Application of ClO2-loaded oral sustained-release hydrogel in the preparation of drugs for treating periodontitis

Abstract

The application belongs to the field of drug-loaded hydrogel, and particularly relates to application of ClO2-loaded oral sustained-release hydrogel in preparation of a drug for treating periodontitis. A preparation method of the ClO2-loaded oral sustained-release hydrogel comprises the following steps: forming a blank gel by electrostatic interaction of chitosan quaternary ammonium salt and sodium alginate; and placing the blank gel in a ClO2 solution to obtain the ClO2-loaded oral sustained-release hydrogel. The ClO2-loaded oral sustained-release hydrogel has good biological safety and has the characteristics of in-situ sustained release, can prolong the drug administration interval, reduce the drug administration dose, and has certain clinical application advantages in treating periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of drug-loaded hydrogels, specifically relating to the application of ClO2-loaded oral sustained-release hydrogels in the preparation of drugs for treating periodontitis. Background Technology

[0002] Periodontitis is an immune-inflammatory disease mediated by pathogenic microorganisms that causes destruction of periodontal tissues. Clinical manifestations include gingival bleeding, periodontal pocket formation, alveolar bone resorption, and tooth loosening. It is also closely related to a variety of systemic diseases, such as cardiovascular disease, diabetes, rheumatoid arthritis, osteoporosis, and Alzheimer's disease, seriously affecting people's physical and mental health.

[0003] Dental plaque is the initiating factor of periodontal disease. *Porphyromonas gingivalis* (P. gingivalis) is an important periodontal pathogen. It is a Gram-negative obligate anaerobe that, in addition to directly attacking periodontal tissues, can also stimulate the host's immune response, causing periodontal tissue damage. *P. gingivalis* can induce immune cells, fibroblasts, and periodontal stem cells to express various cytokines and chemokines. Increased levels of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 can not only promote inflammation-induced periodontal tissue destruction and interfere with periodontal tissue regeneration, but also induce a systemic inflammatory state, mediating the occurrence of systemic diseases.

[0004] Treatment of periodontal disease mainly includes basic periodontal treatment, surgical treatment, restorative treatment, and supportive treatment. Basic periodontal treatment is a non-surgical treatment, which refers to the use of a combination of mechanical debridement and drug treatment to eliminate tartar and subgingival plaque biofilm, thereby achieving the goal of plaque control.

[0005] Local antibacterial therapy, as an adjunct to periodontal disease treatment, can improve clinical efficacy and avoid adverse reactions caused by systemic medication. Balancing the benefits and side effects of treatment, selecting the appropriate type of drug and route of administration is the foundation for successful periodontitis treatment.

[0006] Periodontal medications commonly used in clinical practice can be divided into antibiotics and disinfectants. Antibiotics include minocycline, doxycycline, and metronidazole. Minocycline is the most commonly used sustained-release antibiotic in clinical practice, with a release period of up to 7 days. In vitro and in vivo experiments investigated the efficacy of methacrylated gelatin (GelMA) hydrogels loaded with minocycline-chitosan nanoparticles (MCN) in the treatment of periodontitis. The results showed that the GelMA / MCN composite hydrogel could stably and slowly release minocycline, inhibit the growth of *P. gingivalis*, exhibit good biocompatibility at effective antibacterial concentrations, and promote the repair of bone defects in periodontitis.

[0007] However, with the widespread use of antibiotics, the number of antibiotic-resistant strains in the body is increasing, and we have entered the era of drug resistance. One study isolated drug-resistant *P. gingivalis* from the plaque of patients with chronic periodontitis, and another study found that the percentage of minocycline-resistant strains in saliva and subgingival plaque samples increased after one month of use of minocycline microspheres during maintenance in periodontitis patients. The number of antibiotic-resistant infections is rising, but the number of new antibiotics is declining. Finding alternative treatments to antibiotics is of profound significance for maintaining health.

[0008] ClO2 has achieved good results in the treatment of oral diseases. It can reduce dental plaque and the number of bacteria in the oral cavity, lower the gingival index, and compared with other mouthwashes, ClO2 mouthwash is significantly more effective in killing oral pathogens. It can improve the oral environment and has certain development and application value. However, due to the volatility of the gas, ClO2 has a very short residence time in aqueous solution, resulting in insufficient effective bactericidal time. Furthermore, the high temperature in the oral cavity accelerates the evaporation of ClO2, which is a significant limitation for its local application in the oral cavity.

[0009] A study has shown that ClO2-loaded polyacrylic acid gel can prolong antibacterial time. This is achieved by directly injecting a pure ClO2 solution into the gel, utilizing the gel diffusion barrier layer and the supramolecular interactions between the internal polymer chains and hydrated ClO2 to prevent ClO2 molecules from diffusing out of the gel, thus achieving sustained release. However, because polyacrylic acid is listed as a Group 3 carcinogen by the World Health Organization, it cannot be used clinically. Therefore, there is an urgent need to find new and safe alternatives. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides the application of ClO2-loaded oral sustained-release hydrogel in the preparation of drugs for treating periodontitis. The preparation of the ClO2-loaded oral sustained-release hydrogel includes:

[0011] An empty-loaded gel was obtained by using the electrostatic interaction between chitosan quaternary ammonium salt and sodium alginate.

[0012] The empty gel was placed in a ClO2 solution and allowed to stand to obtain the ClO2-loaded oral sustained-release hydrogel.

[0013] As a preferred application of the present invention, the preparation of the empty-loaded gel includes the following steps:

[0014] Add the chitosan quaternary ammonium salt solution to the sodium alginate solution, react for 10-12 min, then centrifuge to collect the precipitate and obtain the QCS / Alg polyelectrolyte complex.

[0015] The QCS / Alg polyelectrolyte composite is placed in a mold and allowed to solidify to obtain the empty gel.

[0016] As a further preferred embodiment of the application described in this invention, the mass ratio of the chitosan quaternary ammonium salt to sodium alginate is 1-1.5:1.

[0017] As a further preferred embodiment of the application described in this invention, the concentration of the ClO2 solution is 100-250 ppm.

[0018] As a further preferred embodiment of the application described in this invention, the settling time is 20-24 hours.

[0019] In another aspect, the present invention provides the application of the ClO2-loaded oral sustained-release hydrogel in the preparation of therapeutic drugs for inflammatory diseases.

[0020] Furthermore, the inflammatory disease is characterized by elevated levels of the inflammatory factors TNF-α and IL-1β.

[0021] In another aspect, the present invention provides the application of the ClO2-loaded oral sustained-release hydrogel in the preparation of antibacterial agents, wherein the agents are used to inhibit the growth of *Porphyromonas gingivalis*, inhibit *Porphyromonas gingivalis* biofilm formation, inhibit gingivitis hydrolysis activity, or inhibit the secretion of matrix metalloproteinases by gingival fibroblasts.

[0022] Furthermore, the gingivitis pigment is a lysine-based gingivitis pigment.

[0023] Furthermore, the matrix metalloproteinases are MMP-1 and MMP-2.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The ClO2-loaded oral sustained-release hydrogel prepared by this invention has good biocompatibility and can alleviate experimental periodontitis in rats.

