Antibacterial mouthwash for photodynamic therapy of periodontitis and preparation method and use method thereof

Abstract

The present invention belongs to the technical field of periodontitis treatment and discloses an antibacterial mouthwash for photodynamic therapy of periodontitis, as well as its preparation and use methods. The mouthwash comprises, based on a total mass percentage of 100%, 0.05-1% intercalated molybdenum oxide nanoribbons, with the remainder being water. First, intercalated molybdenum oxide nanoribbons and water are weighed in proportion, and then the intercalated molybdenum oxide nanoribbons are added with water, stirred until completely dissolved, and sterilized to obtain the antibacterial mouthwash. The antibacterial mouthwash is used before or after periodontitis treatment under near-infrared laser irradiation. The present invention significantly improves the antibacterial properties of the mouthwash, reduces the high drug concentration in commonly used clinical mouthwashes, increases drug utilization, and, by photodynamically controlling the drug action time, solves the toxic side effects caused by residual antibacterial drugs. Furthermore, the present invention prevents the problem of antibiotic abuse. The mouthwash of the present invention has excellent antibacterial effects both in vitro and in vivo.

Description

Technical Field

[0001] The present invention belongs to the technical field of treatment of periodontitis, and in particular relates to an antibacterial mouthwash for photodynamic treatment of periodontitis, and a preparation method and a use method thereof. Background Art

[0002] Periodontitis, a chronic, infectious oral disease caused by bacteria, is a leading cause of tooth loss. It not only severely impacts oral health but also serves as a risk factor for numerous systemic diseases. Bacteria in plaque are the initiating factors of periodontal disease. Therefore, effective removal of bacterial aggregates around tooth roots is crucial for successful periodontitis treatment. Current periodontal treatments primarily utilize instruments such as ultrasonic scalers, three-way scalers, and periodontal scalers to mechanically remove periodontal bacteria. Due to the unique physiological and anatomical characteristics of the oral cavity, localized salivary irrigation results in a very short duration of antimicrobial efficacy, making it difficult to rapidly eliminate bacteria. Furthermore, the three-dimensional structure of the tooth root limits the removal of plaque from the root's surface, limiting the ability of mechanical procedures to remove plaque from the innermost areas of the root and furcations. This is one of the reasons for the current poor efficacy of periodontitis treatment.

[0003] In addition, during the treatment of periodontitis, it is usually necessary to use it in a water spray or air jet state to ensure cooling and cleaning effects. Within the operating range, there will be a large amount of tooth tissue fragments, dental plaque, tartar, saliva, blood or dental material fragments, and droplets and microbial aerosols will be generated. As one of the four major bacterial reservoirs in the human body (the others are the skin, intestines and urogenital system), the oral cavity normally has a large number of microorganisms. During the treatment process, these microorganisms will remain suspended in the clinic air and stay for a long time. Research on microbial aerosols in the oral diagnosis and treatment environment has shown that dozens of bacteria, fungi and some viruses can be detected in specimens extracted from the oral diagnosis and treatment environment. This undoubtedly poses a potential threat to the health of clinical medical staff and patients, and poses a huge challenge to the public health safety of dental clinics.

[0004] Therefore, the use of antibacterial agents during the treatment of periodontitis further controls periodontal plaque and rapidly reduces the bacterial content in the oral cavity, which is crucial for the prognosis of periodontitis and the prevention and control of oral clinic infections. However, effective commercially available antibacterial mouthwashes, due to the constant flushing of saliva in the oral cavity, greatly limit the duration of drug action, making it difficult to maintain local drug concentrations and ensuring antibacterial effects; while local retention antibacterial agents will have an adverse effect on the regeneration of periodontal tissues after periodontal treatment due to long-term contact. Moreover, as a type of drug that comes into direct contact with the patient's oral cavity, mouthwashes are often swallowed or swallowed accidentally. Some existing high-efficiency antibacterial agents often produce toxic side effects after swallowing, which in turn has an adverse effect on the patient's life safety.

