Antibacterial hydrogel and application thereof in treating acute otitis media

Abstract

The invention relates to an antibacterial hydrogel, which is a gel formed by taking chitosan quaternary ammonium salt as a component and contains protocatechualdehyde. It is verified that the antibacterial hydrogel is good in biocompatibility and high in safety, has the capabilities of resisting bacteria, resisting generation of bacterial biofilms, resisting inflammation, resisting oxidation and the like and immunoregulatory activity, can play a good acute otitis media treatment effect in a local administration mode, is low in drug resistance, facilitates reduction of abuse of antibiotics, and has a good application prospect. Infection and recurrence are effectively prevented, and a novel, safe and ideal treatment method is provided for clinical decision-making of acute otitis media patients. The gel is prepared into an ear drop dosage form, does not flow out after being dropped into the ear canal, and is convenient to take.

Description

Technical Field

[0001] The invention relates to application of a composition in medical treatment, in particular to application of a gel containing chitosan in treating acute otitis media, and a medical device thereof. Background Art

[0002] Acute otitis media is a common infectious disease in infants and young children with a high incidence rate. Patients often experience severe ear pain, fever and other symptoms. If not treated in time, it may cause serious complications such as mastoiditis and meningitis. Currently, antibiotics are often used for targeted treatment of acute otitis media in clinical practice. However, with the widespread use of antibiotics, the problem of drug resistance has become increasingly prominent, resulting in the phenomenon of prolonged illness and increased risk of complications. Summary of the invention

[0003] One object of the present invention is to provide an antibacterial hydrogel, which is a substitute for antibiotics and is beneficial for the treatment of acute otitis media.

[0004] Another object of the present invention is to provide an application of an antibacterial hydrogel in the preparation of a drug for treating acute otitis media.

[0005] Another object of the present invention is to provide an application of an antibacterial hydrogel in the preparation of a medical device for treating acute otitis media.

[0006] An antibacterial hydrogel contains 1.25wt% to 2.5wt% of protocatechualdehyde.

[0007] Another antibacterial hydrogel is a gel formed by chitosan quaternary ammonium salt, containing 1.25wt% to 2.5wt% of protocatechualdehyde.

[0008] Another antibacterial hydrogel is a gel formed by 5wt% to 10wt% chitosan quaternary ammonium salt, containing 1.25wt% to 2.5wt% protocatechualdehyde.

[0009] Chitosan quaternary ammonium salt, also known as hydroxypropyltrimethylammonium chloride chitosan, is substituted on the amino group at position 2, with a degree of substitution of 90% and a molecular weight Mn of 100,000 Da.

[0010] The antibacterial hydrogel of the present invention has a porous (honeycomb) network structure with a porosity of 36±1%.

[0011] The method for preparing the antibacterial hydrogel of the present invention is as follows:

[0012] A 10% chitosan quaternary ammonium salt solution and a 2.5% protocatechuic aldehyde solution are prepared respectively, and the two solutions are mixed in equal volumes and allowed to stand to obtain the antibacterial hydrogel.

[0013] It has been verified that the antibacterial hydrogel of the present invention has good biocompatibility and high safety, and has antibacterial, anti-bacterial biofilm formation, anti-inflammatory, antioxidant and immunomodulatory abilities. It can exert a good therapeutic effect on acute otitis media through local administration, has low drug resistance, and can effectively prevent recurrence of infection.

[0014] The gel of the present invention is made into medicine, such as ear drops, which will not flow out after being dropped into the ear canal, and is convenient for medication. The application of the gel helps to reduce the abuse of antibiotics and provides a new, safe and ideal treatment method for clinical decision-making of patients with acute otitis media. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 For QCS-PA 2.5 SEM electron micrograph of antibacterial hydrogel;

[0016] Figure 2 For QCS-PA 2.5 Viscosity-shear rate curve of antibacterial hydrogel;

[0017] Figure 3 For QCS-PA 2.5 Modulus-strain curves of hydrogels;

[0018] Figure 4 For QCS-PA 2.5 The results of plate counts of the antibacterial hydrogel against Escherichia coli and Staphylococcus aureus;