[0026] Compared with oral administration via rinsing with ClO2 solution, oral sustained-release hydrogel containing ClO2 placed inside the bag has the characteristic of in-situ sustained release, which can prolong the dosing interval and reduce the dosage, and has certain advantages in clinical application. Attached Figure Description

[0027] Figure 1 The cumulative drug release curves of 1,000 g gel placed in ClO2 solutions of different concentrations to reach equilibrium are shown.

[0028] Figure 2 For the gross observation of the hemolysis test.

[0029] Figure 3 To compare the OD values ​​of the supernatant in each group for the hemolysis test.

[0030] Figure 4The images show L929 cells in a near-confluenced state under a light microscope, with scale bars from (a) to (d) of 161 μm, 71 μm, 32 μm, and 16 μm, respectively.

[0031] Figure 5 The growth status of L929 cells in each group is shown under a light microscope, with a scale bar of 200 μm. (a)-(f) show the microscopic cell status of the negative control group, positive control group, empty gel group, drug-loaded gel A group, drug-loaded gel B group, and drug-loaded gel C group in a 96-well plate, respectively.

[0032] Figure 6 Comparison of OD values ​​among groups in the L929(a) and hPDLFs(b) cytotoxicity assays; **** indicates P < 0.0001 between groups.

[0033] Figure 7 The growth states of hPDLFs in each group are shown under a light microscope. The scale bars for (a) and (b) are 150 μm and 75 μm, respectively.

[0034] Figure 8 The growth status of hPDLFs in each group under a light microscope is shown; the scale bars in columns 1, 2, and 3 are 150 μm, 75 μm, and 37 μm, respectively. (a)-(o) show the cell status of the negative control group, empty gel group, drug-loaded gel A group, drug-loaded gel B group, and drug-loaded gel C group under different magnifications in a 96-well plate.

[0035] Figure 9 To model alveolar bone resorption in 0-3 week old SD rats, the scale bar is 500 μm.

[0036] Figure 10 The gingival condition around the maxillary second molar in each group at week 7.

[0037] Figure 11 Compare the body weight (a) and probing bleeding score (b) of each group at weeks 1, 4, and 7.

[0038] Figure 12 HE staining results for liver (a), kidney (b), and periodontal tissue (c) in each group, with a scale bar of 100 μm.

[0039] Figure 13 The expression levels of TNF-α(a), IL-1β(b), and IL-10(c) in gingival tissue of each group were detected by ELISA.

[0040] Figure 14 IHC staining of TNF-α(a), IL-1β(b), and IL-10(c) in periodontal tissues of each group.

[0041] Figure 15The AOD values ​​of TNF-α(a), IL-1β(b), and IL-10(c) in IHC slices of each group were compared.

[0042] Figure 16 Alveolar bone resorption in SD rats in each group after 3W treatment ((a) is Micro-CT image, (b) is CEJ-ABC distance), scale bar is 500μm.

[0043] Figure 17 The curves showing the change in the hydrolytic activity of kgp protein in each group over time are shown. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1: Preparation and characterization of ClO2-loaded oral sustained-release hydrogel

[0046] 1. Preparation method

[0047] 1.1 An empty-loaded gel was obtained by utilizing the electrostatic interaction between chitosan quaternary ammonium salt and sodium alginate; the specific steps are as follows:

[0048] (1) Synthesis of chitosan quaternary ammonium salt

[0049] First, add 800 mL of deionized water and 4 mL of acetic acid to a 2 L round-bottom flask. While stirring, add 20 g of chitosan powder and stir vigorously for 30 min. Then, weigh 60 g of 2,3-epoxypropyltrimethylammonium chloride into a 100 mL beaker and dissolve it in 40 mL of deionized water. Add this solution to the chitosan solution. Stir the reaction mixture in a 55 °C oil bath at 1000 rpm to ensure complete reaction. After 18 h, centrifuge the reaction mixture at 8000 rpm for 10 min. Collect the supernatant and precipitate it in a pre-cooled acetone / ethanol mixture (V:V = 1:1). Dissolve the precipitate in deionized water and dialyze the resulting solution for 3 days. The M of the dialysis bag used for dialysis... WCO The concentration was 10000 Da, and the water was changed every 12 hours during dialysis. The dialyzed solution was then freeze-dried using a vacuum freeze dryer to obtain a sponge-like chitosan quaternary ammonium salt.

[0050] (2) Preparation of chitosan quaternary ammonium salt / sodium alginate hydrogel (QCS / Alg)

[0051] First, the chitosan quaternary ammonium salt / sodium alginate polyelectrolyte complex needs to be prepared. First, prepare an 8% (w / w) chitosan quaternary ammonium salt solution and a 4% (w / w) sodium alginate aqueous solution. Then, add the chitosan solution to a 50 mL wide-mouth bottle, and slowly add the sodium alginate solution while stirring vigorously. The QCS / Alg mass ratio is 1.5:1. After the sodium alginate solution has been added, continue stirring for 10 min. Then, place the mixture in a centrifuge and centrifuge at 10000 rpm for 10 minutes, collect the precipitate, and place it in a sample bottle for later use.

[0052] The QCS / Alg polyelectrolyte composite was placed in a polytetrafluoroethylene mold, and then a layer of plexiglass was placed on top of the mold. The plexiglass was secured to the mold with a long-tail clip for 48 hours to reduce internal defects in the chitosan quaternary ammonium salt / sodium alginate polyelectrolyte composite. The plexiglass was then gently removed, and a few drops of dilute hydrochloric acid were added to the inside of the mold to help the gel solidify. The resulting gel was then demolded and immersed in 1 mol / L dilute hydrochloric acid to enhance its mechanical properties. After immersion for 30 minutes, the gel was removed and dialyzed in deionized water for 48 hours to remove the internal hydrochloric acid, yielding the QCS / ALG hydrogel.

[0053] 1.2 The empty gel was placed in a ClO2 solution and allowed to stand to obtain the ClO2-loaded oral sustained-release hydrogel.

[0054] Table 1 shows the preparation methods for empty gel (BL), drug-loaded gel A (EA), drug-loaded gel B (EB), and drug-loaded gel C (EC), respectively. This grouping will be used for subsequent experiments.

[0055] Group BL is the empty gel group (i.e., QCS / ALG hydrogel without drug loading). The preparation method is as follows: place the QCS / ALG hydrogel without drug loading in sterile double-distilled water (dd water) for 24 hours to reach a completely swollen state.

[0056] Group EA is the drug-loaded gel group A (i.e., oral sustained-release hydrogel loaded with 150ppm ClO2). The preparation method is as follows: place QCS / ALG hydrogel in 100mL of 150ppm ClO2 solution for 24h.

[0057] Group EB is the drug-loaded gel group B (i.e., oral sustained-release hydrogel loaded with 250ppmClO2). The preparation method is as follows: place QCS / ALG hydrogel in 100mL of 250ppmClO2 solution for 24h.

[0058] Group EC is the drug-loaded gel group C (i.e., oral sustained-release hydrogel loaded with 500ppmClO2). The preparation method is as follows: place the QCS / ALG hydrogel in 100mL of 500ppmClO2 solution for 24h.