[0005] Therefore, it is urgent to develop new treatment methods that can overcome the above shortcomings. Summary of the Invention

[0006] In order to overcome the deficiencies in the prior art, the present invention aims to provide an antibacterial mouthwash for photodynamic therapy of periodontitis and a preparation method and a use method thereof.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] An antibacterial mouthwash for photodynamic therapy of periodontitis, wherein the mouthwash comprises, based on 100% by weight, intercalated molybdenum oxide (abbreviated as I-MoO 3-x , I represents Intercalation) nanoribbons 0.05~1%, and the balance is water.

[0009] The inventive point of the present invention is that intercalated molybdenum oxide is used as a photosensitizing antibacterial agent for the first time. Photodynamic therapy is a non-invasive / minimally invasive treatment method that utilizes the optical properties of a substance to generate reactive oxygen species (ROS) by combining a photosensitizing antibacterial agent with light waves of a certain wavelength, thereby killing bacteria.

[0010] A method for preparing an antibacterial mouthwash for photodynamic therapy of periodontitis: first, weigh intercalated molybdenum oxide (I-MoO 3-x ) nanobelts and water, and then intercalated molybdenum oxide (I-MoO 3-x ) The nanobelts are added with water, stirred until completely dissolved, and sterilized to obtain the antibacterial mouthwash.

[0011] A method for using an antibacterial mouthwash for photodynamic treatment of periodontitis: the antibacterial mouthwash is used before or after periodontitis treatment under near-infrared laser irradiation.

[0012] Preferably, the wavelength of the near-infrared laser is 808 nm and the power density is 1 W / cm 2 , the irradiation time is 3~5min.

[0013] The preoperative use process for periodontitis treatment is as follows: before periodontitis treatment, antibacterial mouthwash is poured into the patient's mouth, and after irradiation with near-infrared laser, the preoperative sterilization effect is achieved; the postoperative use process for periodontitis treatment is as follows: after periodontitis treatment, the antibacterial mouthwash is placed on the root surgery wound, and after irradiation with near-infrared laser, the attached bacteria deep in the tooth root that are difficult to remove are killed.

[0014] The antibacterial mouthwash is used before periodontitis treatment, and its main function is to disinfect the oral cavity before treatment and quickly reduce the bacterial content in the oral cavity.

[0015] The antibacterial mouthwash is used after periodontitis treatment. The antibacterial mouthwash flows into the root bifurcation area where plaque is difficult to be removed surgically to sterilize, further control bacterial infection, and improve the treatment effect of periodontitis.

[0016] In the present invention, intercalated molybdenum oxide (I-MoO 3-x ) The preparation steps of nanobelts are as follows:

[0017] S1. Synthesis of molybdenum trioxide (MoO3) nanobelts: 4.54 g of Na2MoO4·2H2O and 2.25 g of NaCl were added to a round-bottom flask (250 mL). Then, 150 mL of deionized water was added to dissolve the inorganic salts. Aqueous hydrochloric acid (15 mL, 5 M) was added, and the mixture was transferred to a 250 mL stainless steel reactor. The stainless steel reactor was then placed in an oven and heated to 180°C for 24 h. After separation by centrifugation (5000 rpm / min, 10 min), the precipitate was washed with deionized water and dried in an oven at 80°C overnight to obtain MoO3 nanobelts.

[0018] S2, synthesis of intercalated molybdenum oxide (I-MoO 3-x ) nanobelts: MoO3 nanobelts (4 g) and deionized water (200 mL) were added to a three-necked flask and ultrasonically treated to obtain a uniform dispersion. Subsequently, nitrogen was used to replace the oxygen in the solution, and Na2MoO4·2H2O (48 g) and Na2O4S2 (0.8 g) were added under nitrogen protection. After stirring at room temperature for 3 h, the blue precipitate was separated by centrifugation and rinsed with deionized water several times. Finally, the mixture was dried in an oven at 80°C overnight to obtain intercalated molybdenum oxide (I-MoO 3-x ) nanoribbons.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention significantly improves the antibacterial properties of mouthwash, reduces the high drug concentration of commonly used clinical mouthwashes (the concentration of chlorhexidine mouthwash commonly used in clinical practice is 0.12 wt% to 0.2 wt%, while the antibacterial mouthwash of the present invention only needs 0.05 wt% to achieve a very good antibacterial effect), and improves the utilization of drugs;