[0019] Figure 5 For QCS-PA 2.5 The results of the antibacterial hydrogel inhibiting the biofilm formation of Escherichia coli and Staphylococcus aureus;

[0020] Figure 6 For ABTS ·+ QCS-PA 2.5 The results of the inhibition rate of antibacterial hydrogels are shown in the figure. **** indicates p < 0.0001;

[0021] Figure 7 For QCS-PA 2.5 The relationship between antibacterial hydrogel concentration and cell activity rate;

[0022] Figure 8 For QCS-PA 2.5 The relationship between the concentration of antibacterial hydrogel and the concentration of ROS, ** indicates p < 0.01, *** indicates p < 0.001;

[0023] Fig. 9 For QCS-PA 2.5 The results of antibacterial hydrogel inhibiting inflammatory factors, **** in the figure indicates p < 0.0001;

[0024] Fig.10 The results of H&E staining of each experimental group on day 1 (D1) and day 7 (D7), **** in the figure indicates p < 0.0001;

[0025] Fig.11 The results of inflammatory factor detection in each experimental group are shown in the figure. In the figure, ** indicates p < 0.01, and **** indicates p < 0.0001;

[0026] Fig.12 Bright field photos of the bacterial colonies obtained by culturing the middle ear cavity of mice in each experimental group on day 1 (D1) and day 7 (D7);

[0027] Fig.13 The SEM images of bacterial biofilms on the middle ear mucosa of animals in each experimental group on the first day (D1) and the seventh day (D7). DETAILED DESCRIPTION

[0028] The technical solution of the present invention is described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0029] Example 1 Antibacterial hydrogel (QCS-PA 2.5 ) preparation and characterization

[0030] After preheating 10mL of deionized water to 80℃, add 0.25g of protocatechuic aldehyde and stir in the dark. At the same time, add 1g of chitosan quaternary ammonium salt to 10mL of deionized water and stir slowly. After both powders are fully dissolved, add the protocatechuic aldehyde solution dropwise to the chitosan quaternary ammonium salt solution at 50℃, stir slowly for 30min in the dark, and let it stand at room temperature to form gel.

[0031] The gel morphology under scanning electron microscopy (SEM) is shown in Figure 1 As shown, the QCS-PA2.5 hydrogel obtained in this example presents a loose and porous (honeycomb) network structure.

[0032] Rheological tests were performed to investigate the viscosity-shear rate curve of the hydrogel to evaluate the performance of QCS-PA. 2.5 The results show that the hydrogel is injectable. Figure 2 As shown, QCS-PA 2.5 The viscosity of the hydrogel gradually decreases with increasing shear rate, indicating that the gel has shear thinning properties and can be injected through an injection needle.

[0033] Testing and detecting QCS-PA using a rheometer 2.5 The self-healing performance of the hydrogel, and this instrument can detect QCS-PA 2.5 The modulus of the hydrogel changes with the strain force, reflecting the changes in the gel morphology and internal covalent bonds. When the storage modulus (G′) is greater than the loss modulus (G″), the material is in a gel state and there are stable covalent bonds inside; when G′ is less than G″, the material is in a liquid state and the internal covalent bonds are broken. The dynamic change process of the gel covalent bonds reflects the self-healing characteristics of the gel.

[0034] Figure 3 For QCS-PA 2.5 The modulus-strain force curve of the QCS-PA hydrogel is as shown. The results show that when the strain force changes from 1% to 1000%, G′ < G″, and the dynamic bonds inside the gel break; when the strain force changes from 1000% to 1%, G′ > G″, and the dynamic bonds inside the gel regenerate. QCS-PA 2.5 The dynamic change of the covalent bonds inside the gel indicates that QCS-PA 2.5 The gel has self-healing performance.