[0059] Table 1. Preparation method of the gel to be tested

[0060]

[0061] 2 drug loading

[0062] The initial state of the empty gel is its fully swollen state in distilled water. The empty gel was placed in 100 mL of ClO2 solutions of different concentrations for 24 hours to obtain ClO2 oral sustained-release hydrogels. The loading principle is as follows: when the gel is immersed in the ClO2 solution, the internal ClO2 concentration is lower than the concentration in the solution. ClO2 molecules diffuse into the gel, and ClO2 is gradually loaded into the gel. After 24 hours, an equilibrium state is reached. At this point, the ClO2 concentration inside the gel is equal to the concentration of the ClO2 solution in the external system, and the liquid content inside the gel is consistent with the initial liquid content (multiple measurements showed that the liquid content inside the gel after complete swelling was approximately 84.20%). Therefore, the drug loading capacity of the gel is equal to the concentration of the ClO2 solution in the external system × the liquid volume inside the gel (since the density of the ClO2 solution is approximately equal to the density of water, the liquid volume inside the gel is approximately equal to the liquid mass (mL) / the density of water (g / mL)).

[0063] m1(g) = m(g) × 84.20% (1-1)

[0064]

[0065] m2(μg)=V1(mL)×c(μg / mL) (1-3)

[0066] Where: m1—liquid content in the gel at equilibrium / g; m—mass of the gel at equilibrium / g; V1—liquid volume in the gel at equilibrium / mL; ρ 水 —Density of water / g / mL; m2 —Drug loading in the gel at equilibrium / μg; c = Concentration of ClO2 solution in the system outside the gel / μg / mL.

[0067] The results calculated according to the formula are shown in Table 2, which shows the drug loading in gels of different masses when placed in ClO2 solutions of different concentrations to reach equilibrium. As can be seen from Table 2, the ClO2 loading is positively correlated with the concentration of the ClO2 solution and the mass of the empty gel during loading.

[0068] Table 2. Drug loading in gels of different masses when placed in ClO2 solutions of different concentrations to reach equilibrium.

[0069]

[0070] Note: 1 μg / mL = 1 mg / L = 1 ppm

[0071] 3. Sustained-release curves

[0072] 1.000g of gel, which had reached equilibrium after being placed in ClO2 solutions of different concentrations for 24 hours, was placed in 2mL of distilled water, and the drug release was measured at fixed time intervals. The sustained-release principle is as follows: when the drug-loaded gel is placed in water, ClO2 molecules migrate from the high-concentration gel interior to the low-concentration gel exterior. The polymer network inside the gel, and supramolecular interactions such as hydrogen bonds and coordination bonds between ClO2 and polymers within the gel, reduce the migration rate of ClO2 in the gel, thereby achieving a sustained-release effect.

[0073] Table 3 shows the time-point and cumulative drug release amounts of 1.000 g drug-loaded gel that reached equilibrium in ClO2 solutions of different concentrations. Figure 1 The cumulative drug release curves of 1,000 g drug-loaded gel placed in ClO2 solutions of different concentrations to reach equilibrium are shown.

[0074] Formula 1-4 shows the calculation method for "average drug release rate of drug-loaded gel over a certain period of time": divide "cumulative drug release amount of drug-loaded gel at that time point" by "cumulative drug release time over that period of time". Formula 1-5 shows the calculation method for "cumulative drug release percentage of drug-loaded gel": divide "cumulative drug release amount of drug-loaded gel at a certain time point" by "drug loading amount inside the gel".

[0075] The drug-loaded gels containing 150 μg / mL (ppm), 250 μg / mL (ppm), and 500 μg / mL (ppm) ClO2 showed the fastest average drug release rates within 12 h, at 7.02 μg / h, 11.46 μg / h, and 24.49 μg / h, respectively, with cumulative release percentages of 66.8%, 65.3%, and 69.8% at 12 h. After 12 h, the release rate of ClO2 from the three drug-loaded gels gradually slowed down. The average ClO2 release rates within 24 h were 3.79 μg / h, 6.16 μg / h, and 12.96 μg / h, respectively, with cumulative release percentages of 72.1%, 70.2%, and 73.9% at 24 h; and cumulative release percentages within 96 h were 84.4%, 81.6%, and 87.3%, respectively, with a small amount of release continuing until day 7.

[0076]

[0077]

[0078] Where: v—average drug release rate over time t / μg / h; m2—drug loading in the gel at equilibrium / μg; t = cumulative drug release time of the drug-loaded gel / h; m3—cumulative drug release amount of the drug-loaded gel at time t / μg; q—cumulative drug release percentage of the drug-loaded gel / %.

[0079] Table 3. Time-point release and cumulative release of 1.000 g drug-loaded gel at equilibrium in ClO2 solutions of different concentrations.

[0080]

[0081]

[0082] Note: 1 μg / mL = 1 mg / L = 1 ppm

[0083] Example 2: In vitro biosafety assessment of ClO2-loaded oral sustained-release hydrogel

[0084] 1 Experimental Methods

[0085] 1.1 Hemolysis test

[0086] 1) Experimental grouping

[0087] There were a total of 6 groups: positive control group (PC), negative control group (NC), empty gel group (BL), drug-loaded gel A group (EA), drug-loaded gel B group (EB), and drug-loaded gel C group (EC). The preparation methods of empty gel, drug-loaded gel A, drug-loaded gel B, and drug-loaded gel C are shown in Table 1.

[0088] 2) Preparation of the extract of the sample to be tested

[0089] According to the preparation standard of GB / T16886.12-2017, empty gel, drug-loaded gel A, drug-loaded gel B, and drug-loaded gel C were placed in sterile physiological saline (0.9% NaCl solution) at an extraction ratio of 0.2 g / mL, incubated in a cell culture incubator at 37℃ for 24 h, filtered through a 0.22 μm bacterial filter to obtain sterile gel extract, which was then stored in a refrigerator at 4℃ for use within 24 h.

[0090] 3) Preparation of red blood cell diluent

[0091] SD rats were given general anesthesia by intraperitoneal injection of 3% (v / v) sodium pentobarbital (10 mL / kg). The abdomen was disinfected with 75% alcohol. After opening the abdomen, 5 mL of blood was collected from the abdominal aorta using a single blood collection tube and placed in an anticoagulant tube. 5 mL of sterile physiological saline was drawn and slowly injected into a centrifuge tube containing 1 mL of blood. The blood was gently blown and mixed with a pipette to thoroughly wash the red blood cells. The mixture was centrifuged at 1500 rpm for 10 min, and the supernatant was discarded. This process was repeated 3 times until the supernatant was clear and colorless. The supernatant was then discarded, and 1 mL of the washed red blood cell suspension was retained. 19 mL of physiological saline was added to the suspension to prepare a 5% (v / v) red blood cell suspension for later use.