[0021] (2) The present invention can quickly reduce the bacterial content in the oral cavity in a short period of time before periodontitis treatment, and reduce the generation of microbial aerosols during the treatment process; at the same time, after the periodontitis treatment, the antibacterial mouthwash in the form of an aqueous solution flows into the root bifurcation area where it is difficult to surgically remove plaque to sterilize, thereby improving the treatment effect of periodontitis;

[0022] (3) The antibacterial properties of the antibacterial mouthwash of the present invention are stimulated by near-infrared laser of a specific wavelength. Unstimulated mouthwash has no antibacterial properties, thus avoiding the toxic and side effects caused by accidental swallowing. Therefore, the present invention can control the drug action time by photodynamics, thus solving the toxic and side effects caused by the residues of other antibacterial drugs.

[0023] (4) The present invention utilizes intercalated molybdenum oxide (I-MoO 3-x ) Nanobelts can be used as photosensitive antimicrobial agents to replace antibiotic therapy, thus preventing the problem of antibiotic abuse;

[0024] (5) The antibacterial mouthwash of the present invention has excellent antibacterial effect in vitro and exhibits excellent efficacy of a first-line clinical drug (chlorhexidine) in vivo;

[0025] (6) The antibacterial mouthwash of the present invention has the advantages of rapid effect, being effective against a large number of bacteria, having no immune response, no side effects, and being reusable. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :MoO3 and I-MoO 3-x Scanning electron microscopy image of the nanoribbons.

[0027] Figure 2 :I-MoO with different concentrations 3-x Antibacterial mouthwash was exposed to near-infrared laser (808 nm, 1 W / cm 2 ) Photos of Staphylococcus aureus colonies on agar plates after irradiation for different times (a) and quantitative statistical graphs (b). Figure 2 The vertical axis of a represents the near-infrared laser irradiation time, and the horizontal axis represents the I-MoO 3-x concentration; Figure 2 Dark in a and 2b represents natural light without near-infrared laser irradiation, and NIR represents near-infrared laser.

[0028] Figure 3 :80 ppm I-MoO 3-x SEM images of Staphylococcus aureus after treatment with antibacterial mouthwash and PBS under different treatment conditions. Dark represents natural light and no near-infrared laser irradiation, and NIR represents near-infrared laser.

[0029] Figure 4 : Photograph of gingival bleeding in rats (a) and quantitative statistical graph of bleeding index GBI (b).

[0030] Figure 5 : Images of bacterial reculture in rat mouth (a) and quantitative analysis of the number of recultured colonies (b).

[0031] Figure 6: Comparison of Micro-CT scans of rat maxilla. DETAILED DESCRIPTION

[0032] In the description of the specific embodiments below, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Example 1 - Synthesis of intercalated molybdenum oxide (I-MoO 3-x ) Nanoribbons

[0034] S1. Synthesis of molybdenum trioxide (MoO3) nanobelts: 4.54 g of Na2MoO4·2H2O and 2.25 g of NaCl were added to a round-bottom flask (250 mL). Then, 150 mL of deionized water was added to dissolve the inorganic salts. Aqueous hydrochloric acid (15 mL, 5 M) was added, and the mixture was transferred to a 250 mL stainless steel reactor. The stainless steel reactor was then placed in an oven and heated to 180°C for 24 h. After separation by centrifugation (5000 rpm / min, 10 min), the precipitate was washed with deionized water and dried in an oven at 80°C overnight to obtain MoO3 nanobelts.

