Antibacterial cheek patch and preparation method thereof

Abstract

The invention relates to the technical field of oral patches, and discloses an antibacterial cheek patch and a preparation method thereof.The antibacterial cheek patch is prepared from antibacterial medicine and gelatin, the antibacterial medicine comprises golden basidiosin A and magnesium hydroxide, and the golden basidiosin A and the magnesium hydroxide synergistically exert the antibacterial effect; the effect has obvious advantages compared with single action of the gold basidiosin A and the magnesium hydroxide; and the composition does not cause drug resistance of candida albicans, and is simple and convenient to use and low in cost. The antibacterial cheek patch disclosed by the invention is prepared in a manner of firstly preparing the gelatin and the medicine patch, and then performing crosslinking and coating the polydopamine coating; the preparation cost is low and the medicine treatment efficiency is high.

Description

Technical Field

[0001] The invention relates to the technical field of oral patches, and in particular to an antibacterial cheek patch and a preparation method thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Oral fungal infection is a common fungal infection in clinical practice. With the high incidence of immunodeficiency diseases, the incidence of oral fungal infection has been increasing year by year. At present, clinical treatment usually uses oral antibacterial drugs in combination with sodium bicarbonate mouthwash, but this will lead to low drug concentration in the oropharynx and increase the burden on the liver and kidneys. Therefore, how to solve the drug resistance of antifungal drugs and the toxicity caused by systemic medication is an urgent problem that needs to be solved in the treatment of oral candidiasis.

[0004] In the past, antifungal treatment usually used drugs in combination to reduce drug resistance, but this method has limitations and may cause drug interactions. Therefore, antifungal drugs based on natural products have gradually gained attention. New antibacterial drugs reduce drug resistance by targeting new targets and using low doses. We screened out a potential natural product: Aureobasidin A (AbA), which is a cyclic lipopeptide antibiotic extracted from the filamentous fungus Aureobasidium pullulans No.R106. It has good research prospects as a natural inhibitor of IPC synthase. Oral buccal mucosal administration has attracted attention due to its advantages in local and systemic administration, by increasing local concentrations, rapidly entering the blood and avoiding first-pass effects. The high lipophilicity of AbA makes it more suitable for local application. In addition, most existing antifungal drugs are small molecule organic compounds with low water solubility, which limits the antibacterial activity. Therefore, the use of lipophilic drugs and alkaline substances encapsulated in polymers can effectively increase the local drug concentration and design for the characteristics of local pH reduction in Candida albicans infection, providing ideas for new antifungal strategies.

[0005] Due to its special properties, MH (Mg (OH) 2, magnesium hydroxide) can be decomposed into Mg in a slightly alkaline and neutral environment. 2+ and OH - This property can neutralize the weak acidic environment created by Candida albicans infection, and at the same time can catalyze the decomposition of MH to generate metal ions, thereby enhancing the permeability of the Candida albicans cell wall. Among them, the fungal cell wall is an important protective structure of Candida albicans, which can effectively resist external stimuli and drug attacks to form a physical barrier. This increases the pathogenicity of Candida albicans during the infection process when facing the host's immune system and external drugs. Because MH releases Mg2+ The integrity of the outer layer of the cell wall is first destroyed, so the natural inhibitor of IPC synthase AbA wrapped in gelatin can directly penetrate the cell wall and exert its effect directly. 2+ The release of antioxidants enhances the anti-inflammatory effect.

[0006] At present, there are inventions for preparing gelatin sustained-release patches, but they only use traditional release mechanisms, such as simple diffusion; and many antibacterial agents only rely on a single mechanism to fight bacteria, leading to an increase in fungal resistance. Summary of the invention

[0007] The purpose of the present invention is to address the problem that current antifungal drugs are prone to drug resistance, and to provide an antibacterial buccal patch and its preparation method. Through multiple mechanisms of combined antibacterial, the oral patch can produce significant synergistic antifungal effects and avoid systemic toxic side effects. This study aims to address the increasingly serious problem of antibiotic resistance by combining multiple mechanisms of antibacterial and reducing the risk of drug-resistant infection. The special chemical properties of MH are used to improve the local acidic environment while establishing a slightly alkaline environment to release Mg. 2+ , establish an intelligent release system and exert a synergistic antibacterial effect. In addition, the buccal delivery method can avoid the first-pass effect of the liver and kidneys and reduce systemic toxic side effects. Finally, a mouse oral Candida albicans model was established for further verification. On the one hand, it can evaluate its antibacterial activity and anti-inflammatory ability in vivo, and on the other hand, it can lay the foundation for clinical application and further provide new ideas for antifungal treatment.

