An antibacterial hydrophobic dressing, its preparation method and application

Abstract

The present invention belongs to the field of medical materials, and particularly relates to an antibacterial hydrophobic dressing, a preparation method thereof, and an application in the treatment of skin wound repair. The medical dressing prepared by the present invention is successfully synthesized through characterization. Mesoporous hydrophobic silica adheres to the surface of cotton fibers, and its morphology and average particle size are the same as those of bare nanoparticles, endowing the fabric surface with a nanoscale structure, improving the surface roughness of the cotton fabric, and endowing it with hydrophobicity, antibacterial property, and good biosafety, and it is very stable; in vivo studies show that the synthesized antibacterial hydrophobic dressing can significantly inhibit bacterial infection, inhibit the expression of pro-inflammatory factors in the skin, promote wound healing, and can treat the healing of refractory drug-resistant skin wounds.

Description

Technical Field

[0001] The present invention belongs to the field of medical materials, and particularly relates to an antibacterial hydrophobic dressing, a preparation method thereof, and an application in the treatment of skin wound repair. Background Art

[0002] Medical dressings are medical materials used to cover sores, wounds or other damages. When the skin is damaged, if it cannot heal in time, the wound surface needs to be covered with a dressing in time. The therapy of systemic application of antibiotics often fails to reach the effective range for preventing and treating infections at the local drug concentration of the wound, and in addition, it will increase the drug dosage and increase side effects. Using a suitable antibacterial dressing can fully reduce the infection of the wound and comprehensively promote wound healing. Clinically, antibiotic drug dressings are generally used for antibacterial treatment of skin wounds. Although antibiotics take effect quickly in antibacterial treatment, long-term use of antibiotics will cause bacteria to develop drug resistance.

[0003] Ordinary dressings have poor hydrophobicity and are prone to adhesion to the wound after covering the wound, resulting in secondary injury to the wound and being unfavorable for wound healing.

[0004] With the in-depth study of the pathophysiology of the wound healing process, people's understanding of the wound healing process has become deeper and deeper, which has led to the continuous improvement and development of medical wound dressings. Compared with the early stage, the new type of wound care dressings has undergone revolutionary changes, and a variety of medical dressings with different performances are available for clinical nursing staff to choose.

[0005] Therefore, in order to better meet the clinical treatment needs, it is an urgent market need to develop a new antibacterial hydrophobic dressing, which has important clinical significance in the treatment of skin wounds, especially in the repair of drug-resistant skin wound traumas. Summary of the Invention

[0006] The purpose of the present invention is to provide an antibacterial hydrophobic dressing, which has both antibacterial and hydrophobic properties. The surface of the dressing is covered with micro-silica nanospheres, whose morphology and average particle size are the same as those of bare nanospheres, making the fabric surface have a nanoscale structure, improving the surface roughness of the fabric, endowing it with hydrophobicity, and being very stable; the anti-inflammatory and antibacterial effects are obvious.

[0007] Another purpose of the present invention is to provide a preparation method of an antibacterial hydrophobic dressing.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A preparation method of an antibacterial hydrophobic dressing, the steps of which include:

[0010] 1), cleaning and drying a cotton fabric; treating it with a sodium periodate solution to prepare an oxidized cotton fabric;

[0011] The cleaning process of the cotton fabric is as follows: A 5*5 cm cotton fabric is ultrasonically cleaned with a 5% sodium hydroxide solution for 1 - 2 hours; rinsed 2 - 3 times with absolute ethanol; and cleaned 2 - 3 times with deionized water. Then it is dried at a temperature above 80°C for 12 hours and reserved for use.

[0012] 2) Put the oxidized cotton fabric into a glycerol solution, let it stand, wash, and dry at room temperature; then place the cotton fabric in a mixed oxidation solution composed of deionized water, concentrated sulfuric acid, and potassium dichromate, and after washing, obtain carboxylated cotton fabric for reserve.

[0013] 3) Dissolve cetyltrimethylammonium p-toluenesulfonate and triethanolamine, which are surfactants, in deionized water. After the surfactants are completely dissolved, add tetraethyl orthosilicate and 3-aminopropyltriethoxysilane, and stir; after centrifugation, collect the product, wash it 2 - 3 times with ethanol to remove residual reactants; extract with an ammonium nitrate ethanol solution to obtain aminated mesoporous silica.

[0014] 4) Immerse the aminated mesoporous silica in step (3) and the antibacterial drug in a dimethyl sulfoxide solution, stir magnetically, and perform centrifugal separation to obtain drug-loaded aminated mesoporous silica.

[0015] 5) Add the drug-loaded aminated mesoporous silica in step (4) to ethanol and mix well to form a mixed solution. Control the pH of the mixed solution to be 6, and repeatedly add an equal volume of octadecyltriethoxysilane and stir at room temperature to obtain a hydrophobic sol system.

