test of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate The mass change of four electrospun nanofiber dressings with temperature was tested using a thermal analyzer. PSA, PBAC, PSA+BAC and PSA-BAC dressing samples (6 mg ± 2 mg) were placed in an aluminum crucible and heated from 25°C to 600°C at a heating rate of 10.0°C / min under nitrogen. The data were recorded and a thermogravimetric analysis (TGA) curve was plotted. The results are shown in Figure 6. Figure 4 As shown in Figure e, the two stages in the TGA curve of the dressing show significant fluctuations. The first shows smaller fluctuations, occurring in the range of 60.0-180.0°C, which may be caused by the evaporation of water in the dressing, with a weight loss of 5wt%. The second stage of the curve has larger fluctuations, occurring between 260.0-500.0°C, mainly caused by the carbonization and cracking of PVA. The curve of the PSA-BAC dressing shows a slight improvement in the thermal stability of the dressing. The thermal decomposition temperature of all dressings remained above 90.0°C, indicating that the dressing can maintain the durability and consistency of its performance during storage and use even under temperature fluctuations.

[0091] Example 7: Mechanical properties test of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0092] The relationship between force and elongation of electrospun nanofiber dressings was investigated using an electronic universal testing machine. 100 μm thick dressings of PSA, PBAC, PSA+BAC, and PSA-BAC electrospun nanofiber dressings were measured using a thickness gauge. These dressings were then cut into 20 mm x 2 mm rectangular sections (at least three samples of each dressing were retained). These sections were then mounted on the machine and subjected to a tensile test at a rate of 10 mm / min. Stress-strain curves were recorded to determine tensile strength and elongation at break. Three measurements were performed on each dressing to ensure reliability.

[0093] We used an electronic universal tensile testing machine to investigate the mechanical properties of PSA-BAC. Figure 4 As shown in Figure f, the tensile strengths of PBAC, PSA, and PSA-BAC were 12.29±1.09MPa, 5.37±1.12MPa, and 10.31±1.68MPa, respectively. Compared with the PBAC dressing, BAC and SA grafting slightly reduced the elongation at break and tensile strength of the dressing. This is due to the increased viscosity of the electrospinning fluid, which results in a less continuous jet during the electrospinning process. This results in a decrease in the tensile strength and mechanical properties of the dressing, but its mechanical properties are improved compared to those of the PSA dressing. That is, although the mechanical properties and tensile strength of the PSA-BAC dressing are not as good as those of PBAC, they are better than those of PSA. Judging from the results, the reduction in the mechanical properties of PSA-BAC is within an acceptable range.

[0094] Example 8: Blood compatibility test of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0095] Blood samples were extracted from the eyeballs of 8-week-old BALB / c male mice weighing approximately 20 grams to determine the compatibility of the dressing with the blood. The blood was removed, centrifuged, and the supernatant was removed. The separated red blood cells were washed three times with PBS and diluted. PSA, PBAC, PSA+BAC, and PSA-BAC dressings were then cut into circular shapes with a diameter of 9 mm and placed at the bottom of a 12-well plate. 1 mL of diluted red blood cell solution was added to each well, and 0.1% Triton X-100 was set as a positive control and PBS as a negative control. After incubation for 1 hour in a carbon dioxide incubator at 37°C and 5% CO2, the solutions of each group were collected, centrifuged, and the supernatant was retained. The absorbance of the supernatant was then measured at 540 nm.

[0096] The hemolysis rate of the material is Figure 5 Figure A and Figure 5As shown in Figure b, this application used a 10% (w / v) Triton solution as a positive control and a PBS solution as a negative control, with the hemolysis rate of the positive control group set to 100%. The results showed that the hemolysis rates of PBAC, PSA, PSA+BAC, and PSA-BAC dressings were 0.69±0.1%, 0.44±0.022%, 1.46±0.06%, and 0.15±0.04%, respectively. The international standard (ISO 10993-4) stipulates that a good dressing should have a hemolysis rate of less than 5%. The dressing prepared in this application meets this national standard.