[0035] Example 2: In vitro antibacterial ability test

[0036] Testing the ability of QCS-PA hydrogel against Escherichia coli and Staphylococcus aureus using the plate counting method 2.5 The experimental method is as follows: Add 100 μL of the QCS-PA hydrogel extract (4 mg / mL) to a 48-well plate, and use LB liquid medium as the control group. Then add 10 μL of the bacterial suspension (10 2.5 CFU / mL), and place it in an incubator at 37°C for 2 h. Add 1 mL of the medium to resuspend the bacterial suspension, take 100 μL of the resuspended liquid and spread it on an agar plate, and place it in an incubator at 37°C for 18 - 24 h. Observe the colony-forming units on the plate and count. Bactericidal rate % = (CFU 6 -CFU 对照 -CFU 材料 ) / CFU 对照 × 100%.

[0037] The plate counting results are shown in Figure 4 . QCS-PA 2.5 The QCS-PA hydrogel has a significant inhibitory effect on both Escherichia coli and Staphylococcus aureus. The inhibition rate of Escherichia coli reaches 100%, and the inhibition rate of Staphylococcus aureus reaches 61%.

[0038] Example 3: Evaluation of in vitro anti-bacterial biofilm performance

[0039] Detecting QCS-PA using crystal violet staining 2.5Effect of hydrogel on bacterial biofilm. The experimental method is as follows: add 1 mL of bacterial solution and 1 mL of LB liquid culture medium to a 6-well plate and place it in a 37°C incubator for 2-3 days. Discard the old culture medium and add QCS-PA 2.5 The hydrogel extract (4 mg / mL) and LB liquid culture medium were used as the control group and placed in a 37°C incubator for further culture for 3 days. The culture medium was discarded and 2 mL of PBS was added for washing 2-3 times to wash away the floating bacteria. 1 mL of 10% methanol was added for fixation for 15 minutes, and 1 mL of 0.1% crystal violet was added for staining for 15 minutes after natural air drying. PBS was washed 3-4 times, and 2 mL of 33% glacial acetic acid was added after natural drying. The dye was allowed to stand in a 37°C incubator for 20 minutes to completely dissolve the dye, and its OD value was measured at 595 nm using a multifunctional microplate reader. Inhibition rate % = (1-A 材料 / A 对照 )×100%.

[0040] The results showed that QCS-PA 2.5 The gel can destroy biofilms secreted by Escherichia coli and Staphylococcus aureus (see Figure 5 ), indicating that it has an anti-bacterial biofilm effect.

[0041] Example 4 In vitro scavenging ability of ABTS free radicals (ABTS + ) determination

[0042] The total antioxidant capacity (T-AOC) colorimetric assay kit (ABTS enzyme catalysis method) is used. The assay principle is that ABTS is oxidized to green ABTS under the action of appropriate oxidants. + , in the presence of antioxidants ABTS + The production of will be inhibited, according to ABTS + The change in the characteristic absorption peak at 734 nm can be used to measure and calculate the total antioxidant capacity of the sample.

[0043] The results are as follows Figure 6 As shown, with QCS-PA 2.5 As the concentration of hydrogel extract increased, the total antioxidant capacity increased, and ABTS + The clearance rate can reach 88%, indicating that the QCS-PA 2.5 The hydrogel is able to scavenge ABTS free radicals.

[0044] Example 5 QCS-PA 2.5 Cytotoxicity testing of hydrogels

[0045] Evaluation of different concentrations of QCS-PA by CCK-8 assay 2.5 The toxicity of hydrogel extract to L929 cells. 2.5The hydrogel was immersed in DMEM culture medium and placed in a 37°C incubator for 24 hours. After filtering through a filter head, gel extracts of different concentrations (0, 1, 2, 3 and 4 mg / mL) were obtained. L929 cells were inoculated in a 96-well plate and cultured overnight. The culture medium was removed, and gel extracts of different concentrations were added to the 96-well plate and incubated with fibroblast L929 cells for 24 hours. After reaching the set time point, the culture medium was aspirated, the cells were incubated with CCK-8 solution for 30 minutes, and then the absorbance at 450nm was detected by an enzyme marker to evaluate cell viability.

[0046] The results are as follows Figure 7 As shown, different concentrations of QCS-PA 2.5 After the hydrogel extract was incubated with L929 cells for 24 h, the cell activity was always maintained at a high level (more than 70%), indicating that the QCS-PA 2.5 The hydrogel itself has negligible toxicity to cells and exhibits good biocompatibility.