[0016] 6) Disperse the drug-loaded aminated mesoporous silica in step (4) in an absolute ethanol solution, ultrasonically disperse it, and then add N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), and triethylamine to form a reaction solution.

[0017] 7) Put the carboxylated cotton fabric in step (2) into the hydrophobic sol system in step (5) and stir, wash, and dry; then put the cotton fabric into the reaction solution in step (6) to react, wash, and dry.

[0018] Preferably, in step (2), the glycerol solution is composed of ethanol and glycerol with an addition ratio of 100 mL: 0.7 - 1.2 g.

[0019] Preferably, in step (3), the addition ratio of cetyltrimethylammonium p-toluenesulfonate, triethanolamine, deionized water, and tetraethyl orthosilicate is 1 - 5 g: 0.1 - 0.8 g: 100 mL: 10 - 30 mL.

[0020] Preferably, the antibacterial drug is effective against both Gram-negative and Gram-positive bacteria, and can carry drugs such as ciprofloxacin, norfloxacin, doxycycline hydrochloride, vancomycin, etc. More preferably, it can carry ciprofloxacin or norfloxacin, with a high drug loading rate.

[0021] The drug concentration with better antibacterial effect is: norfloxacin NOR (30 μg mL -1 ), ciprofloxacin CIP (20 μg mL -1 )

[0022] The effect of different contents of 3-aminopropyltrimethoxysilane (APTES) on the pore size of amino-functionalized mesoporous silica (NH2-MSN). As the content of APTES increases, the average particle size and PDI also show an increasing trend. When the volume ratio of TEOS to TES = 5:1, the particle size distribution is the most stable and the standard deviation is the smallest. At this time, the PDI can reach about 0.1.

[0023] Preferably, in the step (5), the addition ratio of the drug-loaded aminated mesoporous silica to ethanol is 1 g: 30 - 50 mL.

[0024] Preferably, in the step (6), the volume ratio of the octadecyltriethoxysilane added each time to ethanol in the mixed solution is 1:150 - 250, stirred at room temperature for 1 - 3 hours, and the rotation speed is 250 rpm.

[0025] Preferably, in the step (7), the addition ratio of the drug-loaded aminated mesoporous silica, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and triethylamine is 2 - 7 mg: 0.5 - 4 mg: 0.1 - 1.5 mg: 1 μL.

[0026] The antibacterial hydrophobic dressing can be obtained through the above preparation method.

[0027] The application of the above antibacterial hydrophobic dressing in the repair of skin wound trauma.

[0028] The beneficial effects of the present invention are

[0029] 1. The antibacterial hydrophobic dressing prepared by the present invention is used to inhibit the growth of bacteria at the skin wound and relieve the pain of dressing change, thereby promoting wound healing.

[0030] 2. The cotton fiber surface of the antibacterial hydrophobic dressing prepared by the present invention is covered with micro-silica nanospheres, which improves the surface roughness of the cotton fabric and significantly improves its hydrophobicity. It still has good hydrophobicity after 10 washes.

[0031] 3. The antibacterial hydrophobic dressing prepared by the present invention can significantly reduce the inflammatory reaction at the wound, regulate anti-inflammatory and pro-inflammatory factors, and promote wound healing. Brief Description of the Drawings

[0032] Figure 1 It is the SEM image of the original cotton fabric in Example 1.

[0033] Figure 2 It is the SEM image of the original mesoporous silica in Example 2.

[0034] Figure 3 It is the comparison chart of the influence of different APTES contents on the particle size and PDI of NH2-MSN in Example 2.

[0035] Figure 4 It is the comparison chart of the influence of different APTES contents on the Zeta potential of NH2-MSN in Example 2.

[0036] Figure 5 It is the chart of the content of CIP and NOR loaded on NH2-MSN in Example 3.

[0037] Figure 6 It is the SEM image of the antibacterial hydrophobic dressing in Example 5.

[0038] Figure 7 It is the comparison chart of the hydrophobicity of the antibacterial hydrophobic dressing in Example 5.

[0039] Figure 8 It is the characterization chart of the inhibition zone of the antibacterial hydrophobic dressing in Example 7.

[0040] Figure 9 It is the comparison chart of the healing of full-thickness skin wounds of rats infected with SA by the antibacterial hydrophobic dressing loaded with CIP in Example 9.

[0041] Figure 10 It is the comparison chart of HE staining of wound tissue samples in Example 9.