[0097] Example 9: Adhesion performance test of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0098] A digital push-pull tester was used. PSA, PBAC, PSA+BAC, and PSA-BAC dressings were cut into rectangles approximately 10 cm long and 3 cm wide and applied to fresh pigskin. A digital push-pull tester was used to firmly grasp a corner of each dressing and gradually lift it until it completely separated from the skin. The maximum reading displayed on the digital tester during the separation process was considered the adhesion force. Five experiments were conducted for each dressing to ensure the accuracy and reliability of the results, and the data were recorded.

[0099] We used fresh pig skin to simulate human skin and used a digital push-pull force gauge to measure the adhesion of the dressing. The dressing was applied to the pig skin and then gently pulled up at a constant speed. Each dressing was repeated five times and the data was recorded. Figure 5 As shown in Figures c and d, the forces applied to PBAC, PSA, PSA+BAC, and PSA-BAC during the pull-up process were 0.13 ± 0.02 N, 0.3 ± 0.01 N, 0.2 ± 0.01 N, and 0.29 ± 0.02 N, respectively. The force applied to the dressing with SA was greater than that applied to the dressing without SA, indicating that SA increased the adhesion of the dressing to the skin.

[0100] Example 10: Moisturizing performance test of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0101] The hydrophilic properties of the dressings were measured using a contact angle tester. PSA, PBAC, PSA+BAC, and PSA-BAC dressings were cut into 2cm×5cm rectangles and placed on the machine, adjusting the placement and lighting position. The contact angle value and ultrapure water penetration time of the dressings were measured using the standard sessile drop method. 10μL of ultrapure water was injected into each droplet. When the water droplet contacted the dressing, a photo was taken and the contact angle was calculated. Once the water droplet was completely absorbed, the time it took to absorb the water droplet was recorded. To ensure data accuracy, each sample was measured five times in parallel at different locations.

[0102] We used a contact angle measuring instrument such as Figure 5 As shown in FIGS. e and f, the water droplet penetration times of the four dressings were 3.18 ± 0.31 s (PBAC), 12.29 ± 0.41 s (PSA), 10.95 ± 1.11 s (PSA+BAC), and 10.836 ± 0.72 s (PSA-BAC), respectively. Although the penetration time of the water droplet increased after the addition of SA, the four dressings still exhibited good penetration performance, ensuring rapid absorption of wound exudates in a moist microenvironment and accelerating wound healing. Figure 5 As shown in FIG. g, the contact angles of the four dressings were 43.81 ± 2.96° (PBAC), 64.78 ± 3.21° (PSA), 56.43 ± 1.85° (PSA+BAC), and 57.17 ± 2.1° (PSA-BAC), respectively. The results showed that the contact angle of the PSA-BAC dressing significantly increased with the addition of SA, from an average of 43.81° to 57.71°. In addition, the contact angles of all dressings were less than 90°, indicating that the dressings were hydrophilic, keeping the wound moist and avoiding dryness and adhesion. This property helps to maintain a moist environment in the wound, thereby promoting cell proliferation and tissue repair.

[0103] Example 11: Cytotoxicity study of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0104] In this experiment, L929 cells and HaCaT cells were selected as experimental objects. When the cells were cultured to 3 to 5 passages, they were removed, trypsinized, centrifuged, and resuspended in DMEM culture medium to obtain a new cell stock solution. The stock solution was diluted to a concentration of 1 × 10 5 The bottom of the 12-well plate was placed into the cell climbing sheet, and then 10,000 cells per well were inoculated into the well plate containing the climbing sheet. Three control groups were set up in each group. After 24 h and 72 h of incubation at 37°C in a 5% CO2 carbon dioxide incubator, the cells were observed to adhere, the liquid in the well plate was discarded, and the cells were washed once with PBS. Then, 1 mL of prepared Calcein-AM / PI detection solution was added, and the plate was placed in a 37°C, 5% CO2 carbon dioxide incubator for further incubation for 30 min. Then, the climbing sheet was removed and the surface was immersed in microscope immersion oil, and then placed under an inverted fluorescence microscope for observation.