[0008] The technical solution of the present invention is as follows:

[0009] On the one hand, the present invention provides an antibacterial buccal patch, which is made of antibacterial drugs and gelatin. The antibacterial drugs include aurobacitin A and magnesium hydroxide, and the chemical cross-linking agent of the gelatin is, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS).

[0010] The oral patch of the present invention is cross-linked after being fully dried to enhance its hydrophobicity, so as to effectively resist the moist environment in the oral cavity and prolong the service life. At the same time, the outer layer is coated with polydopamine (PDA) to improve the surface tightness and hydrophobicity of the membrane to ensure the sustained release ability of the drug. The addition of magnesium hydroxide can neutralize the weak acidic environment caused by Candida albicans, make the surrounding environment tend to be neutral or weakly alkaline, promote the release of magnesium ions, and further enhance the synergistic antibacterial effect of the membrane. The membrane has a wide range of application prospects in the field of oral health.

[0011] According to a preferred embodiment, the amount of aurobacitin A is 9-11 μg / patch, the amount of magnesium hydroxide is 190-210 mg / patch, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 0.47 g, and the amount of N-hydroxysulfosuccinimide is 0.28 g.

[0012] According to a preferred embodiment, polydopamine is further included, and the amount of polydopamine used is 0.08-0.12 g / patch.

[0013] According to a preferred embodiment, the mass ratio of gelatin to magnesium hydroxide is 45-55:1, so as to ensure the response characteristics of the prepared buccal patch to pH.

[0014] According to a preferred embodiment, the ratio of the cross-linking agent EDC / NHS to gelatin is 0.70-0.80:1, ensuring sufficient cross-linking without affecting drug release.

[0015] Another aspect of the present invention provides a method for preparing an antibacterial cheek patch, comprising the following steps:

[0016] (1) Mixing: Mix gelatin and deionized water and dissolve and stir evenly;

[0017] (2) Adding drugs: Add aurobacitin A and magnesium hydroxide according to the ratio and continue stirring to ensure that the drugs are evenly dispersed;

[0018] (3) Molding: pouring the mixed solution prepared in step (2) and adding the drug into a mold, placing it at room temperature, and drying it;

[0019] (4) Cross-linking: EDC and succinimide NHS are weighed according to the ratio, dissolved in anhydrous ethanol, and then ultrapure deionized water is added to fully dissolve into a uniform solution; the membrane dried in the above step (3) is placed in the uniform solution to fully cross-link;

[0020] (5) Drying: The cross-linked patch is taken out and dried at room temperature to obtain a cross-linked membrane sheet;

[0021] (6) Addition of surface coating: Dopamine was placed in TRIS-HCL and mixed evenly, and the membrane was placed in the mixed solution to prepare a polydopamine coating. Then, 100 μL of 30% hydrogen peroxide solution was added, and 2 μL of CAT enzyme was added to react for 30 minutes. The surface of the membrane changed from white to the black color of polydopamine, indicating that the membrane had a polydopamine coating on the surface.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. An antibacterial cheek patch and a preparation method thereof. The composite material Gel / AbA / MH@PDA of the present invention further improves the antibacterial efficiency through the synergistic effect of multiple mechanisms such as pH response release and intelligent drug release; the antibacterial effect has significant advantages over the single AbA treatment and MH treatment, and effectively reduces the occurrence of fungal resistance;