[0035] S2, synthesis of intercalated molybdenum oxide (I-MoO 3-x ) nanobelts: MoO3 nanobelts (4 g) and deionized water (200 mL) were added to a three-necked flask and ultrasonically treated to obtain a uniform dispersion. Subsequently, nitrogen was used to replace the oxygen in the solution, and Na2MoO4·2H2O (48 g) and Na2O4S2 (0.8 g) were added under nitrogen protection. After stirring at room temperature for 3 h, the blue precipitate was separated by centrifugation and rinsed with deionized water several times. Finally, the mixture was dried in an oven at 80°C overnight to obtain intercalated molybdenum oxide (I-MoO 3-x ) nanoribbons.

[0036] MoO3 and I-MoO 3-x SEM images of nanoribbons Figure 1 As shown in the figure, the length of the synthesized MoO3 nanobelts is in the micrometer scale, and the I-MoO 3-x The nanoribbons are approximately 500 nm, indicating that intercalation can effectively regulate the nanosize of the material.

[0037] Example 2 - Preparation of 0.05 wt% Antibacterial Mouthwash

[0038] Weigh the I-MoO prepared in Example 1 3-x 50 mg of nanobelts were added to 100 mL of water for injection and stirred until completely dissolved to obtain a 0.05 wt% antibacterial mouthwash. The entire process was performed at room temperature.

[0039] Application Example 1--Preoperative use for periodontitis treatment

[0040] 10 mL of the 0.05 wt% antibacterial mouthwash prepared in Example 2 was poured into the mouth of a patient who was about to undergo periodontal treatment. The patient was asked to rinse vigorously so that the mouthwash could flow into the gaps between the teeth and evenly cover the entire oral cavity. A near-infrared laser (808 nm, 1 W / cm 2 ) After irradiating the patient's oral cavity for 3 minutes, the patient was asked to spit out the mouthwash and then conventional periodontal treatment was started.

[0041] Application Example 2 - Use after periodontitis treatment

[0042] After the periodontal scaling instrument operation was completed, 5 mL of the 0.05 wt% antibacterial mouthwash prepared in Example 2 was drawn with a sterile syringe and injected into the periodontal pocket in the periodontal scaling area, the root bifurcation area, and around the exposed tooth root. A near-infrared laser (808 nm, 1 W / cm 2 ) After irradiating the above-mentioned surgical area of ​​the patient's mouth for 3 minutes, use a strong suction device to absorb the remaining mouthwash, and then start routine postoperative suture to stop bleeding.

[0043] In vitro antibacterial effect

[0044] Weigh the I-MoO prepared in Example 1 3-x 100 mL of water for injection was added to the nanobelts and stirred until completely dissolved to obtain I-MoO nanobelts with concentrations of 10 ppm, 20 ppm, 40 ppm, 50 ppm, 60 ppm, 80 ppm, and 100 ppm, respectively. 3-x Antibacterial mouthwash (concentration as I-MoO 3-x The entire operation was carried out at room temperature.

[0045] Detection of I-MoO using Staphylococcus aureus (S. aureus) 3-x Antibacterial activity of nanobelts: In bacterial solution (10 5 CFU / mL) were added with equal volumes of I-MoO at different concentrations. 3-x Antibacterial mouthwash, followed by near-infrared laser (808 nm, 1 W / cm 2 ) were irradiated for different times to obtain bacterial suspensions; the treated bacterial suspensions (0.1 mL) were evenly spread on Luria-Bertani (LB) agar plates and cultured at 37 °C for 16 h. The survival rate of Staphylococcus aureus was calculated by plate counting method. 3-x Antibacterial mouthwash was used as a control according to the above process.

