An antibacterial cheek patch and a method of preparing the same

Abstract

The present application relates to the technical field of oral patch, and discloses an antibacterial buccal patch and a preparation method thereof, wherein the antibacterial buccal patch is made of bacteriostatic drugs and gelatin, the bacteriostatic drugs include cymadicine A and magnesium hydroxide, and the two play a synergistic bacteriostatic effect, which has a significant advantage compared with the single effect of cymadicine A and magnesium hydroxide; and the antibacterial buccal patch does not cause drug resistance of Candida albicans, is easy to use, and has low cost. The antibacterial buccal patch is prepared by the method of first preparing a gelatin and drug patch, then cross-linking and coating a polydopamine coating layer; the preparation cost is low, and the drug treatment efficiency is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oral patches, in particular to an antibacterial buccal patch and a preparation method thereof. BACKGROUND

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

[0003] Oral fungal infection is a common fungal infectious disease in clinical practice. With the high incidence of immunodeficiency diseases, the incidence of oral fungal infection is increasing year by year. Currently, oral antifungal drugs are usually used in combination with sodium bicarbonate gargle, but this can result in low drug concentration in the oropharynx and increased liver and kidney burden. Therefore, how to solve the drug resistance of antifungal drugs and the toxicity caused by systemic administration is an urgent problem that needs to be solved in the treatment of oral candidiasis.

[0004] Previous antifungal treatment usually uses drug combination to reduce drug resistance, but this method has limitations and can cause drug interactions. Therefore, antifungal drugs based on natural products have gradually attracted attention. New antifungal drugs target new targets and use low-dose methods to reduce drug resistance. We screened a potential natural product: Aureobasidin A (AbA), which is a cyclic depsipeptide antibiotic extracted from the filamentous fungus Aureobasidiumpullulans No.R106, and has good research prospects as a natural inhibitor of IPC synthetase. Oral buccal mucosa administration is concerned due to its advantages in local and systemic administration, which can improve local concentration, rapidly enter the blood and avoid first-pass effect. The high lipophilicity of AbA makes it more suitable for local application. In addition, existing antifungal drugs are mostly small-molecule organic compounds with low water solubility, which limits the antifungal activity. Therefore, the encapsulation of liposoluble drugs and basic substances in polymers can effectively improve the local drug concentration and design for the characteristics of local ph reduction in Candida albicans infection, providing a new antifungal strategy.

[0005] MH (Mg(OH)2, magnesium hydroxide) can decompose into Mg2+ and OH- in slightly alkaline and neutral environments due to its special properties. This property can exactly neutralize the weakly acidic environment created by Candida albicans infection, and at the same time catalyze the decomposition of MH to generate metal ions, thereby enhancing the permeability of 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 makes Candida albicans increase its pathogenicity in the infection process when facing the host's immune system and external drugs. Because MH releases Mg2+ and OH-, it can increase the permeability of the cell wall of Candida albicans and increase the permeability of the cell wall of Candida albicans. 2+ - This property can exactly neutralize the weakly acidic environment created by Candida albicans infection, and at the same time catalyze the decomposition of MH to generate metal ions, thereby enhancing the permeability of 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 makes Candida albicans increase its pathogenicity in the infection process when facing the host's immune system and external drugs. Because MH releases Mg​2+ The pre-attack destroys the integrity of the outer layer of the cell wall, so that the IPC synthetase natural inhibitor AbA wrapped in the gelatin can directly penetrate the inside of the cell wall and directly play a role. It plays a multi-effect synergistic antibacterial role, and the release of Mg 2+ improves the anti-inflammatory effect.

[0006] There are currently inventions to prepare gelatin sustained-release patches, but only traditional release mechanisms such as simple diffusion are used, and many antibacterial agents only use a single mechanism to resist bacteria, leading to an increase in fungal drug resistance. SUMMARY

[0007] The purpose of the present application is to address the problem of current antifungal drugs being prone to drug resistance, and to provide an antibacterial buccal patch and a preparation method thereof, which uses multiple mechanisms to resist bacteria and is prepared into an oral patch that can produce significant synergistic antifungal effects while avoiding systemic toxic side effects. This study aims to address the increasingly serious problem of antibiotic resistance by using multiple mechanisms to resist bacteria while reducing the risk of drug-resistant infections. The special chemical properties of MH are used to improve the local acidic environment while establishing a slightly alkaline environment to release Mg 2+ , establishing an intelligent release system and exerting a synergistic antibacterial effect. In addition, the use of buccal drug delivery can avoid the first-pass effect of the liver and kidneys, reducing systemic toxic side effects. Finally, a mouse oral Candida albicans model is established to verify it again, which can evaluate its in vivo antibacterial activity and anti-inflammatory ability, and lay the foundation for clinical application and further provide new ideas for antifungal treatment.