[0042] Figure 11 It is the comparison chart of the mRNA of inflammatory factors IL-β, IL-17, IL-6 and Tnf-α in Example 9. Detailed Description of the Invention

[0043] The present invention will be further described below in conjunction with the embodiments:

[0044] Example 1

[0045] Surface modification of cotton fabric:

[0046] Take a 5*5 cm cotton fabric weighing approximately 0.3 g, ultrasonically clean it with a 5% sodium hydroxide solution for 1 hour, then rinse it with anhydrous ethanol 2 - 3 times; finally, wash it with deionized water 2 - 3 times; dry it at a temperature above 80 °C for 12 hours for standby.

[0047] Immerse the above cotton fabric in a 0.05 mL·L-1 sodium periodate solution and ultrasonically treat it at room temperature for 20 minutes. When the sodium periodate is completely dissolved, place the reaction mixture in a constant-temperature oil bath at 40 °C for 4 h. Then wash the oxidized cotton fabric with deionized water 2 - 3 times, and put it into a glycerol solution (100 ml ethanol and 0.9209 g glycerol), and let it stand at room temperature for 1 hour to remove the excess sodium periodate. Wash and dry again to obtain an aldehyde-containing fiber material. For further oxidation, place the cotton fabric in a mixed solution at 40 °C (50 mL deionized water + 1 drop of concentrated sulfuric acid + potassium dichromate solution with a concentration of 0.4 mol·L-1) for 30 minutes, and obtain a carboxyl fiber material after thorough washing.

[0048] Figure 1 SEM image of the original cotton fabric, the results are shown in Figure 1 as follows.

[0049] Example 2

[0050] Preparation and modification of aminated mesoporous silica nanoparticles (MSN):

[0051] Dissolve 1.92 g of cetyltrimethylammonium p-toluenesulfonate (CTATos) and 0.374 g of triethanolamine (TEAH3) in 100 mL of deionized water, and stir at 80 °C for 1 hour. After the surfactant is completely dissolved, quickly add 15.67 mL of tetraethyl orthosilicate (TEOS) and 3.134 mL of 3-aminopropyltriethoxysilane (APTES), and stir at 80 °C at a stirring speed of 1200 rpm for another 2.5 hours; collect the product by centrifugation, wash it with ethanol 2 - 3 times to remove the residual reactants. Extract it with a 0.6 wt% ammonium nitrate (NH4NO3) ethanol solution at 60.0 °C for 6 h, and repeat 2 times to obtain aminated mesoporous silica (MSN). Its SEM image is as shown in Figure 2 as follows.

[0052] Use APTES to carry out amino modification on MSN. During this process, since the content of APTES will affect the particle size, PDI and Zeta potential of MSN, its addition amount is explored. When the addition amount is 3.134 mL, that is, (the volume ratio of TEOS:APTES = 5:1), the obtained Zeta potential is positive, and the particle size and PDI are the smallest. For details, see Appendix Figure 3 and Appendix Figure 4As shown. At this time, the PDI can reach about 0.1. This is due to the increase in the APTES concentration and the enhancement of the solution charge effect. Due to the electrostatic effect, the intermolecular force between particles increases, resulting in aggregation, which also increases the particle size; After amination, the obtained MSN was used for drug loading, and the corresponding pore size distribution was calculated through the adsorption - desorption isotherm. The pore size of all samples was about 10 nm, which was beneficial for drug loading.

[0053] Example 3

[0054] Drug loading process of aminated mesoporous silica

[0055] Two commonly used antibacterial drugs, ciprofloxacin (CIP) and norfloxacin (NOR), were selected for drug loading. The drug loading processes of ciprofloxacin and norfloxacin are as follows:

[0056] 200 mg of the aminated mesoporous silica sample was immersed in 20 mL of DMSO (10 mg·mL-1) drug solution, and magnetically stirred for 24 hours. Then, centrifugal separation was carried out. Finally, the supernatant concentration was measured by liquid chromatography. The drug loading calculation formula is:

[0057] W(%) = m / M * 100% (1)

[0058] Where: W is the drug loading rate of the drug in the drug loading system;

[0059] m is the amount of the drug encapsulated in the carrier;

[0060] M represents the total amount of the injected drug.

[0061] The contents of CIP and NOR loaded on the aminated mesoporous silica are as Figure 5 shown.

[0062] Example 4

[0063] Hydrophobic modification of aminated mesoporous silica after drug loading:

[0064] Weigh 1 g of aminated mesoporous silica (powder), add 40 mL of ethanol, and mix well. Adjust the pH = 6 with 1 mol hydrochloric acid (HCl), and add 200 μL of octadecyltriethoxysilane (ODTES) to the mixture. After stirring at room temperature for 2 h, add the same volume of ODTES at 250 rpm and continue stirring for 2 h. Repeat the addition several times to obtain a hydrophobic sol system.