[0105] Calcein-AM in the live and dead staining dye can dye live cells green, so the stronger the green light, the better the cell proliferation effect. Therefore, we evaluated the cell live and dead staining of PBAC, PSA, PSA+BAC, and PSA-BAC dressings on day 1 and day 3, as shown in FIGS. h and i. Figure 6As shown in Figures a, b, c, and d, the green fluorescence intensity on day 3 was greater than that on day 1, indicating that the cells were in a normal growth state during this period. Under the influence of the dressing, the green fluorescence intensity in the PSA-BAC group was greater than that in the blank group on days 1 and 3, indicating that the PSA-BAC group had a positive effect on promoting cell proliferation.

[0106] Example 12: Study on the antibacterial properties of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0107] The concentration was 1×10 6 CFU / mL of bacterial solution was added to each well of a 24-well plate at 500 μL and a blank control group was set up. The plate was then placed in a 37°C constant temperature shaker and shaken for 6 h before being taken out. The bacterial solution of the experimental group and the blank control group in the plate was diluted with PBS to a concentration of 1×10 3 CFU / mL. Then, take 100 μL of each sample and drip it into LB medium. Spread it evenly, then place it upside down in a biochemical incubator overnight. The next day, observe and record the number of colonies growing on the LB solid medium. Substitute this into the following formula to calculate the bacterial inhibition rate of the experimental group.

[0108] Bacterial inhibition rate (%) = [1-(K sample / K control )]×100%

[0109] Where: K sample represents the number of colonies in the experimental group, K control Represents the number of colonies in the blank control group.

[0110] This experiment used the dilution coating method to explore the antibacterial ability of PBAC, PSA, PSA+BAC and PSA-BAC dressings against Staphylococcus aureus and Escherichia coli.

[0111] The dressings were co-cultured with bacteria, and the number of colonies in each dressing group was recorded using the dilution smear method, with the control group's data set as 100%. Figure 6As shown in Figures e, f, and g, the average inhibition rates against Staphylococcus aureus for PBAC, PSA, PSA+BAC, and PSA-BAC were 97.93%, 75.12%, 85.71%, and 99%, respectively. With the exception of the PSA dressing, the average inhibition rates against S. aureus for all dressing groups were greater than 80%. This indicates that the dressings have a significant inhibitory effect on S. aureus. The average inhibition rates against E. coli for PBAC, PSA, PSA+BAC, and PSA-BAC dressings were 89.68%, 27.71%, 58.71%, and 85.48%, respectively. At the same dressing dosage, the inhibition rates against E. coli in each dressing group were lower than those against S. aureus. These results indicate that PSA-BAC dressing has a good inhibitory effect on Staphylococcus aureus, but has no obvious inhibitory effect on Escherichia coli, so PSA-BAC will have good antibacterial activity against wound infections caused by Staphylococcus aureus.

[0112] Example 13: Bioactivity test of electrospun nanofiber dressing containing sodium alginate-bacitracin (SA-BAC) conjugate

[0113] As a topical preparation that comes into direct contact with wounds, an ideal wound dressing must possess excellent bioactivity, primarily to ensure its practical biological functions in promoting wound healing, antibacterial properties, anti-inflammation, and tissue regeneration, thereby better meeting clinical needs. A dressing with excellent bioactivity can release antimicrobial components to effectively inhibit bacterial growth and reduce the risk of infection. This inhibition is not only reflected in already infected wounds, but also in its ability to resist bacteria in the external environment while the wound dressing is in use. A good dressing should also promote cell proliferation, accelerating this process to shorten wound healing time, accelerate wound re-epithelialization and tissue repair, and reduce inflammation and the risk of infection. Furthermore, some dressings have anti-inflammatory properties, inhibiting inflammatory factors, thereby reducing inflammation and tissue damage, creating a favorable microenvironment for wound healing. Crucially, a dressing with excellent bioactivity can mimic the properties of the extracellular matrix, providing an excellent scaffold for cell attachment and growth, promoting tissue regeneration and remodeling, and reducing scarring.