[0008] The technical solutions of the present application are as follows:

[0009] The present application provides an antibacterial buccal patch made of bacteriostatic drugs and gelatin. The bacteriostatic drugs include aurantiamine 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 application enhances its hydrophobicity through a cross-linking process after sufficient drying, effectively resisting the moist environment in the oral cavity and prolonging the service life. At the same time, the outer layer is coated with polydopamine (PDA) to improve the surface tightness and hydrophobicity of the film, ensuring the sustained-release ability of the drug. The addition of magnesium hydroxide can neutralize the weak acidic environment caused by Candida albicans, making the surrounding environment tend to be neutral or weakly alkaline, promoting the release of magnesium ions, and further enhancing the synergistic antibacterial effect of the film. The film has a wide application prospect in the field of oral health.

[0011] According to a preferred embodiment, the amount of Aureobasidin 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 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 prepared buccal patch to have a pH response characteristic.

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

[0015] Another aspect of the present application provides a preparation method of an antibacterial buccal patch, comprising the following steps:

[0016] (1) mixing: mixing gelatin with deionized water and stirring until the gelatin is dissolved and uniform;

[0017] (2) adding drugs: adding Aureobasidin A and magnesium hydroxide according to the ratio and continuing to stir to ensure uniform dispersion of the drugs;

[0018] (3) forming: pouring the drug-added mixed solution prepared in step (2) into a mold and placing it at room temperature for drying;

[0019] (4) cross-linking: weighing EDC and succinimide NHS according to the ratio, dissolving them in anhydrous ethanol, and then adding ultrapure deionized water to fully dissolve into a uniform solution; placing the dried film prepared in step (3) above into the uniform solution for cross-linking;

[0020] (5) drying: taking out the cross-linked patch and drying it at room temperature to obtain the cross-linked film;

[0021] (6) adding a surface coating: placing dopamine in TRIS-HCL and mixing until uniform, placing the film in the mixed solution to prepare a polydopamine coating. Then adding 100 μL of 30% hydrogen peroxide solution and 2 μL of CAT enzyme to perform an enzymatic reaction for 30 min, and the surface of the film changes from white to black, indicating the presence of a polydopamine coating on the surface.

[0022] Compared with the existing technology, the present application has the following beneficial effects:

[0023] 1. An antibacterial buccal patch and its preparation method, the composite material Gel / AbA / MH@PDA of the application further improves the antibacterial efficiency through the synergistic effect of multiple mechanisms such as pH-responsive release and intelligent drug release; the bacteriostatic effect is significantly superior to that of AbA treatment and MH treatment alone, effectively reducing the generation of fungal drug resistance;

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

[0025] 3. An antibacterial buccal patch, the existing technology of oral drug and other systemic drug delivery methods often accompanied by significant systemic toxic side effects, and the application can significantly reduce systemic drug exposure through buccal local drug delivery, thereby reducing the risk of systemic toxic 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 and difficult to operate, the patient's compliance is low; the application of Gel / AbA / MH@PDA is simple and easy for patients to operate, improving the convenience of treatment and patient compliance, thereby enhancing the therapeutic effect;

[0026] 4. An antibacterial buccal patch, the use cost is reduced, the use of traditional antibiotics is reduced, the selection of materials is optimized, the preparation cost is reduced, the therapeutic efficiency of the drug is improved and the systemic side effects of the drug are reduced, to a certain extent, the medical cost is reduced, and the economic benefit of the drug is improved. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a preparation flow chart of Gel / AbA / MH@PDA;

[0028] Figure 2 is a patch surface element mapping;

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

[0030] Figure 4 is the effect of spot plate experiment of different component patches;

[0031] Figure 5 is the bacteriostatic ring and growth curve of different component patches (synergistic antibacterial ability);

[0032] Figure 6The tongue photos of the model mice of the Candida albicans infection model group of the present application;

[0033] Figure 7 The results of the Candida albicans plate coating of the model mice treated by different groups;

[0034] Figure 8 The results of the pathological section staining of the mice treated by different groups;

[0035] Figure 9 The duration effect of the different groups of drugs. DETAILED DESCRIPTION

[0036] The specific examples listed in the present application are only as examples of the present application, and the present application is not limited to the specific examples described below. Any equivalent modifications and substitutions of the examples described below by those skilled in the art are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be included in the scope of the present application. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If no manufacturer of all reagents or instruments is specified, it is a conventional product that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some other examples, methods, means, instruments and steps well known to those skilled in the art are not described in detail in order to highlight the main idea of the present application.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, the units used in the specification are international standard units, and the numerical values and numerical ranges appearing in the present application should be understood to include the systematic errors inevitable in industrial production.