[0092] 4) Group treatment and calculate hemolysis rate

[0093] Add 500 μl of sterile double-distilled water, sterile physiological saline, and sterile gel extraction solution to 1.5 mL sterile EP tubes, respectively, as the positive control (PC), negative control (NC), empty gel group (BL), drug-loaded gel group A (EA), drug-loaded gel group B (EB), and drug-loaded gel group C (EC). Then add 500 μl of 5% (v / v) red blood cell suspension and gently pipette to mix thoroughly. Incubate the 6 groups of EP tubes in a cell culture incubator at 37°C for 1 h. After centrifugation, add 100 μl of the supernatant to each well of a 96-well plate. Set the wavelength to 545 nm and measure the absorbance (optical density, OD) using a multi-mode microplate reader. This experiment was repeated 3 times. The hemolysis rate was calculated using the following formula:

[0094]

[0095] In the formula: A is the OD value of the experimental group; B is the OD value of the negative control group; C is the OD value of the positive control group.

[0096] According to the YY / T0127.1-1993 standard, a hemolysis rate of <5% meets the requirements.

[0097] 1.2 In vitro cytotoxicity test

[0098] 1) L929 cells

[0099] (1) Experimental grouping

[0100] There were a total of 6 groups: negative control group (NC), positive control group (PC), empty gel group (BL), drug-loaded gel A group (EA), drug-loaded gel B group (EB), and drug-loaded gel C group (EC). The preparation methods of empty gel, drug-loaded gel A, drug-loaded gel B, and drug-loaded gel C are shown in Table 1.

[0101] (2) Preparation of the extract of the sample to be tested

[0102] According to the preparation standard of GB / T16886.12-2017, empty gel, drug-loaded gel A, drug-loaded gel B, and drug-loaded gel C were immersed in MEM complete culture medium at a ratio of 0.2 g / mL, incubated at 37°C for 24 h in a cell culture incubator, filtered through a 0.22 μm filter to obtain sterile gel extract, which was then stored at 4°C for later use.

[0103] (Use within 24 hours)

[0104] (3) L929 cell resuscitation

[0105] Prepare several tubes of MEM complete medium containing 1% penicillin-dextrose antibody (V / V), 10% Gibico fetal bovine serum (V / V), and 89% MEM basal medium (V / V), and store at 4°C (use within one week). Take one tube of L929 cells from liquid nitrogen, incubate at 37°C with shaking to rapidly thaw them, add 6 volumes of complete medium in a biosafety cabinet, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fresh MEM complete medium, mix well, and seed into a culture dish with a moistened bottom. Place the culture dish in a cell culture incubator (37°C, 5% CO2), change the medium after 24 h, and continue culturing.

[0106] (4) L929 cell plating

[0107] Once the L929 cells in the dish have reached near-confluence, discard the supernatant, wash three times with sterile PBS, add 2 mL of trypsin, and digest in a cell culture incubator for 1-1.5 min. Observe the cells under a microscope until they appear like quicksand. Add twice the volume of MEM culture to stop the digestion. Centrifuge the cell suspension for 5 minutes at 1000 rpm. After removing the supernatant, add MEM culture and gently pipette to mix. Count the cells and adjust the cell density to 1 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates, with 6 replicates per group. Cell seeding was observed under a microscope to ensure uniformity. After 24 hours of cell culture, the cells adhered to the plates, and the number and morphology of cells were observed under a microscope to ensure consistency in the initial state of each group.

[0108] (5) CCK-8 experiment

[0109] The liquid in the wells was aspirated, and the preheated extracts of each group (100 μl / well) were slowly added along the well wall as BL, EA, EB, and EC groups. The negative control group (NC) was added with MEM complete culture medium (100 μl / well) without other substances. The positive control group (PC) was added with 100 μl of MEM complete culture medium containing 10% dimethyl sulfoxide (DMSO) per well. The well plates were placed in a cell culture incubator at 37°C with 5% CO2 and incubated for 24 h. The cell growth and morphological changes of each group were observed under a microscope and photographed.

[0110] Cell proliferation and viability assay kit (Cell Counting Kit-8, CCK-8) and MEM culture medium were mixed at a volume ratio of 1:9. The liquid in the wells was aspirated, and 100 μL of the mixture was added to each well. 100 μL of the mixture was added to each cell-free well as a zeroing well. To avoid the influence of evaporation, 100 μL of sterile PBS was added to the periphery of each well. The 96-well plate was incubated in a cell culture incubator for 2 hours. The OD value of each well at λ = 450 nm was measured using a microplate reader, and the relative growth rate (RGR) was calculated.

[0111]

[0112] In the formula: A is the OD value of the experimental group; B is the OD value of the zeroing hole; C is the OD value of the negative control group.

[0113] According to GB / T 16886.5-2017, if RGR < 70%, there is potential cytotoxicity.

[0114] 2) Human periodontal ligament fibroblasts (hPDLFs)

[0115] (1) Experimental grouping

[0116] Five groups were included: negative control group (NC), empty gel group (BL), drug-loaded gel A group (EA), drug-loaded gel B group (EB), and drug-loaded gel C group (EC). The preparation methods of empty gel, drug-loaded gel A, drug-loaded gel B, and drug-loaded gel C are shown in Table 1.

[0117] (2) Preparation of the extract of the sample to be tested

[0118] According to the preparation standard of GB / T16886.12-2017, empty gels, drug-loaded gels A, B, and C were immersed in α-MEM complete medium at a ratio of 0.2 g / mL, incubated at 37°C for 24 h in a cell culture incubator, and filtered through a 0.22 μm filter to obtain sterile gel extracts, which were then stored at 4°C for later use (within 24 h).

[0119] (3) hPDLFs cell resuscitation

[0120] Prepare several tubes of α-MEM complete medium containing 1% penicillin-dextrose antibody (V / V), 10% Gibico fetal bovine serum (V / V), and 89% α-MEM basal medium (V / V), and store at 4°C (use within one week). Remove one tube of hPDLFs from liquid nitrogen, incubate at 37°C with shaking to rapidly thaw it, add 6 volumes of complete medium in a biosafety cabinet, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fresh α-MEM complete medium, mix well, and inoculate into culture dishes with moistened bottoms. Place the culture dishes in a cell culture incubator (37°C, 5% CO2), change the medium after 24 h, and continue culturing.

[0121] (4) hPDLFs cell plating

[0122] Once the hPDLFs cells in the dish reached a near-confluence state, the supernatant was aspirated, and the cells were washed three times with sterile PBS. 2 mL of trypsin was added, and the cells were digested in a cell culture incubator for 2 minutes. After observing the cells under a microscope until they resembled quicksand, twice the volume of α-MEM was added to terminate the digestion. The cell suspension was centrifuged for 5 minutes at 1000 rpm. The supernatant was aspirated, and α-MEM was added to complete the culture. The cells were gently mixed by pipetting. After cell counting, the cell density was adjusted to 1 × 10⁶ cells / year. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates, with 6 replicates per group. Cell seeding was observed under a microscope to ensure uniformity. After 24 hours of cell culture, the cells adhered to the plates, and the number and morphology of cells were observed under a microscope to ensure consistency in the initial state of each group.

[0123] (5) CCK-8 experiment

[0124] The liquid in the wells was aspirated, and the preheated extracts of each group (100 μl / well) were slowly added along the well wall as BL, EA, EB, and EC groups. The negative control group (NC) was added with α-MEM complete medium (100 μl / well) without other substances. The well plates were placed in a cell culture incubator at 37°C with 5% CO2 and incubated for 24 h. The cell growth and morphological changes of each group were observed under a microscope and photographed.