[0065] Example 5

[0066] Preparation of antibacterial hydrophobic dressing:

[0067] Take 500 mg of drug-containing aminated hydrophobic mesoporous silica, disperse it in absolute ethanol and mix well to form a mixed solution. After ultrasonic dispersion, add 200 mg of NHS, 100 mg of EDCHCl, and 100 μL of triethylamine. Put the cotton fabric in Example 1 into the sol solution and stir for 8 hours; wash the cotton fabric several times with absolute ethanol and dry it at a constant temperature of 100 °C for 12 hours; put the cotton fabric into the reaction solution again and continue to react for 8 hours, wash it several times with absolute ethanol and dry it under the same conditions to prepare an antibacterial hydrophobic dressing, and its SEM image is as Figure 6 shown.

[0068] It can be seen from the electron micrograph that the surface of the cotton fiber is covered with micro-silica nanospheres, whose morphology and average particle size are the same as those of the bare nanospheres, making the surface of the fabric have a nanoscale structure, improving the surface roughness of the fabric, and endowing it with hydrophobicity. Hydrophobic NH2-MSN can be tightly combined with the oxidized cotton fabric. After 10 washes, it is still in a hydrophobic structure, proving that the mesoporous silica has been successfully grafted onto the surface of the cotton fiber;

[0069] EDX elemental scanning analysis of the surface of the functional cotton fabric shows that the surface of the fiber is covered with mesoporous silica nanoparticles. Compared with untreated cotton fibers, the surface of the functionalized cotton fabric is very rough. It is also known that there are a large number of C, O, and Si elements on the surface of the functionalized cotton fabric; since many silicon particles adhere to the surface of the oxidized fabric, it forms a hydrophobic structure. Therefore, the reduction of the toughness of the cotton fabric will affect the physical and mechanical properties of the cotton fabric. Antibacterial and hydrophobic cotton fabrics are mainly used for the application direction of medical dressings. Therefore, it is necessary to ensure the dryness of the dressing surface and the repellency of liquids so that the liquid cannot adhere to and contaminate the cotton fabric;

[0070] The contact angles of the cotton fabric before and after functional modification were measured, as detailed in the appendix Figure 7As shown, the contact angle after the reaction on the surface of the hydrophobic cotton fabric can reach 135.21 (meeting the hydrophobic range (a contact angle greater than 120 can be called hydrophobic)). Hydrophobic NH2-MSN has been successfully grafted onto the cotton fabric, and the surface of the functionalized cotton fabric has changed from hydrophilic to hydrophobic, endowing the fabric with anti-fouling properties. Compared with the untreated cotton fabric, the properties of the functionalized cotton fabric have changed. To explore the stability of the antibacterial and hydrophobic cotton fabric, the functionalized cotton fabric was washed repeatedly with absolute ethanol. As the number of washing cycles increased, the contact angle of the modified cotton fabric decreased slightly. After 10 washes, the fabric contact angle decreased from 142.35 initially to 137.06, but the fabric still maintained a good hydrophobic state. During the hydrophobic reaction, most of the hydrophobic NH2-MSN may undergo a chemical bonding reaction with the oxidized cotton fabric. At the same time, some hydrophobic NH2-MSN was adsorbed on the oxidized cotton fabric. Therefore, after the hydrophobic reaction, the functionalized cotton fabric has superhydrophobicity. After washing, the unreacted NH2-MSN was washed away, so the hydrophobicity decreased slightly.

[0071] Example 6

[0072] In vitro drug release of the antibacterial and hydrophobic dressing:

[0073] Study the release characteristics of different drugs such as ciprofloxacin or norfloxacin. The prepared antibacterial and hydrophobic dressing was cut into 2.5*5 rectangles and placed in a dialysis bag. It was immersed in PBS buffer solution with PH = 7.4 / PH = 5.4, and the solution temperature was maintained at 37 °C. Shake and dialyze using a dialysis bag (MWco = 3500, MD = 44 mm). Samples were aspirated at different time intervals. The sampling interval will gradually increase with time. But the sampling volume must be the same. Then calculate the release rate according to the formula:

[0074]

[0075] Formula Ep: Cumulative drug release Ve: Replacement of PBS Vo: Total volume of the release medium C: Concentration of the liquid released during the i-th replacement sampling Mdrug: Total mass of the drug in the nanoparticles n: Number of times PBS is changed.

[0076] Using the same release process, repeat the release experiment 3 times and take the average of the measurement results. Calculate the cumulative drug release at each sampling point according to the above formula.