[0038] The features and properties of the present application are further described in detail below in conjunction with the examples.

[0039] Example 1 Preparation of an antibacterial buccal patch

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

[0041] (1) Mixing: 1 g of gelatin is mixed with 10 ml of deionized water, and then stirred at 65°C for 20 min to mix uniformly; a gelatin solution is obtained.

[0042] (2) Drug addition: 10 μg of aureobasidin A and 200 mg of magnesium hydroxide are added and continue to stir to ensure that the drugs are uniformly dispersed in the polymer matrix.

[0043] (3) Molding: Pour the mixed solution into a circular mold and dry at room temperature for 48 h.

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

[0045] (5) Drying: Take out the cross-linked patch and dry it at room temperature for 24 h to obtain the cross-linked membrane piece;

[0046] (6) Addition of surface coating: Place 1.0 mg of dopamine in TRIS-HCL (pH = 8.5) and mix well. Place the membrane piece in the mixed solution to prepare a polydopamine coating. Then add 100 μL of 30% hydrogen peroxide solution and 2 μL of C AT enzyme to perform enzymatic reaction for 30 min. The surface of the membrane piece changes from white to black, indicating the presence of a polydopamine coating.

[0047] The Gel / AbA / MH@PDA patch prepared was detected by gas chromatography mass spectrometry, and the results are shown in Figure 3 . The presence of amide bonds indicates the successful loading of AbA.

[0048] The surface element mapping of the patch was performed, and the results are shown in Figure 2 . It can be seen that the patch surface has elements such as C, O, and Mg.

[0049] Example 2 in vitro experiment

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

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

[0052] Each group of materials was prepared by the following method: Gel@PDA: gelatin solution (without antibacterial drugs) was dried and then PDA coating was directly performed on the surface; Gel / AbA@PDA: 10 μg of aureobasidin A was mixed in the gelatin solution to form a sheet, and then PDA coating was performed on the surface; Gel / MH@PDA: 100 mg of magnesium hydroxide was mixed in the gelatin solution, and then PDA coating was performed.

[0053] The results are shown in Table 1. Figure 4 As shown in Table 1, the bacteria in different treatment groups had certain antibacterial effect compared with the control group; 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, and was one order of magnitude higher.

[0054] (2) Antibacterial ring experiment: overnight cultured white Candida albicans was uniformly coated on YPD agar plates, and 5 mm x 5 mm patches of different components were placed on the plates for co-culture for 48 h. ImageJ was used to calculate the area of the antibacterial ring, and the control group was a simple gelatin film. The results are shown in Table 2. Figure 5 As shown in Table 2, the size of the antibacterial ring of different components was calculated, and it was found that under the same action time, the addition of PDA coating could make the internal structure compact, thereby releasing the drug, so Figure 5 A could find that the size of the antibacterial ring after adding PDA coating was larger than that without adding coating, indicating that the addition of PDA had the ability to release and enhance the antibacterial effect. Figure 5 B: It was proved that the patch with MH had a larger antibacterial ring size than the patch without MH. Therefore, it could be indirectly explained that AbA and MH had a synergistic antibacterial effect. The effect of the Gel / AbA / MH@PDA group was significantly better than that of the Gel / AbA@PDA and Gel / MH@PDA groups with AbA or MH alone.

[0055] (3) To explore the drug release ability of Gel / AbA / MH@PDA, simple AbA and Gel / AbA / MH@PDA were co-cultured with white Candida albicans (1 x 10 5 CFU / mL) for spot plate experiment on the first day, the third day, and the fifth day. The results are shown in Table 3. Figure 9 As shown in Table 3, AbA could completely inhibit the growth of white Candida albicans on the first day, but its antibacterial effect gradually failed as time went on. However, Gel / AbA / MH@PDA could continuously exert antibacterial effect for up to 5 days, thus indirectly indicating that Gel / AbA / MH@PDA had good sustained-release antibacterial effect.