[0024] 2. An antibacterial buccal patch that optimizes drug release. Traditional drug delivery methods often have unstable drug release rates, resulting in unstable efficacy. The patch of this application uses an intelligent release system that can adjust drug release according to changes in the oral environment (such as pH and temperature). This controlled release improves drug utilization and ensures effective drug release when needed; ensuring the efficiency and reliability of the product in clinical applications;

[0025] 3. An antibacterial buccal patch. The existing systemic administration methods such as oral drugs are often accompanied by significant systemic toxic and side effects. However, the local buccal administration method of the present application can significantly reduce systemic drug exposure, thereby reducing the risk of systemic toxic and side effects. This feature makes the patch more suitable for the treatment of high-risk patients (such as the elderly and children); at the same time, compared with some traditional treatment methods that are complex, difficult to operate, and have low patient compliance; the application method of Gel / AbA / MH@PDA of the present application is simple and easy for patients to operate by themselves, which improves the convenience of treatment and patient compliance, thereby enhancing the treatment effect;

[0026] 4. An antibacterial cheek patch has a lower cost of use, reduces the use of traditional antibiotics, optimizes the selection of materials, reduces preparation costs, and reduces medical costs and improves the economic benefits of drugs to a certain extent by improving the therapeutic efficiency of drugs and reducing the systemic side effects of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Flow chart for the preparation of Gel / AbA / MH@PDA;

[0028] Figure 2 Mapping of patch surface elements;

[0029] Figure 3 is a graph of gas chromatography-mass spectrometry data (showing successful loading of AbA);

[0030] Figure 4 The results of the spot board experiment of different component patches;

[0031] Figure 5 The antibacterial rings and growth curves (synergistic antibacterial ability) of patches with different components are shown;

[0032] Figure 6This is a photo of the mouse tongue in the Candida albicans infection model group of this application;

[0033] Figure 7 The results of plate spreading of Candida albicans in the mouths of model group mice treated with different groups;

[0034] Figure 8 The staining results of pathological sections of mice treated in different groups;

[0035] Fig. 9 The duration of action of different groups of drugs. DETAILED DESCRIPTION

[0036] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0038] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0039] Example 1 Preparation of an antibacterial cheek patch

[0040] Reference Figure 1 , including the following steps:

[0041] (1) Mixing: Mix 1 g of gelatin with 10 ml of deionized water, and stir at 65° C. for 20 min to obtain a gelatin solution.

[0042] (2) Adding drugs: Add 10 μg of aurobacitin A and 200 mg of magnesium hydroxide and continue stirring to ensure that the drugs are evenly dispersed in the polymer matrix.

[0043] (3) Molding: Pour the mixed solution into a round mold and place it at room temperature to dry for 48 hours.

[0044] (4) Cross-linking: Weigh 0.47 g EDC and 0.28 g succinimide NHS on an analytical balance, dissolve in 78 g anhydrous ethanol, and then add 10 g ultrapure deionized water to fully dissolve into a uniform solution. Place the dried membrane in the mixed solution and fully cross-link for 30 min at room temperature.

[0045] (5) Drying: The cross-linked patch is taken out and dried at room temperature for 24 hours to obtain a cross-linked membrane sheet;

[0046] (6) Addition of surface coating: 1.0 mg of dopamine was placed in TRIS-HCL (PH=8.5) and mixed evenly, and the membrane was placed in the mixed solution to prepare a polydopamine coating. Then, 100 μL of 30% hydrogen peroxide solution was added, and 2 μL of CAT enzyme was added to react for 30 minutes. It can be seen that the surface of the membrane changed from white to the black color of polydopamine, indicating that the surface of the membrane already had a polydopamine coating.

[0047] The prepared Gel / AbA / MH@PDA patch was subjected to gas chromatography-mass spectrometry detection, and the results were as follows: Figure 3 The appearance of amide bonds indicates successful loading of AbA.

[0048] Map the surface elements of the patch, the result is as follows Figure 2 It can be seen that the surface of the patch has elements such as C, O, and Mg.