[0077] The in vitro drug release was as follows: the release amount of ciprofloxacin (CIP) increased first and then decreased over time. Within the first 36 hours, the drug release amount increased rapidly. During the period of 36 - 84 h, the release amount reached approximately 9.3 mg g-1, and at this time the overall release trend was relatively small. However, when the time exceeded 84 h, the release of CIP began to decrease. But the decreasing rate was much lower than the increasing rate and finally tended to be flat. Therefore, the optimal time period for CIP drug release was 36 - 84 hours. The drug release amount of NOR increased rapidly within the first 12 h and remained basically unchanged within 12 - 72 h. After 72 hours, the drug release amount increased again. It reached the peak at about 96 hours, and the release amount was approximately 1.8 mg g-1. After more than 96 hours, the release amount continued to decrease, and finally the release amount remained at about 1 mg g-1. It can be seen that the NOR drug release of the antibacterial cotton fabric reached the maximum at 96 hours. Therefore, this time period was most favorable for the antibacterial experiment.

[0078] Example 7

[0079] In vitro antibacterial experiment

[0080] (1) Bacterial culture: Staphylococcus aureus (ATCC35556) was inoculated in tryptic soy broth (TSB) at 37 °C and cultured overnight. Escherichia coli (ATCC12408) was inoculated in Luria - Bertani (LB) and cultured overnight. 1 mL of each bacterial suspension was taken for sub - culture and harvested at the exponential growth stage. Subsequently, the optical density values of 100 μl of Staphylococcus aureus and Escherichia coli solutions were monitored at 600 nm using a microplate spectrophotometer on a 96 - well plate, and samples with an optical density (OD600) of 0.08 - 0.1 were taken for the experiment.

[0081] (2) Extracting drugs from oxidized cotton fabrics loaded with ciprofloxacin and norfloxacin drugs:

[0082] Each 0.3 g of ciprofloxacin gauze and norfloxacin gauze was immersed in 5 mL of TSB and LB media respectively. Under the condition of pH = 7.4, the maximum drug release amount of ciprofloxacin gauze after soaking for 72 h was 300 mg, and the maximum drug release amount of norfloxacin gauze after soaking for 96 h was 300 mg. The concentrations of ciprofloxacin and norfloxacin were 300 mg / 5 mL.

[0083] (3) OD600 determination

[0084] Dilute the extracts of ciprofloxacin and norfloxacin gauzes into 5 concentration gradients: ciprofloxacin 0 μg mL-1, 1 μg mL-1, 5 μg mL-1, 10 μg mL-1, 20 μg mL-1; norfloxacin 0 μg mL-1, 1 μg mL-1, 2 μg mL-1, 10 μg mL-1, 30 μg mL-1. Take the bacterial suspension (500 μL) with OD600 = 0.08 and treat it with drugs of different gradients. Inoculate the bacterial suspension into a 96-well plate, with 4 replicates for each concentration gradient, and place 90 μl in each well. Monitor the OD600 values of each concentration gradient at 0, 1, 2, and 3 h respectively. Obtain its minimum inhibitory concentration based on the optical density and plot the bacterial growth curve.

[0085] (4) CCK-8 analysis of bacterial viability experiment

[0086] Use the CCK-8 detection method to detect the viability of this bacterium. According to the bacterial kinetic analysis, inoculate the bacterial suspension containing each concentration gradient into a 96-well plate, 90 μl per well. After culturing in an incubator at 37°C for 120 min, add CCK-8 solution (10 μL / well) to the 96-well plate. After co-incubating for 90 min at room temperature in the dark, place the 96-well plate into a microplate spectrophotometer to measure the absorbance value at 450 nm, so as to reflect the number of live bacteria in each well. Each group contains 3 replicates. Bacterial viability is expressed as the mean ± standard deviation (SD) of the absorbance of 3 wells in each group.

[0087] (5) Bacterial CFU determination

[0088] The antibacterial activities of ciprofloxacin gauze and norfloxacin gauze extracts against Staphylococcus aureus and Escherichia coli were determined by the CFU counting method on agar plates. Count the colonies obtained to determine the inhibition of bacterial growth by the drug. Incubate the bacterial suspension with extracts of different concentrations for 3 h. Subsequently, take 30 μL of bacteria at different concentrations, dilute them 10-fold continuously, and drop them onto TSB and LB agar plates, spread them evenly, and incubate them at 37°C for 24 h. Count the colonies. Coat three agar plates with different drug concentrations. And use GraphPad Prism 9 to calculate the CFU number. This experiment was repeated three times.

[0089] (6) Antibacterial ring experiment

[0090] 1 mL of Escherichia coli and Staphylococcus aureus were subcultured and harvested at the exponential growth phase. At the same time, the OD values of each bacterium were adjusted by the optical density value to make the OD value OD600 = 0.5. 1 mL of the bacterial suspension with OD600 = 0.5 was evenly distributed on TSB agar plates and LB agar plates. The sterilized ciprofloxacin gauze and norfloxacin gauze were cut into large circles. The diameter of the gauze was 1 cm. Then the gauze was attached to the agar plates and cultured at 37 °C for 24 h, and then the size of the inhibition zone was measured. This experiment was repeated three times.