[0125] Prepare a mixture of CCK-8 reagent and α-MEM complete culture medium at a volume ratio of 1:9. Discard the liquid in the wells and add 100 μL of the CCK-8 mixture to each well. Add 100 μL of the CCK-8 mixture to the cell-free wells as a zeroing well. To avoid the influence of evaporation, add sterile PBS (100 μL / well) around the perimeter of the test wells. Incubate the 96-well plate in a cell culture incubator for 2 hours. Measure the OD value of each well at λ = 450 nm using a microplate reader and calculate the relative growth rate (RGR) using the same method as before. According to GB / T16886.5-2017, if the RGR < 70%, there is potential cytotoxicity.

[0126] 2 Experimental Results

[0127] 2.1 Results of the hemolysis test

[0128] Figure 2 For a general observation of the hemolysis test, the supernatant showed a progressively changing color: red, completely clear and colorless, clear and colorless, mostly clear and colorless, slightly red, and pale red. The erythrocyte sedimentation rate was also progressively bright red, bright red, bright red, bright red, dark red, and reddish-brown. The color of the supernatant increased progressively from the NC group, BL group, EA group, EB group, EC group to the PC group.

[0129] 2) Hemolysis rate

[0130] Table 4 shows the OD values, hemolysis rates, and toxicity evaluations of the supernatants in each group of the hemolysis test. The hemolysis rate was 0% in the NC group and 100% in the PC group. The hemolysis rates of the BL, EA, and EB groups increased sequentially, reaching 0.14%, 1.42%, and 2.16%, respectively, all <5%, meeting the national standard for hemolysis testing. The hemolysis rate of the EC group was 14.30%, >5%, and did not meet the national standard for hemolysis testing. The hemolysis rate increased sequentially from NC, BL, EA, EB, EC, to PC groups, and the hemolysis rate of erythrocytes increased with increasing gel drug concentration.

[0131] like Figure 3 Statistical analysis showed that there were no statistically significant differences in hemolysis rates between the NC group and the BL group, and between the EA group and the EB group (P>0.05), while there were statistically significant differences between the remaining groups (P<0.05).

[0132] Table 4. OD values, hemolysis rates, and toxicity evaluations of supernatants in each group of the hemolysis test (n=3)

[0133]

[0134] 2.2 Mouse fibroblasts (L929 cells)

[0135] (1) Observation under a microscope

[0136] a) Normal morphology of L929 cells in cell culture dish

[0137] like Figure 4 The image was taken of L929 cells in a cell culture dish in a near-confluenced state using an intelligent biological image navigator. Under normal conditions, the cells are spindle-shaped and polygonal, with clear nucleoli, and some are round cells undergoing mitosis. Figure 4 The scales for (a)-(d) are 161 μm, 71 μm, 32 μm and 16 μm respectively.

[0138] b) Morphology of L929 cells in each group in 96-well plates

[0139] like Figure 5 (a)-(f) show the microscopic cell morphology of the negative control group (NC), positive control group (PC), empty gel group (BL), drug-loaded gel group A (EA), drug-loaded gel group B (EB), and drug-loaded gel group C (EC) in a 96-well plate, respectively. Cells in the NC and BL groups were polygonal or spindle-shaped, with a small number of elliptical cells, and a density of approximately 75%. Cells in the PC group were mostly shrunken and round, with a density of approximately 40%. Cells in the EA and EB groups were mostly round and spindle-shaped, with a small number of elliptical cells, and a density of approximately 75%. Cells in the EC group were mostly round and spindle-shaped, with a density of approximately 70%. The PC group had the fewest cells and the worst growth condition. From the NC, BL, EA, EB, and EC groups, the growth condition of L929 cells deteriorated sequentially, but the cell number did not differ significantly.

[0140] (2) CCK-8 test

[0141] The OD values, RGR, and cytotoxicity evaluations for each group are shown in Table 5. The RGRs of the BL, EA, and EB groups were all greater than 70%, meeting the national standards for cytotoxicity testing; the RGR of the EC group was less than 70%, failing to meet the national standards for cytotoxicity testing. From the NC, BL, EA, EB, EC, and PC groups, the RGR decreased sequentially, indicating that the cytotoxicity of the drug-loaded gel to L929 cells increased with increasing drug concentration. Figure 6 (a) Statistical analysis showed that the differences in OD values ​​between groups were statistically significant (P < 0.0001).

[0142] Table 5. OD values, RGR (%), and toxicity evaluation of L929 cells in each group at 24 h (n=6)

[0143]

[0144] 2) Human periodontal ligament fibroblasts (hPDLFs)

[0145] (1) Observation under a microscope

[0146] a) Normal morphology of hPDLFs in cell culture dish

[0147] like Figure 7 The hPDLFs (generation 5) in the cell culture dish were photographed under an inverted microscope. Under normal conditions, the cells are spindle-shaped or elongated spindle-shaped, growing radially or in a spiral pattern; under high magnification, the cells are plump, the cytoplasm is uniform, the cell processes are slender, the nucleus is oval or round and centrally located, and the nucleolus is clear.

[0148] b) Morphology of hPDLFs in each group in a 96-well plate

[0149] like Figure 8(a)-(o) show the microscopic cell states at different magnifications for the negative control group (NC), empty gel group (BL), drug-loaded gel group A (EA), drug-loaded gel group B (EB), and drug-loaded gel group C (EC) in a 96-well plate, respectively.

[0150] There was no significant difference in cell density among the groups. Under high magnification, the cells in the NC and BL groups were elongated spindle-shaped with smooth cell membrane curves, uniform cytoplasm, and clear nucleoli. The cells in the EA group had normal morphology. The cell membrane curves in the EB group were not smooth. In the EC group, a small number of cells changed from stretched elongated spindle shapes to curled shapes. In addition, the EA, EB, and EC groups all showed a gradual increase in degenerated cells, which appeared honeycomb-like. In severe cases, the cell membrane dissolved, and the cellular contents flowed out, with cell bifurcation gradually increasing. The growth status of hPDLFs deteriorated sequentially from the NC, BL, EA, EB, and EC groups, but there was no significant difference in cell number. Figure 8 The scale bars in columns 1, 2, and 3 are 150μm, 75μm, and 37μm, respectively.

[0151] (2) CCK-8 test

[0152] The OD values, RGR, and cytotoxicity evaluations for each group are shown in Table 6. The RGRs of the BL, EA, and EB groups were all greater than 70%, meeting the national standards for cytotoxicity testing; the RGRs of the EC group were all less than 70%, failing to meet the national standards for cytotoxicity testing. From the NC, BL, EA, EB, and EC groups, the RGR decreased sequentially, indicating that the toxicity of the drug-loaded gel to hPDLFs increased with increasing drug concentration. Figure 6 (b) Statistical analysis showed that the differences in OD values ​​between groups were statistically significant (P < 0.0001).