[0049] Example 2 In vitro experiment

[0050] Fungal spot plate experiment after co-culture of composite materials with different components and Candida albicans and antibacterial ring experiment of composite materials with different components:

[0051] (1) Spot plate experiment: Candida albicans in the logarithmic growth phase was diluted to 1×10 using Sabouraud liquid medium. 6 CFU / mL, and then different components (control group, Gel@PDA, Gel / AbA@PDA, Gel / MH@PDA, Gel / AbA / MH@PDA) were co-cultured with white beads and diluted to different multiples (10 1 , 10 2 , 10 3 , 10 4 , 10 5 ) and evenly spread on YPD agar plates and count the number of colonies.

[0052] Each group of materials was prepared using the following methods: Gel@PDA: the gelatin solution (without antibacterial drugs) was dried and then directly coated with PDA on the surface; Gel / AbA@PDA: 10 μg of aureobasidin A was mixed in the gelatin solution and then made into sheets and then coated with PDA on the surface; Gel / MH@PDA: 100 mg of magnesium hydroxide was mixed in the gelatin solution and then coated with PDA.

[0053] The results are as follows Figure 4 As shown, compared with the control group, the different treatment groups had a certain antibacterial effect; among them, the antibacterial effect of the Gel / AbA / MH@PDA group was more significant than that of the Gel / AbA@PDA and Gel / MH@PDA groups, which was one order of magnitude higher.

[0054] (2) Inhibition ring experiment: Take the overnight cultured Candida albicans and evenly spread it on the YPD agar plate, and place 5 groups of 5 mm × 5 mm patches of different components on it for co-culture for 48 hours. Use ImageJ to calculate the area of ​​the inhibition ring. The control group is a simple gelatin film. The results are as follows Figure 5 By calculating the size of the inhibition zone of different components, it can be found that under the same action time, the addition of PDA coating can make the internal structure compact, thereby allowing the drug to be released slowly. Figure 5 A It can be found that the size of the inhibition zone after adding PDA coating is larger than that of the group without coating, indicating that the addition of PDA has the ability to slow release and enhance the antibacterial effect. Figure 5 B: This proves that the patch with MH added has a larger inhibition zone size than the patch without MH added. Therefore, it can be indirectly shown that AbA and MH have a synergistic antibacterial effect. The effect of the Gel / AbA / MH@PDA group is significantly better than that of the Gel / AbA@PDA and Gel / MH@PDA groups with AbA or MH acting alone.

[0055] (3) To explore the drug sustained release ability of Gel / AbA / MH@PDA, AbA alone and Gel / AbA / MH@PDA were mixed with Candida albicans (1×10 5 CFU / mL) co-culture was performed on the first, third, and fifth days. Fig. 9 As shown in the figure, AbA can completely inhibit the growth of Candida albicans on the first day, but its antibacterial effect gradually loses its effectiveness over time. However, Gel / AbA / MH@PDA can continue to exert its antibacterial effect for up to 5 days, which indirectly indicates that Gel / AbA / MH@PDA has a good sustained-release and long-lasting antibacterial effect.

[0056] Example 3 In vivo experiment

[0057] Data on the use of this composite material in the treatment of oral Candida albicans infections:

[0058] It was applied to the oral Candida albicans mouse model to further observe the number of strains, hyphae attachment, and inflammation-related factor indicators.

[0059] (1) After destroying the immunity of mice by intraperitoneal injection of cortisone, the oral Candida albicans mouse model was established by intraoral smear method:

[0060] 220mg / kg cortisone acetate (immunosuppressant) was injected subcutaneously 3 days before infection, 1 day and 2 days after infection, and 28.3mg / ml tetracycline hydrochloride was added to the drinking water to prevent it from damaging the mouse immune system. A saturated cotton ball dipped in Candida albicans liquid (C.albicans) was placed under the tongue of the mouse and removed after the anesthetic wore off (2.5h). After 3 days of continuous infection, the size of the white plaque on the tongue and the in vivo growth of Candida albicans were observed.

[0061] The Candida albicans infection model was constructed as follows Figure 6 shown.