[0091] Experimental results:

[0092] Bacterial infections at the wound site may trigger an inflammatory response, hindering the wound healing process. Therefore, first, the antibacterial activity of the dressing prepared in the present invention was evaluated by the inhibition zone test of Escherichia coli and Staphylococcus aureus.

[0093] Both drug-loaded discs had obvious inhibition zones against Escherichia coli, and the effect of CIP was more obvious. In this study, the antibacterial properties of the extract of functionalized cotton fabric in direct contact with active bacteria were evaluated using the typical Gram-positive bacterium Staphylococcus aureus and Gram-negative bacterium Escherichia coli. A large number of colonies were observed on the plates of the control group. In contrast, the number of colonies in the plates of the functionalized cotton fabric group decreased. As the drug concentration increased, its antibacterial effect became more and more obvious. The effects of different drug concentrations on bacterial growth were analyzed by the optical density OD value at 600. A positive control group was established using metronidazole (MNZ, 10 mg / mL) solution. According to the analysis of the bacterial growth curve, when the extract of norfloxacin gauze was 30 μg / mL, the bacterial growth curve tended to be flat compared with the control group. Similarly, the extract of ciprofloxacin gauze at 5 μg / mL had an effect on the growth of both bacteria, and the effect at a concentration of 20 μg / mL on bacterial growth was the greatest. The extracts of both gauzes inhibited the growth of Escherichia coli and Staphylococcus aureus at different concentrations.

[0094] Example 8 Cytotoxicity experiment

[0095] The HGF-1 cells are a normal human gingival fibroblast cell line, purchased from the Shanghai Institute of Biological Sciences Center (ATCC CRL-2014). The HGF-1 cells are adherent cells. The cells grow in DMEM medium containing 10% fetal bovine serum (Gibco), 100 U / mL penicillin, and 100 mg / L streptomycin. They are cultured at 37 °C and 5% carbon dioxide. The HGF-1 cells are seeded into a 96-well plate at a density of 5000 cells per well. After stimulating the HGF-1 cells with different concentrations of ciprofloxacin and norfloxacin gauze extracts for 4 h, the cell viability is detected by the cck-8 method. There are six replicates for each concentration. The cell viability is measured using the CCK-8 solution (1:10). After co-incubation for 90 min, the 96-well plate is placed into a microplate spectrophotometer to measure the absorbance value at 450 nm.

[0096] The control group is the extract of medical gauze, and the experimental group is the extract of the drug-loaded gauze. The extract in the experimental group is diluted into different concentrations and acts on human gingival fibroblasts. Compared with the control group, the OD450 of the low drug concentration has no obvious change, indicating that the two drugs have good biocompatibility at low concentrations. However, the CIP gauze extract has a significant effect on the fibroblast activity under the condition of a concentration of 20 μg / mL, and the NOR gauze extract has a significant effect on the fibroblast activity under the condition of a concentration of 30 μg / mL. Therefore, the drug concentration of CIP cannot exceed 20 μg / mL, and the drug concentration of NOR cannot exceed 30 μg / mL, which will produce certain cytotoxicity.

[0097] Example 9 Animal Skin Infection Experiment on the Body Surface

[0098] All 10-week-old female rats (200 - 230 g) are from the Shanghai Vital River Laboratory Animal Center. All animal experiment equipment and procedures are inspected and confirmed by the Animal Care and Use Committee of Shanghai Tenth People's Hospital, Tongji University School of Medicine (ID: SHDSYY-2022-3063).

[0099] (1) In Vivo Assessment of Skin Defect Wound Closure

[0100] Rats were anesthetized with 2% pentobarbital (0.2 ml / 100 g), and a defect was created on the back of each rat. Using a perforator with a diameter of 7 mm, 4 skin defects of the same size were evenly created on the back of the rats. At the same time, infection treatment was carried out. 50 μl of Staphylococcus aureus with OD600 = 0.5 was added to the 4 defects on the back of each rat and left stationary on the back of the rat for 10 min. Then, a 1 cm * 1 cm ciprofloxacin gauze and a 1 cm * 1 cm sterile medical gauze were cut and applied to the skin defect infected with Staphylococcus aureus. The control group was the medical sterile gauze, and the anti-infection group was the ciprofloxacin-loaded gauze. In the anti-infection group, 2 pieces of 1 cm * 1 cm ciprofloxacin gauze were applied at each skin incision. There were 2 cases in the control group and 2 cases in the anti-infection group for the 4 skin defect wounds on the back. These gauzes were fixed with medical tape. Each mouse was placed in a separate cage to prevent the gauze from falling off. The gauzes of the rats were changed daily for three consecutive days, and then the skin defects were allowed to heal naturally. The experiment lasted for 7 days. On the 7th day, the rats were asphyxiated with carbon dioxide, and the infected skin defect tissues were collected.