[0047] Example 6 Detection of QCS-PA 2.5 Effect of hydrogel on ROS level in L929 cells

[0048] Detection of QCS-PA using reactive oxygen species probe DCFH-DA 2.5 Effect of hydrogel on ROS level in L929 cells. The experimental method is as follows: L929 cells were seeded in a 24-well plate and incubated for 24 hours and divided into three groups. The control group was normal growth cells; the H2O2 group was incubated with fresh DMEM containing 450μM H2O2 for 6 hours; the treatment group was incubated with QCS-PA 2.5 After pretreatment with hydrogel extract (0, 1, 2, 3 and 4 mg / mL) for 3 h, fresh DMEM containing 450 μM H2O2 was added and incubated for another 6 h. Subsequently, the culture medium was aspirated, washed 2-3 times with PBS, and 100 μL of DMEM culture medium containing 10 μM DCFH-DA was added and incubated at 37 °C in the dark for 30 min. Hoechst dye was added for nucleus staining during the last 10 min of probe incubation. The probe and dye were aspirated, washed 2 times with PBS, and then photographed by confocal microscopy, and DCF fluorescence was semi-quantitatively analyzed.

[0049] The results are as follows Figure 8 As shown, compared with the H2O2 group, QCS-PA 2.5 The hydrogel significantly reduced the level of ROS in L929 cells.

[0050] Example 7 Detection of QCS-PA 2.5 Effects of hydrogel on the expression of inflammatory factors in Raw264.7 cells

[0051] ELISA kit was used to detect QCS-PA 2.5 Effect of hydrogel on the expression of inflammatory factors in mononuclear macrophage cell line Raw 264.7 cells. The experimental method is as follows: Raw 264.7 cells were inoculated in a 6-well plate and incubated for 24 hours, and then divided into three groups: the control group was normal growth cells; the LPS group was incubated with fresh DMEM containing 10μg / mL lipopolysaccharide (LPS) for 24 hours; the treatment group was incubated with QCS-PA 2.5 The hydrogel extract (0, 1, 2, 3 and 4 mg / mL) and fresh DMEM containing 10 μg / mL LPS were incubated for 24 hours. Subsequently, the cells were collected and centrifuged at 1000 rpm for 5 minutes, and the supernatant was taken for subsequent ELISA tests of inflammatory factors TNF-α and IL-6. 100 μL of standard solution and the cell supernatant to be tested were added to the ELISA plate and incubated at room temperature for 90 minutes. After washing 5 times with the washing solution, 100 μL of biotin-labeled antibody was added and incubated at 37°C for 60 minutes. After washing 5 times again, 100 μL of enzyme-labeled reagent was added and incubated at 37°C for 45 minutes. Wash 5 times, add 100 μL of color developer, and incubate at 37°C in the dark for 30 minutes. Finally, 100 μL of terminator was added to terminate the reaction, and the reaction solution turned yellow. The absorbance at 450 nm was detected by an ELISA instrument to evaluate the protein concentration of inflammatory factors.

[0052] The results are as follows Fig. 9 As shown, compared with the LPS group, QCS-PA 2.5 The hydrogel significantly inhibited the secretion of inflammatory factors in Raw 264.7 cells.

[0053] Example 8 QCS-PA 2.5 Hydrogel treatment of lipopolysaccharide-induced acute otitis media in mice

[0054] Fifteen 4-week-old male C57BL / 6J mice were randomly divided into three groups, with 5 mice in each group, including a control group, a lipopolysaccharide-induced acute otitis media group, and a QCS-PA group. 2.5 The three groups of mice were treated differently as follows:

[0055] Control group: raised under normal conditions and not treated.

[0056] Lipopolysaccharide-induced acute otitis media group (LPS): 15 μL LPS (1 mg / mL) was injected into the tympanic membrane to induce acute otitis media in mice.

[0057] Drug treatment group (LPS+QCS-PA 2.5 ): One day after LPS injection, 15 μL QCS-PA was injected into the tympanic membrane 2.5 Hydrogel.