[0114] This example evaluates the antibacterial effect of PSA-BAC on wounds and its effect on cell proliferation around tissues by setting up in vitro antibacterial and cytotoxicity experiments. In vivo experiments are also conducted on mice with back infected wound models to evaluate the anti-inflammatory and tissue regeneration promotion effects of PSA-BAC.

[0115] The healing of infected wounds is often accompanied by tissue repair and inflammatory cell changes. Hematoxylin-eosin (H&E) and Masson trichrome staining can clearly show these pathological changes. HE staining can observe the infiltration of inflammatory cells, necrotic tissue fragments, and morphological changes of the epidermis and granulation tissue in the wound tissue, and can visually evaluate the inflammatory response and healing process of the wound. Masson trichrome staining is mainly used to observe the content and arrangement of collagen fibers. In infected wounds, the synthesis and arrangement of collagen fibers are crucial for wound healing. Through Masson trichrome staining, it is possible to evaluate whether the synthesis of collagen fibers in the wound site is blocked and whether its arrangement is disordered. Therefore, we stained the wound tissue specimens on the 3rd, 5th and 7th days. As Figure 7 Figure A and Figure 7 As shown in Figure 2(b), the wound healed over time, and the PSA-BAC and 3M dressing groups showed more significant healing than the PBAC and control dressing groups. This may be due to the addition of SA to the dressing, which improved the adhesion of the final dressing and thus increased the duration of action of the dressing on the infected wound.

[0116] To further explore the tissue repair of the wound, we continued to slice and stain the wound on day 14. Figure 7 Figure C and Figure 7 As shown in Figure (e), H&E staining of wound tissue specimens on day 14 revealed that protein and connective tissue production in the dressing group was superior to that in the blank control group. The dressing group showed more pronounced changes in the layered structure and morphology of the wound tissue, accompanied by the formation of skin pores. This indicates that the wound tissue structure is being repaired, with cells arranged more neatly, which promotes a reduction in tissue necrosis and degeneration. Masson trichrome staining of wound tissue specimens on day 14 revealed a darker blue staining in the dressing group compared to the control group and other dressing groups. This indicates that collagen fiber production is more pronounced in the dressing group than in the blank control group, indicating that the wound is effectively entering the tissue repair and remodeling phase. Increased collagen deposition is typically accompanied by a decrease in inflammatory cell infiltration, indicating that the inflammatory response at the wound site is effectively controlled, creating favorable conditions for tissue repair. This also enhances the mechanical strength and structural integrity of the wound, promoting rapid wound closure and functional recovery.

[0117] TNF-α, also known as "tumor necrosis factor α", is an important pro-inflammatory cytokine that plays a key role in inflammatory response, immune regulation and cell apoptosis. TNF-α is one of the core cytokines of inflammatory response, which can activate neutrophils and macrophages, enhance their bactericidal ability, and promote the production of other inflammatory mediators (such as IL-1, IL-6). It can also induce vascular endothelial cells to express intercellular adhesion molecules, promote the adhesion and migration of leukocytes, and the results of TNF-α staining of mouse wounds are shown in Figure 2. Figure 7 As shown in Figure d.