[0046] Different concentrations of I-MoO 3-x Antibacterial mouthwash was exposed to near-infrared laser (808 nm, 1 W / cm 2 ) After irradiation for different time periods, the photos (a) and quantitative statistical graphs (b) of Staphylococcus aureus colonies on the agar plate are shown in Figure 2. Figure 2 As shown, Figure 2 The vertical axis of a represents the near-infrared laser irradiation time, and the horizontal axis represents the I-MoO 3-x concentration; Figure 2 Dark in a and 2b represents natural light without near-infrared laser irradiation, NIR represents near-infrared laser, and the following value represents the near-infrared laser irradiation time. Figure 2 a shows that: for 80 ppm~100 ppm I-MoO 3-x Antibacterial mouthwash and near-infrared laser (808 nm, 1 W / cm 2 ) After irradiation for 3 to 10 minutes, the bacterial solution was re-cultured and no colonies were observed; Figure 2 b Statistical results show: 80 ppm~100 ppm I-MoO 3-x Antibacterial mouthwash combined with near-infrared laser (808 nm, 1 W / cm 2 ) After irradiation for 3 to 10 minutes, the antibacterial rate reaches 99% ( Figure 2 b) indicates that the sterilization is successful.

[0047] 80 ppm I-MoO 3-x SEM images of Staphylococcus aureus after treatment with antibacterial mouthwash and PBS under different treatment conditions are shown in Figure 2. Figure 3 As shown in the figure, Dark represents natural light without near-infrared laser irradiation, and NIR represents near-infrared laser (808 nm, 1 W / cm 2 ) for 3 minutes. Figure 3 The results showed that compared with “PBS, NIR”, “80 ppm I-MoO 3-x Antibacterial mouthwash, I-MoO visible after NIR treatment 3-x The nanoribbons remained and attached to the bacteria; compared with the "80 ppm I-MoO 3-x Antibacterial mouthwash, Dark" compared to "80 ppm I-MoO 3-x Antibacterial mouthwash and NIR treatment showed visible damage on the bacterial surface and deformation of the bacteria, indicating that the bacteria had died.

[0048] The above in vitro antibacterial results show that when I-MoO 3-x I-MoO in antibacterial mouthwash 3-x The concentration is 80 ppm and the near-infrared laser (808 nm, 1 W / cm2 ) irradiated for 3 min, it has achieved 99% bactericidal effect. 3-x I-MoO in antibacterial mouthwash 3-x When the concentration is above 80 ppm, the near-infrared laser (808 nm, 1 W / cm 2 ) Irradiation for more than 3 minutes can have a more effective bactericidal effect.

[0049] Antibacterial effect in vivo

[0050] SD male rats were used as research subjects. The rats were treated with "∞-ligation + local application of Porphyromonas gingivalis liquid (liquid concentration 10 9 CFU / mL) + high sugar diet (100 mg / mL high sugar water, the diet is rat feed soaked in high sugar water) to establish a rat periodontitis model to study the in vivo effect of mouthwash. The 0.05 wt% antibacterial mouthwash prepared in Example 2 of the present invention was combined with near-infrared laser (808 nm, 1 W / cm 2 ) irradiated for 3 min (abbreviated as: I-MoO 3-x +NIR) as the experimental group, and the 0.05 wt% antibacterial mouthwash prepared in Example 2 of the present invention (abbreviated as: I-MoO 3-x ) and commercially available 0.05 wt% chlorhexidine antibacterial mouthwash (abbreviated as: CHX) were used as positive control groups, and normal saline was used as a blank drug control group (abbreviated as: Control); at the same time, healthy rat teeth were used as a healthy control group (abbreviated as: Healthy, this group neither established a rat periodontitis model nor received any drug or normal saline treatment). The specific process is as follows: SD male rats were anesthetized with 10% chloral hydrate (4 mL / kg). Under a surgical microscope, a 5-0 silk thread was tied to the neck of the upper left first and second molars. The silk thread was inserted as deep as possible into the gingival sulcus, and then a blade was used to cut the gums at the ligature site. After that, a sterile bamboo stick was used to apply the Porphyromonas gingivalis liquid to the ligature area. Two days later, 20 μL of drugs were instilled into the rat's mouth, and I-MoO 3-x The +NIR experimental group was additionally treated with near-infrared laser (808 nm, 1 W / cm 2 ) irradiated for 3 minutes (other groups did not require near-infrared laser irradiation). After 14 days of administration, the redness, swelling and bleeding of the gingiva in the ligature area of ​​the rats were observed and the gingival bleeding index (GBI) score was calculated. Bacterial plaques in the surgical area of ​​the rats were collected using a sterile cotton swab and diluted 10% in sterile saline. -6 Micro-CT was used to scan the maxilla of rats to evaluate the alveolar bone loss.