[0058] H&E staining was used to detect the morphological changes of the middle ear tissues of mice in different groups, and the thickness of the middle ear mucosa and the inflammatory infiltration area in the middle ear cavity were quantitatively analyzed. Fig.10 As shown, compared with the LPS group, QCS-PA 2.5 The hydrogel significantly alleviated the inflammatory changes in the middle ear tissue caused by lipopolysaccharide, reduced the thickness of the middle ear mucosa, and reduced the area of ​​inflammatory infiltration in the middle ear cavity. Seven days after administration, the morphology of the middle ear tissue in the drug treatment group basically returned to normal.

[0059] The levels of inflammatory factors in the middle ear tissues of mice in different groups were detected by ELISA. Fig.11 As shown, compared with the LPS group, QCS-PA 2.5 The hydrogel significantly inhibited the secretion of inflammatory factors induced by lipopolysaccharide. 2.5 The hydrogel can effectively treat lipopolysaccharide-induced acute otitis media.

[0060] Example 9 QCS-PA 2.5 Hydrogel treatment of acute otitis media induced by Staphylococcus aureus in mice

[0061] Fifteen 4-week-old male C57BL / 6J mice were randomly divided into three groups, with 5 mice in each group, including a control group, a group with acute otitis media induced by Staphylococcus aureus, and a group with QCS-PA. 2.5 The three groups of mice were treated differently as follows:

[0062] Control group: raised under normal conditions and not treated.

[0063] Acute otitis media induced by Staphylococcus aureus (S. aureus): 15 μL of Staphylococcus aureus solution (10 6 CFU / mL), induced mice with acute otitis media.

[0064] Drug treatment group (S. aureus + QCS-PA 2.5 ): One day after the tympanic membrane injection of bacterial solution, 15 μL QCS-PA was injected into the tympanic membrane 2.5 Hydrogel.

[0065] The changes in the number of bacterial colonies in the middle ear cavity of mice in different groups were detected by plating the middle ear lavage fluid. The results are as follows Fig.12 As shown, compared with the S. aureus group, QCS-PA 2.5 The number of bacteria in the middle ear cavity of mice in the hydrogel treatment group was significantly reduced, and 7 days after administration, the bacteria in the middle ear of the drug treatment group were almost completely eliminated.

[0066] At the same time, scanning electron microscopy was used to detect the morphological changes of bacterial biofilms on the middle ear mucosa of mice in different groups. Fig.13 As shown, compared with the S. aureus group, QCS-PA 2.5 The hydrogel removed the bacterial biofilm structure on the middle ear mucosa, and 7 days after administration, the morphology of the middle ear mucosa tissue in the drug treatment group basically returned to normal. 2.5 The hydrogel can effectively treat acute otitis media induced by Staphylococcus aureus.

Claims

1. An antibacterial hydrogel, characterized in that It has a porous network structure and contains 1.25wt% to 2.5wt% of protocatechuic aldehyde.

2. The antibacterial hydrogel according to claim 1, characterized in that The gel is formed with chitosan quaternary ammonium salt as the component.

3. The antibacterial hydrogel according to claim 1, characterized in that The gel is formed by taking 5wt% to 10wt% of chitosan quaternary ammonium salt as a component.

4. The antibacterial hydrogel according to claim 2 or 3, characterized in that The chitosan quaternary ammonium salt is substituted on the 2-amino group.

5. The antibacterial hydrogel according to claim 2 or 3, characterized in that The chitosan quaternary ammonium salt substitution degree is 90%.

6. The antibacterial hydrogel according to claim 2 or 3, characterized in that The molecular weight of the chitosan quaternary ammonium salt is 100,000 Da.

7. The antibacterial hydrogel according to claim 2 or 3, characterized in that The porosity of the gel was 36±1%.

8. Use of the antibacterial hydrogel according to claim 1 in preparing a drug for treating acute otitis media.

9. The use according to claim 8, characterized in that The dosage form of the medicine is drops.

10. Use of the antibacterial hydrogel according to claim 1 in preparing medical devices for treating acute otitis media.

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