[0153] Table 6. OD values, RGR (%), and toxicity evaluation of hPDLFs in each group at 24 h (n=6)

[0154]

[0155] Example 3: Evaluation of the anti-inflammatory effect and safety of topical application of ClO2-loaded oral sustained-release hydrogel within the periodontal pocket

[0156] 1 Experimental Methods

[0157] 1.1 Grouping of experimental animals, establishment of the model, and administration of drugs

[0158] 1) Animal grouping

[0159] Thirty healthy male Sprague-Dawley (SD) rats (200g ± 10g) of SPF grade were purchased from the Animal Experiment Center of Xi'an Jiaotong University School of Medicine. The research protocol has been approved by the Biomedical Ethics Committee of Xi'an Jiaotong University School of Medicine (Application Acceptance No.: 2022-1356, Date: 2022-03-15). All SD rats used in the experiment were housed in the experimental animal center in a quiet environment with a room temperature of 20-26℃. The animal room was regularly disinfected with ultraviolet light, and the cages and water bottles were regularly cleaned and disinfected. The bedding was changed once a week, and the animals were fed a normal diet. During the intervention period, there were no restrictions on the food or water intake of the experimental animals (the experimental animals were male SD rats housed in the animal experiment center).

[0160] After one week of acclimatization, the rats were randomly divided into a healthy control group (Group A), a negative control group (Group B), a positive control group (Group C), a ClO2 flushing group (Group D), and a drug-loaded gel group (Group E). Among them, Group A was the untreated group, Groups B, C, D, and E were the treated groups, and Groups C, D, and E were the treatment groups (Table 7 shows the drug administration of each group of rats).

[0161] 2) Establish an experimental rat model of periodontitis

[0162] Group A received no treatment. Groups B, C, D, and E established an experimental periodontitis model in the bilateral maxillary second molars (M2). Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (5 mL / kg) according to a standard anesthetic dose-to-body weight ratio of 50 mg / kg. After the anesthetic took effect, the rats were placed in a supine position, and their limbs and heads were immobilized. A dental examination probe was inserted into the mesial and distal proximal surfaces of the bilateral maxillary M2 molars. 4-0 surgical sutures were wrapped and fixed around the bilateral maxillary M2 molars, placed as close to the gingival sulcus as possible, and tied on the palatal side. Subsequently, for 3 weeks, P. gingivalis bacterial solution (ATCC33277, OD=1.0) was applied weekly to the gingival sulcus around the bilateral maxillary M2 molars using a small cotton swab. Periodontal condition was checked every 4 days during the modeling period. If ligation sutures fell off or were damaged, they were re-ligated promptly. Three weeks after modeling, bilateral maxillary M2 gingival redness and swelling, periodontal pocket formation, and positive bleeding on probing (BOP+) were observed in rats (as shown in the image, bleeding on probing of the periodontal pocket in rats). M2 was significantly loose. One SD rat was randomly selected from each group and sacrificed. Maxillary bone samples from each group were scanned using a Micro-CT imaging system. No alveolar bone resorption was observed in the healthy control group. The alveolar bone resorption in M2 of the modeling group was basically consistent, with exposed root bifurcation, indicating that the experimental periodontitis model in SD rats was successfully established and the baseline was basically consistent (as shown in the image). Figure 9 As shown, this represents the alveolar bone resorption in SD rats during the 0-3 week modeling period.

[0163] 3) Administration method

[0164] After successful modeling, the surgical sutures were removed, and different treatments were administered over a period of 3 weeks:

[0165] Group A (n=5): No treatment;

[0166] Group B (n=5): Rinse the bilateral maxillary M2 buccal and palatal periodontal pockets of rats with 200μL of physiological saline using a 0.5mL insulin syringe. The rinsing volume is 50μL per side, and the rinsing is performed once every 3 days for a total of 7 times.

[0167] Group C (n=5): Commercial 2% minocycline hydrochloride ointment (Perio) was injected into the periodontal pockets of the buccal and palatal surfaces of the bilateral maxillae of rats until it just overflowed the edge of the pocket. A probe was used to assist in the administration to ensure that the drug was completely inserted into the pocket. The injection was repeated once every 7 days for 3 times.

[0168] Group D (n=5): Rinse the bilateral maxillary M2 buccal and palatal periodontal pockets of rats with 200 μL of 60 ppm ClO2 pure solution using a 0.5 mL insulin syringe. The rinsing volume is 50 μL per side, and the rinsing is performed once every 3 days for a total of 7 times.

[0169] Group E (n=5): The drug-loaded gel B was cut into crescent-shaped thin slices with a mass of about 0.01g, which were suitable for the shape of the periodontal pockets of rats. The slices were placed in the buccal and palatal periodontal pockets of the M2 maxilla of the rats, with one slice placed on the buccal and one on the palatal surface, for a total of 4 slices. The slices were placed once every 7 days for a total of 3 times. (The preparation method of drug-loaded gel B is shown in Table 1.)

[0170] 4) Experimental Records

[0171] During the experiment, record the general condition and periodontal clinical examination of the rats: 1) Record the mortality of rats throughout the experiment; 2) Compare the body weight of rats in each group when the model was successfully established and 3 weeks after drug administration; 3) Compare the periodontal condition and bleeding on probing (BOP) scores of rats in each group 3 weeks after drug administration, as follows: Divide the bilateral M2 of the maxilla of the rats into 4 surfaces (left buccal-palatal surface and right buccal-palatal surface). No bleeding on probing on any four surfaces is scored as 0 points, bleeding on any one surface is scored as 1 point, bleeding on any two surfaces is scored as 2 points, bleeding on any three surfaces is scored as 3 points, and bleeding on all four surfaces is scored as 4 points (Note: At the baseline of treatment, group A is scored as 0 points, and groups B, C, D, and E are all scored as 4 points).

[0172] Table 7. Drug administration details for rats in each group.

[0173]

[0174] 1.2 ELISA detection of inflammatory factor expression in gingival tissue

[0175] 1) Gingival tissue separation

[0176] All experimental rats (n=5) were euthanized by sodium pentobarbital overdose. 10 mg of the free gingiva from the distal aspect of the second molar (M2), the first molar (M1), and the mesial buccal-lingual aspect of the third molar (M3) on ice was collected from each side. The collected samples were rinsed thoroughly with sterile PBS, blotted dry, and stored at -80°C for later use. The remaining alveolar bone on the ipsilateral side (for measuring alveolar bone resorption, see below) and the alveolar bone with gingival attachment on the contralateral side (for preparing tissue sections, see below) were washed with physiological saline, blotted dry, and placed in tissue fixative (4% paraformaldehyde solution) for later use.

[0177] 2) Gingival tissue protein extraction

[0178] Take out a 1.5 mL EP tube containing gingival tissue from a -80°C freezer, equilibrate to 25°C, add grinding beads and pre-cooled PBS, place the EP tube in a tissue homogenizer to obtain tissue homogenate, centrifuge, and transfer the supernatant to a new EP tube and label it.

[0179] 3) Determination of total protein content

[0180] Follow the instructions for use of the BCA protein quantification kit to detect the total protein content in the sample.

[0181] 4) Measurement of inflammatory factor expression

[0182] Follow the instructions for use of the ELISA kit to detect the levels of TNF-α, IL-1β, and IL-10 in the sample.