[0056] Example 3 in vivo experiment

[0057] The data of the composite material for treating oral Candida albicans infection:

[0058] The application of the mouse model of oral Candida albicans was further observed in the number of strains and hyphae attachment, inflammation-related factor indicators.

[0059] (1) The mouse model of oral Candida albicans was established by applying the mouse immune system to be destroyed by intraperitoneal injection of cortisone and applying Candida albicans in the mouth:

[0060] Three days before infection, one day before infection, and two days after infection, 220 mg / kg of cortisone acetate was subcutaneously injected (immunosuppression) and 28.3 mg / ml of tetracycline hydrochloride was added to the drinking water to prevent the mouse immune system from being destroyed. A saturated cotton ball dipped in Candida albicans solution (C. albicans) was placed under the mouse's tongue, and was removed after the anesthetic wore off (2.5 h). 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 constructed Candida albicans infection model is as shown in Figure 6 .

[0062] Treatment process and subsequent sample staining experiment method:

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

[0064] The experimental groups are as follows: (oral administration group: ① fluconazole + NaHCO3 gargle ② basidiomycete A + NaHCO3 gargle; buccal administration group: ③ Gel / Mg(OH)2 / AbA@PDA ④ Gel / AbA@PDA ⑤ untreated control group) After 5 days of treatment, the size of the white plaque on the tongue was observed macroscopically to investigate the synergistic antibacterial effect of the slightly alkaline basidiomycete A patch in the mouse disease model in vivo and the difference in treatment effect of different administration methods. Subsequently, the mice were euthanized, and their tongue pathological tissue sections were taken for HE and PAS staining to observe whether the inflammation level, epithelial abnormal hyperplasia, hyphae attachment, and growth and reproduction changed. Subsequently, immunofluorescence staining was performed to detect the inflammation-related gene IL-6 marker for immunohistochemical staining to further evaluate the anti-inflammatory ability of each group.

[0065] After the mouse model of oral Candida albicans was established, different administration routes were used for treatment for 5 days, and the samples were taken:

[0066] The fungal samples from each group of mice were diluted and plated, and the results are shown in Figure 7 . The number of fungi in the Gel / Mg(OH)2 / AbA@PDA group was significantly reduced. As shown in Figure 8The pathological section results showed that PAS staining found that the model control group had obvious hyphal PAS positive staining, and the epithelial hyperplasia was obvious. A small amount of fungi still colonized in the oral administration group. No hyphae were attached in the Gel / Mg(OH)2 / AbA@PDA group, and the epithelial pegs did not disappear. HE and immunohistochemical results showed that the inflammatory factor level of the Gel / Mg(OH)2 / AbA@PDA group was significantly reduced. These results showed that magnesium hydroxide and AbA synergistically enhanced the antibacterial effect, and the comprehensive treatment improved the Candida albicans infection.

[0067] The above-described embodiments only express the specific implementation of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are within 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 agents of the gelatin include 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide; The concentrations of the antibacterial drugs are: aurobacitin A is 9-11 μg / patch; magnesium hydroxide is 190-210 mg / patch; The antimicrobial cheek patch is coated with a polydopamine coating; The mass ratio of gelatin to magnesium hydroxide is 45-55:1; The antibacterial cheek patch is prepared by the following method: The steps include: (1) Mixing: Mix gelatin with deionized water and dissolve and stir evenly; (2) Add drugs: Add aurobacitin A and magnesium hydroxide according to the ratio and continue stirring to ensure that the drugs are evenly dispersed; (3) Molding: Pour the mixed solution prepared in step (2) and added with the drug into a mold, place it at room temperature, and dry it to obtain a dried membrane; (4) Cross-linking: EDC and succinimide NHS were weighed according to the ratio, dissolved in anhydrous ethanol, and then added with ultrapure deionized water to fully dissolve into a uniform solution; the membrane dried in step (3) above was placed in the uniform solution and fully cross-linked; (5) Drying: The cross-linked patch is taken out and dried at room temperature to obtain a cross-linked membrane; (6) Addition of surface coating: Dopamine is placed in TRIS-HCl and mixed evenly. The cross-linked membrane in step (5) is placed in the mixed solution to prepare a polydopamine coating.

2. An antibacterial buccal patch according to claim 1, characterized in that: The concentration of the polydopamine is 0.08-0.12 g / patch.

3. The antibacterial buccal patch according to claim 1, characterized in that: The ratio of the cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide to gelatin is 0.70-0.80:

1.

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

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