[0062] Treatment process and subsequent sampling and staining experimental methods:

[0063] Detection of the antibacterial activity of slightly alkaline aurobasidin A patch in the oral Candida albicans mouse model.

[0064] The experimental groups were as follows: (Oral administration group: ① Fluconazole + NaHCO3 mouthwash ② Gold Basidin A + NaHCO3 mouthwash; Buccal administration group: ③ Gel / Mg(OH)2 / AbA@PDA ④ Gel / AbA@PDA ⑤ No treatment control group) After 5 days of treatment, the size of the white plaque on the tongue was roughly observed to explore the synergistic antibacterial effect of the slightly alkaline gold basidiin A patch in the mouse disease model and the difference in the therapeutic effect of different administration methods. The mice were then euthanized, and the pathological tissue sections of the tongue were taken for HE and PAS staining to observe whether the inflammation level, epithelial hyperplasia, hyphae adhesion, and growth and reproduction changed. Immunofluorescence staining was performed later, and the inflammation-related gene IL-6 marker was detected for immunohistochemical staining to further evaluate the anti-inflammatory ability of each component.

[0065] After establishing the oral Candida albicans model in mice, samples were collected after 5 days of treatment using different administration routes:

[0066] The fungi in the mouths of mice in each group were diluted and spread on the plates. The results were as follows: Figure 7 As shown in the figure, the number of oral fungi in the Gel / Mg(OH)2 / AbA@PDA group was significantly reduced. Figure 8The results of the pathological sections shown in the figure show that PAS staining found obvious hyphae PAS positive staining in the model control group, and obvious epithelial abnormal proliferation. There was still a small amount of fungal colonization in the oral administration group. No hyphae were observed in the Gel / Mg(OH)2 / AbA@PDA group, and the epithelial spikes did not disappear. HE and immunohistochemistry results showed that the level of inflammatory factors in the Gel / Mg(OH)2 / AbA@PDA group was significantly reduced. These results show that magnesium hydroxide and AbA work synergistically, significantly enhancing the antibacterial effect, and comprehensive treatment improves Candida albicans infection.

[0067] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. An antibacterial cheek patch, characterized in that: The invention is prepared from antibacterial drugs and gelatin, wherein the antibacterial drugs include aurobacitin A and magnesium hydroxide, and the cross-linking agent of the gelatin includes 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide.

2. An antibacterial cheek patch according to claim 1, characterized in that: The concentrations of the antibacterial drugs are: aurobacitin A is 9-11 μg / patch; magnesium hydroxide is 190-210 mg / patch.

3. An antibacterial cheek patch according to claim 1, characterized in that: The outer layer is coated with a polydopamine coating.

4. An antibacterial cheek patch according to claim 3, characterized in that: The concentration of the polydopamine is 0.08-0.12 g / patch.

5. The antibacterial cheek patch according to claim 1, characterized in that: The mass ratio of the gelatin to magnesium hydroxide is 45-55:

1.

6. An antibacterial cheek patch according to claim 1, characterized in that: The ratio of the cross-linking agent EDC / NHS to gelatin is 0.70-0.80:

1.

7. A method for preparing an antibacterial buccal patch according to any one of claims 1 to 6, characterized in that: The steps include: (1) Mixing: Mix gelatin and deionized water and dissolve and stir evenly; (2) Adding drugs: Add aurobacitin A and magnesium hydroxide according to the ratio and continue stirring to ensure that the drugs are evenly dispersed; (3) Molding: pouring the mixed solution prepared in step (2) and adding the drug into a mold, placing it at room temperature, and drying it; (4) Cross-linking: EDC and succinimide NHS are weighed according to the ratio, dissolved in anhydrous ethanol, and then ultrapure deionized water is added to fully dissolve into a uniform solution; the membrane dried in the above step (3) is placed in the uniform solution to fully cross-link; (5) Drying: The cross-linked patch is taken out and dried at room temperature to obtain a cross-linked membrane sheet; (6) Addition of surface coating: dopamine is placed in TRIS-HCl and mixed evenly, and the membrane is placed in the mixed solution to prepare a polydopamine coating.

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