[0183] 1.3 HE staining to observe the morphology of liver, kidney, and periodontal tissues

[0184] 1.4 IHC staining to observe the expression of inflammatory factors in periodontal tissues

[0185] 1.5 Micro-CT Measurement of Alveolar Bone Resorption

[0186] Alveolar bone samples were removed from the fixative, and alveolar bone resorption in the maxillary second molar (M2) of SD rats was evaluated using a Micro-CT imaging system. Scans were performed at 90 kVp and 200 μA, with a slice thickness of 4.5 μm and an exposure time of 300 ms. Bone resorption was analyzed using Mimics 21.0. Before analysis, all samples were rotated to the same position to ensure that the coordinate axes of the axial, sagittal, and coronal planes were identical. Three measurement points were taken on the palatal sagittal section: mesial, intermediate (bifurcation zone), and distal. The distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) (CEJ-ABC) was measured, and the average value was used for intergroup comparison.

[0187] 2 Experimental Results

[0188] 2.1 Observation and Recording Results of General Condition of SD Rats

[0189] Changes in rat body weight and periodontal condition were recorded during the experiment, and no rats died.

[0190] 1) Weight changes

[0191] From Table 8, Figure 11 (a) It can be seen that the body weight of rats in each group gradually increased in weeks 1, 4 and 7. Before modeling, there was no statistically significant difference in body weight among the groups (P>0.05), indicating that the baseline was comparable. At week 7, the body weight of rats in group A was the heaviest, and the difference was statistically significant (P<0.05). There was no statistically significant difference in body weight among the other groups (P>0.05).

[0192] 2) Periodontal condition

[0193] like Figure 10 The images show the gingival condition around the maxillary second molars in each group at week 7 (due to thorough cleaning, physical signs are not obvious). The black circle indicates the maxillary second molar and its periodontal tissues, and the arrows point to the mesial and distal gingival papillae and the free gingival margin.

[0194] Group A: The gingival surface is smooth, the gingiva is light pink, the gingival margin and gingival papillae have regular contours, and they are tightly attached to the tooth surface. The probe can hardly penetrate into the gingival sulcus, and there is no bleeding on probing. Group B: The gingiva is significantly red and swollen, the gingival margin and gingival papillae have serrated edges, the damage is obvious, the probe can easily penetrate into the periodontal pocket, and there is more bleeding on probing. Groups C, D, and E: The gingiva is basically not red and swollen, the gingival margin and gingival papillae have irregular contours, the probe cannot easily penetrate into the periodontal pocket, and there is less bleeding on probing.

[0195] From Table 8, Figure 11 (b) It can be seen that at week 7, there was no bleeding on probing in group A rats. The bleeding on probing scores of groups C, D and E were all lower than those of group B, and the differences were statistically significant (P < 0.05). However, there were no statistically significant differences between groups C, D and E (P > 0.05).

[0196] Table 8. Changes in rat body weight and scores for bleeding upon probing (n=5)

[0197]

[0198] 2.2 HE staining results of liver, kidney, and periodontal tissues from SD rats

[0199] HE staining was used to observe the pathological morphology of liver, kidney, and periodontal tissues of rats after 3 weeks of treatment. The results are as follows:

[0200] 1) Liver tissue

[0201] like Figure 12As shown in (a), no liver injury was observed in any group. The hepatic cords were arranged radially around the central vein, with uniform cytoplasm, round nuclei located in the center of the cytoplasm, and no pathological changes such as inflammatory infiltration, tissue degeneration, or necrosis were observed.

[0202] 2) Kidney tissue

[0203] like Figure 12 As shown in (b), no kidney injury was observed in any group. The glomeruli and tubules were structurally normal, with no proliferation of mesangial cells and matrix, no inflammatory cell infiltration or fibrosis, and no pathological changes such as degeneration or necrosis.

[0204] 3) Periodontal tissues

[0205] like Figure 12 (c) shows that the periodontal tissue structure in group A is normal, the gingival epithelium margin is continuous, the junctional epithelium is tightly attached to the tooth structure and attached to the cementoenamel junction, the fibroblasts and collagen fibers are neatly arranged, and very few inflammatory cells are observed. In group B, the periodontal tissue is severely damaged, the gingival epithelium margin is discontinuous, the junctional epithelium is completely destroyed down to the lamina propria, the fibroblasts and collagen fibers are loosely and disorderly arranged, and a large number of inflammatory cells are infiltrated. The periodontal tissue structure in groups C, D, and E is less damaged than that in group B, the gingival epithelium margin is more continuous, the junctional epithelium is destroyed but does not reach the lamina propria, the fibroblasts and collagen fibers are more neatly arranged, and the inflammatory cell infiltration is less than that in group B, but there is no significant difference between the groups.

[0206] 2.3 SD rat gingival tissue expression results

[0207] The expression of inflammatory factors TNF-α, IL-1β, and IL-10 in the gingival tissue of rats after 3 weeks of treatment was detected by ELISA. The results are as follows:

[0208] As shown in Table 9, Figure 13 As shown, TNF-α and IL-1β were expressed at the lowest level in group A (P < 0.05), the highest level in group B (P < 0.05), and the expression levels were comparable in groups C, D, and E (P > 0.05).

[0209] IL-10 expression was lowest in group A (P < 0.05), and slightly higher in group B than in group A (P > 0.05); the expression levels in groups C, D, and E were comparable (P > 0.05), and higher than in groups A and B (P < 0.05).

[0210] Table 9 shows the levels of TNF-α, IL-1β, and IL-10 in gingival tissue of each group as determined by ELISA (pg / mL, n=5).

[0211]

[0212] 2.4 SD rat periodontal tissue IHC staining results

[0213] Immunohistochemical (IHC) staining was used to observe the expression of inflammatory factors TNF-α, IL-1β and IL-10 in the periodontal tissues of rats after 3 weeks of treatment.

[0214] Figure 14 This indicates the binding of inflammatory cytokine proteins to specific antibodies. The darker the brown color, the stronger the positive result and the higher the expression level of the inflammatory cytokine. Figure 15 The value represents the average optical density (AOD) of each group of slices. The higher the AOD value, the stronger the positivity and the higher the expression level of inflammatory factors.

[0215] Group A showed the lightest TNF-α and IL-1β staining and the lowest AOD value (P < 0.05); Group B showed the darkest TNF-α and IL-1β staining and the highest AOD value (P < 0.05); Groups C, D, and E showed TNF-α and IL-1β staining and AOD values ​​between Groups A and B, and no significant differences were observed among Groups C, D, and E (P > 0.05).

[0216] Group A had the lightest IL-10 staining and the lowest AOD value (P < 0.05); Group B had slightly darker IL-10 staining and a slightly higher AOD value than Group A (P < 0.05); Groups C, D, and E had higher IL-10 staining and AOD values ​​than Groups A and B, with Group E being higher than Group C and Group D, but the differences among the three groups were small (P < 0.05).

[0217] All groups showed deep staining of the gingival epithelium and light staining of the lamina propria.