[0101] (2) Collection and processing of skin infection tissues

[0102] The infected skin tissues were excised along the edges of the four defects on the back of each rat. One of the tissues in each group was stored at -80 °C, and the other was fixed with 4% paraformaldehyde for 24 hours, then paraffin-embedded, sectioned, and stained. Subsequently, under sterile and enzyme-free conditions, each frozen tissue in each group was ground into fragments, and each frozen tissue was 0.1 g. Then, the fragmented tissues were treated with 300 μL of RNA lysis buffer for 20 min. After centrifugation (4 °C, 12,000 rpm, 10 min), the supernatant was taken and stored at -80 °C to extract RNA.

[0103] (3) Histopathological analysis

[0104] The skin tissues were fixed with 4% paraformaldehyde, paraffin-embedded, and sectioned (4 μm thick). Then, the sections were dewaxed in xylene, hydrated in gradient alcohol, and stained with hematoxylin and eosin. A pathologist evaluated the degree of skin infection, and the stained samples were photographed under a microscope.

[0105] (4) Tissue real-time fluorescence quantitative PCR (qRT-PCR) analysis

[0106] Total RNA of 0.1 g of skin tissue was isolated by TRIzol extraction method. cDNA was reverse transcribed using the PrimeScript RT reagent kit (TaKaRa, Japan). The reverse transcription conditions were: 37 °C for 15 min; 85 °C for 5 s; 12 °C. The RT-qPCR reaction was performed using HieffTM RT-qPCR The reagent was amplified on the ABI 7500 RT-qPCR system. Amplification and detection were carried out under the following conditions: stable stage at 95°C for 5 min; hot start with 40 s cycles at 95°C and 30 s cycles at 60°C; melting stage with 15 s cycles at 95°C and 1 min cycles at 60°C. The relative gene expression was calculated using the 2-ΔΔCt method. The average Ct value of the target gene in each group was normalized to the average Ct value to obtain the ΔCt value, and then normalized to the control sample to obtain the ΔΔCt value. Each measurement method was repeated 3 times. The gene expression rates of 3 independent experiments were all mean ± SD. The primers used are listed in Table S1.

[0107]

[0108] (5) Immunohistochemical analysis

[0109] The skin tissue pathological sections were dewaxed by immersion in xylene and rehydrated through gradient alcohol, and treated with 0.125% trypsin at 37°C for 10 min. The sections were incubated in a hydrogen peroxide blocker at room temperature for 15 minutes to prevent endogenous peroxidase activity.

[0110] They were blocked with a blocking reagent for 10 min. The sections were incubated with primary antibodies, anti-IL-1β antibody (1:200), anti-IL-6 antibody (1:200), and anti-IL-17 (1:300) and incubated overnight at 4°C. Then, the primary antibodies were washed with PBS, and secondary antibody HRP was added and incubated at room temperature for 30 min. A DAB (3,3'-diaminobenzidine) working solution was used for color development. Hematoxylin was used for counterstaining, and observations and photographs were taken under an optical microscope.

[0111] Experimental results:

[0112] A study on the efficacy of the antibacterial hydrophobic dressing loaded with CIP in treating full-thickness skin wounds of rats infected with SA was conducted. The healing process of the wounds was detected within five days, and the wounds treated with medical gauze were used as the control group. On the first day after surgery, there was a slight bacterial infection at the wound treated with medical gauze. In contrast, the wounds treated with the antibacterial hydrophobic dressing prepared by the present invention did not show obvious exudate, indicating effective inhibition of bacterial infection. By the fourth day, the wound edges of both groups began to contract, and scabbing and drying were observed without exudate. Notably, the wounds treated with the antibacterial hydrophobic dressing were significantly smaller than those treated with medical gauze. By the fifth day, all the wounds in both groups had healed, and the group treated with the antibacterial hydrophobic dressing showed excellent healing performance. It was proved that the antibacterial hydrophobic dressing loaded with CIP might be a promising method for treating bacterial infections in full-thickness wounds, as Figure 9 shown.

[0113] Tissue samples were obtained from the wound site 5 days after surgery and subjected to H&E staining to evaluate the efficacy of various treatments in promoting wound healing. As Figure 10 shown, a marked influx of inflammatory cells was detected around the wound site in the medical gauze group. In contrast, wounds treated with the antibacterial hydrophobic dressing were encapsulated in a tissue layer composed of necrotic tissue and exudates, and the abundance of inflammatory cells was significantly lower than that observed in the medical gauze group. These findings highlight the anti-inflammatory properties of the ciprofloxacin-loaded hydrophobic dressing with antibacterial attributes.