[0051] Photos of gingival bleeding in rats (a) and quantitative statistical graph of bleeding index GBI (b) Figure 4 The results show that: I-MoO 3-x The gingival health of rats in the +NIR group was close to that of the Healthy group, and its bleeding index and redness and swelling were significantly lower than those in the other periodontitis treatment groups, and the differences were statistically significant.

[0052] Figure 2: Bacterial reculture images in rat mouth (a) and quantitative analysis of recultured colony counts (b). Figure 5 The results show that: I-MoO 3-x The oral bacterial content of rats treated with +NIR was significantly reduced.

[0053] Comparison of Micro-CT scans of rat maxilla Figure 6 The results showed that compared with the Healthy group, the Control group had a higher degree of bone destruction at the cervical region and more exposed roots; I-MoO 3-x In the +NIR group (using the mouthwash of the present invention combined with near-infrared laser), the exposure of the tooth roots was significantly reduced, and the degree of destruction of the surrounding alveolar bone was significantly alleviated.

[0054] In summary, the antibacterial mouthwash of the present invention can quickly kill bacteria in a short time, significantly improve the antibacterial performance of the mouthwash, reduce the high drug concentration of common clinical mouthwashes, improve the utilization of drugs, and control the drug action time by photodynamics to reduce the toxic and side effects of drugs; at the same time, the present invention utilizes I-MoO 3-x As a photosensitizing antibacterial agent, it can replace antibiotic therapy, thus preventing the problem of antibiotic abuse. It also demonstrates excellent efficacy in vivo compared to the first-line clinical drug (chlorhexidine).

Claims

1. An antibacterial mouthwash for photodynamic therapy of periodontitis, characterized in that: Based on the total mass percentage of 100%, the mass percentage composition of the mouthwash is: 0.05-1% intercalated molybdenum oxide nanoribbons, and the balance is water; The photodynamic therapy is performed at a wavelength of 808 nm and a power density of 1 W / cm 2 Irradiate with near-infrared laser for 3-5 min; The intercalated molybdenum oxide nanobelts are prepared according to the following preparation process: S1. Synthesis of MoO3 nanobelts: 4.54 g of Na2MoO4·2H2O and 2.25 g of NaCl were added to a round-bottom flask. 150 mL of deionized water was then added to dissolve the inorganic salts. 15 mL of 5 M hydrochloric acid was then added, and the mixture was transferred to a 250 mL stainless steel reactor. The stainless steel reactor was then placed in an oven and heated to 180°C for 24 h. After centrifugation, the precipitate was washed with deionized water and dried in an oven at 80°C overnight to obtain MoO3 nanobelts. S2. Synthesis of intercalated molybdenum oxide nanobelts: 4 g of MoO3 nanobelts and 200 mL of deionized water were added to a three-necked flask and ultrasonically treated to obtain a uniform dispersion. Subsequently, nitrogen was used to replace the oxygen in the solution. Under nitrogen protection, 48 g of Na2MoO4·2H2O and 0.8 g of Na2O4S2 were added. After stirring at room temperature for 3 h, the blue precipitate was separated by centrifugation and rinsed several times with deionized water. Finally, the solution was dried in an oven at 80°C overnight to obtain intercalated molybdenum oxide nanobelts.

2. A method for preparing the antibacterial mouthwash for photodynamic therapy of periodontitis according to claim 1, characterized in that: First, intercalated molybdenum oxide nanobelts and water are weighed in proportion, and then the intercalated molybdenum oxide nanobelts are added with water, stirred until completely dissolved, and sterilized to obtain the antibacterial mouthwash.

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