[0118] Tumor necrosis factor is one of the early mediators of inflammatory response, which can activate the production of other inflammatory cytokines. The gray value statistics of the immunohistochemical THF-α staining experimental images of the tissue specimens extracted on the 14th day were as follows: Figure 7 As shown in Figure f, the THF-α levels in the blank group, 3M group, PBAC group, and PSA+BAC group were 31.02±1.92%, 15.49±1.59%, 16.57±1.52%, and 10.97±1.64%, respectively. The THF-α expression levels in the experimental groups were all lower than those in the blank group, and the expression of THF-α in the PSA-BAC dressing was the lowest in the experimental groups. This suggests that the PSA-BAC dressing can significantly reduce inflammation.

[0119] The above is a detailed introduction to a polysaccharide-bacitracin conjugate, an electrospun nanofiber dressing, a preparation method, and an application thereof provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method and core idea of ​​the present application. At the same time, for those skilled in the art, according to the ideas of the present application, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present application.

Claims

1. A polysaccharide-bacitracin conjugate, characterized in that The structural formula of the polysaccharide-bacitracin conjugate is as follows:

2. The polysaccharide-bacitracin conjugate according to claim 1, characterized in that The polysaccharide-bacitracin conjugate is prepared by the following method: EDC and NHS are added to an aqueous solution of sodium alginate, followed by adding bacitracin, and after heat preservation and reaction, the carboxyl group in the sodium alginate is coupled with the amino group in the bacitracin, and finally the obtained reactant is purified to obtain a sugar-peptide conjugate.

3. Use of the polysaccharide-bacitracin conjugate according to any one of claims 1 to 2 in the preparation of antibacterial materials.

4. An electrospun nanofiber dressing, characterized in that: The polysaccharide-Bacitracin conjugate comprising polyvinyl alcohol and any one of claims 1 to 2, wherein the mass ratio of the polyvinyl alcohol to the polysaccharide-Bacitracin conjugate is (15-20):1; or The electrospun nanofiber dressing includes polyvinyl alcohol, sodium alginate and bacitracin, the mass ratio of the polyvinyl alcohol to the sum of the mass of the sodium alginate and the bacitracin is (15-20):1, and the mass ratio of the sodium alginate to the bacitracin is 1:(2-5).

5. The electrospun nanofiber dressing according to claim 4, characterized in that Prepared by the following method: Mixing polyvinyl alcohol and polysaccharide-bacitracin conjugate to obtain a spinning solution, followed by electrospinning to obtain a nanofiber dressing; or, Polyvinyl alcohol, sodium alginate and bacitracin are mixed to obtain a spinning solution, which is then electrospun to obtain a nanofiber dressing.

6. The electrospun nanofiber dressing according to claim 4, characterized in that The electrospinning parameters are as follows: voltage: 20 kV; flow rate: 0.001-0.0013 mm / s; receiving distance: 18 cm; receiving cylinder speed: 100 r / min; environmental parameter temperature: 25.0±2.0°C; Humidity: 27.0±2.0%.

7. A method for preparing the electrospun nanofiber dressing according to claim 4, characterized in that: The following steps are involved: Polyvinyl alcohol and polysaccharide-bacitracin conjugate are mixed to obtain a spinning solution, which is then electrospun to obtain a nanofiber dressing; or, Polyvinyl alcohol, sodium alginate and bacitracin are mixed to obtain a spinning solution, which is then electrospun to obtain a nanofiber dressing; Sodium alginate and said bacitracin.

8. Use of the electrospun nanofiber dressing according to any one of claims 4 to 6 or the electrospun nanofiber dressing prepared by the preparation method of the electrospun nanofiber dressing according to claim 7 in the field of antibacterial materials or medical devices.

9. Use of the electrospun nanofiber dressing according to any one of claims 4 to 6 or the electrospun nanofiber dressing prepared by the preparation method of the electrospun nanofiber dressing according to claim 7 in tissue repair or scar repair.

10. Use of the electrospun nanofiber dressing according to any one of claims 4 to 6 or the electrospun nanofiber dressing prepared by the preparation method of the electrospun nanofiber dressing according to claim 7 in wound dressing and tissue engineering dressing.

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