[0114] To clarify the inflammatory status of the wounds, we performed immunohistochemical analysis on day 5 after injury to evaluate the expression of pro-inflammatory cytokines (i.e., IL-1β, IL-17, IL-6). As Figure 11 shown, immunohistochemical staining for IL-β, IL-17, and IL-6 was observed. Notably, the group treated with medical gauze showed a significantly higher amount of pro-inflammatory cytokines (visualized as brown staining) compared to the antibacterial hydrophobic dressing group (where the expression of IL-1β, IL-17, and IL-6 was significantly reduced).

[0115] After collecting RNA from infected skin tissue, our study showed that the mRNA levels of inflammatory factors (such as IL-β, IL-17, IL-6, and Tnf-α) were significantly reduced after CIP treatment. In addition, the mRNA level of the anti-inflammatory factor IL-10 was significantly increased. These findings were complemented by the observed downregulation of Nfkb mRNA levels, indicating that the CIP-loaded antibacterial hydrophobic dressing has potent in vivo anti-inflammatory properties. Therefore, our study highlights the promising potential of ciprofloxacin-loaded cotton fabric as a mandatory therapeutic intervention for suppressing inflammation.

[0116] The above are the preferred embodiments of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, all equivalent or modified implementations completed without departing from the spirit disclosed in the present invention fall within the protection scope of the present invention.

Claims

1. A preparation method of an antibacterial hydrophobic dressing, the steps of which include: 1), ultrasonically cleaning the cotton fabric with a sodium hydroxide solution with a concentration of 5% for 1-2 hours, rinsing with absolute ethanol 2-3 times; rinsing with deionized water 2-3 times; then drying at a temperature above 80 °C; treating with a sodium periodate solution to prepare an oxidized cotton fabric; 2), putting the oxidized cotton fabric into a glycerol solution, standing, washing and drying at room temperature; then putting the cotton fabric into a mixed oxidation solution composed of deionized water, concentrated sulfuric acid and potassium dichromate, and obtaining carboxylated cotton fabric after washing for standby; 3), dissolving hexadecyltrimethylammonium p-toluenesulfonate and triethanolamine, which are surfactants, in deionized water. After the surfactants are completely dissolved, adding tetraethyl orthosilicate and 3-aminopropyltriethoxysilane, and stirring; collecting the product after centrifugation, washing with ethanol 2-3 times to remove residual reactants; extracting with an ammonium nitrate ethanol solution to obtain aminated mesoporous silica; 4), soaking the aminated mesoporous silica in step (3) and the antibacterial drug in a dimethyl sulfoxide solution, magnetically stirring, and centrifuging to separate to obtain drug-loaded aminated mesoporous silica; 5), adding the drug-loaded aminated mesoporous silica in step (4) to ethanol and mixing evenly to form a mixed solution, controlling the pH of the mixed solution to be 6, repeatedly adding an equal volume of octadecyltriethoxysilane, and stirring at room temperature to obtain a hydrophobic sol system; 6), dispersing the drug-loaded aminated mesoporous silica in step (4) in an absolute ethanol solution, ultrasonically dispersing, and then adding N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and triethylamine to form a reaction solution; 7), putting the carboxylated cotton fabric in step (2) into the hydrophobic sol system in step (5) and stirring, washing and drying; then putting the cotton fabric into the reaction solution in step (6) to react, washing and drying to obtain the product.

2. The preparation method according to claim 1, wherein In the step (2), the glycerol solution is composed of ethanol and glycerol with an addition ratio of 100 mL: 0.7-1.2 g.

3. The preparation method according to claim 1, characterized in that, In the step (3), the addition ratio of hexadecyltrimethylammonium p-toluenesulfonate, triethanolamine, deionized water and tetraethyl orthosilicate is 1-5 g: 0.1-0.8 g: 100 mL: 10-30 mL.

4. The preparation method according to claim 1, wherein In the step (3), the volume addition ratio of tetraethyl orthosilicate and 3-aminopropyltriethoxysilane is 5:

1.

5. The preparation method according to claim 1, characterized in that, In the step (5), the addition ratio of the drug-loaded aminated mesoporous silica and ethanol is 1 g: 30-50 mL.

6. The preparation method according to claim 1, characterized in that, In the step (5), the volume ratio of the octadecyltriethoxysilane added each time in the mixed solution to the volume of ethanol is 1:150-250, and stirring is carried out at room temperature for 1-3 hours at a rotation speed of 250 rpm.

7. The preparation method according to claim 1, characterized in that, In the step (6), the addition ratio of the drug-loaded aminated mesoporous silica, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and triethylamine is 2-7 mg: 0.5-4 mg: 0.1-1.5 mg: 1 μL.

8. An antibacterial and hydrophobic dressing, characterized in that Obtained by any of the preparation methods in claims 1-7.

Images