[0218] Results of alveolar bone resorption in 2.5 SD rats

[0219] Micro-CT was used to measure bone destruction in the mesial, distal, and furcation regions of the maxillary second molar in rats after 3 weeks of treatment. The distance from the cementoenamel junction to the alveolar ridge crest was used as the amount of alveolar bone resorption. Figure 16 (a) shows the three-dimensional (3D) and palatal sagittal (2D) images of the maxillary second molars (M2) in each group; (b) shows the statistical analysis of the distance from the cementoenamel junction to the alveolar ridge crest (CEJ-ABC) in each group.

[0220] M1, M2, and M3 represent the first, second, and third maxillary molars, respectively. In the 3D diagram, the white dashed line represents the cementoenamel junction, and the red dots represent the mesial, intermediate, and distal measurement points on the palatal side of M2. In the 2D diagram, the white vertical line represents the mesial and distal CEJ-ABC distance of M2, and the area within the white circle is the root bifurcation zone.

[0221] The results showed that no alveolar bone resorption was observed in group A (P < 0.05); group B had the greatest alveolar bone resorption (P < 0.05); groups C, D, and E were intermediate between groups A and B (P < 0.05), but there was no statistically significant difference between groups C, D, and E (P > 0.05). In the root bifurcation zone, group A had the densest bone; group B showed obvious radiolucency; groups C, D, and E had comparable bone density, falling between groups A and B.

[0222] Example 4: Mechanism study of ClO2-loaded sustained-release hydrogel for the treatment of periodontitis

[0223] 1 Experimental Methods

[0224] 1.1 Study on the growth-inhibiting effect of ClO2-loaded oral sustained-release hydrogel on P. gingivalis

[0225] The optimal concentration of ClO2-loaded oral sustained-release hydrogel for *P. gingivalis* was determined by micro-broth dilution and plate counting methods, and the MIC and MBC were measured. The ratio of live to dead bacteria in the bacterial culture after application of the ClO2-loaded oral sustained-release hydrogel to *P. gingivalis* was observed using a live / dead bacteria staining method to further investigate the inhibitory and bactericidal effects of the ClO2-loaded oral sustained-release hydrogel on *P. gingivalis*.

[0226] 1.2 Effect of ClO2-loaded oral sustained-release hydrogel on biofilm formation of P. gingivalis

[0227] The logarithmic growth phase of P. gingivalis culture was diluted to 1.0 × 10⁻⁶. 6 CFU / mL was prepared for use. In a clean bench, 200 μL of P. gingivalis bacterial suspension was added to a 48-well plate, followed by 0.04 g of sustained-release gel containing MIC, 1 / 2 MIC, and 1 / 4 MIC concentrations of ClO2, respectively, as the experimental group; another group was selected with only 200 μL of P. gingivalis bacterial suspension added as the blank control group.

[0228] The plate was transferred to an anaerobic jar and cultured anaerobicly at 37°C for 48 hours. After 48 hours, the plate was carefully removed, the gel in the wells was gently removed with tweezers, and the liquid in the wells was gently aspirated with a pipette. Then, 200 μL of sterile PBS was added to each well, and the plate was gently and slowly rinsed three times. Next, 200 μL of methanol solution was added, and the plate was fixed for 15 minutes. The formaldehyde in the wells was then aspirated. The plate was air-dried in a clean bench, and 100 μL of 0.4% crystal violet staining solution was added. After staining for 5 minutes, the staining solution in the wells was aspirated, and the plate was gently rinsed several times with sterile PBS solution until the rinsing solution was colorless and transparent. The plate was air-dried again, and 200 μL of 33% glacial acetic acid was added to each well. One well was selected as a blank control with only 200 μL of 33% glacial acetic acid. The plate was then covered and placed in a 37°C shaker for 1 hour for elution. After 1 hour, the plate was removed and the absorbance value of each well at 550 nm was read using an ELISA reader.

[0229] Calculate OD in the experimental group and the control group 550 Subtract blank group OD 550 Recorded as corrected OD 550 And according to Formula 4-1, the biofilm formation inhibition rate of each experimental group relative to the control group was calculated.

[0230]

[0231] 1.3 Evaluation of Kgp hydrolysis activity using specific chromogenic peptide substrates

[0232] 1) Take P. gingivalis in the logarithmic growth phase, check whether the centrifuge tube is sealed and intact, then put it into a high-speed centrifuge for centrifugation (1000r, 15min), and take the supernatant.

[0233] 2) Use a spectrophotometer to measure the absorbance of the supernatant at 660 nm and dilute it with sterile PBS solution to A600nm = 1.

[0234] 3) Take a 48-well plate, add 100 μL of diluted supernatant, then add 50 μL of the prepared ClO2 hydrogel extract at different concentrations, 25 μL of the prepared substrate solution, and 25 μL of dithiothreitol (DTT) solution. The above steps constitute the experimental group; at the same time, select another well and add only the chromogenic substrate solution as the control group.

[0235] 4) Wrap the well plate with aluminum foil and place it in a 37℃ constant temperature incubator for dark incubation. Measure the absorbance at 405nm using an ELISA reader at 15min, 30min, 45min, 1h, 2h, 3h, and 4h.

[0236] 5) Record the data, calculate the kgp activity of each experimental group relative to the control group, and plot the time change curve.

[0237] 1.4 Effect of ClO2-loaded oral sustained-release hydrogel on the secretion of MMP-1 and MMP-2

[0238] 1) Resuscitate and passage human gingival fibroblasts (HGFs).

[0239] 2) Heat inactivation of P. gingivalis at 60℃ for 1 hour.

[0240] 3) Microscopic observation of the toxicity of P. gingivalis to HGFs.

[0241] Experiments were conducted using HGFs from generations 3-6. First, cells in good growth condition were removed from the incubator, and the supernatant was discarded. Then, the cells were washed 2-3 times with sterile PBS to remove any suspended dead cells. Next, 2 mL of trypsin was added to digest the adherent cells, and the cells were transferred to an incubator and incubated at 37°C for 1 min. Observation was performed under an inverted microscope until all cells were in suspension. Then, 4 mL of serum-containing α-MEM complete culture medium was added to terminate the digestion process, and the cells were thoroughly mixed. The concentration of suspended cells was calculated using a cell counting chamber, and the concentration was adjusted to 1 × 10⁻⁶ using α-MEM complete culture medium. 6 CFU / mL.

[0242] Diluted HGFs were seeded into 6-well plates, 2 mL per well. After observing uniform cell distribution under a microscope, the plates were transferred to a constant temperature incubator and incubated at 37°C for 24 h.

[0243] After observing cell adhesion and growth to confirm good cell condition, the prepared heat-inactivated *P. gingivalis* bacterial culture was removed and thawed in a constant-temperature water bath. After thawing, 40 μL, 20 μL, and 10 μL were added to different wells (corresponding to MOIs of 200, 100, and 50, respectively), serving as the experimental groups; wells containing only HGFs served as the control group. The plate was transferred to a constant-temperature incubator and incubated at 37°C for 24 h. Cell growth and adhesion were observed to determine the appropriate MOI value for HGF infection inactivated *P. gingivalis*.

[0244] 4) Co-culture HGFs infected with P. gingivalis with ClO2-loaded sustained-release hydrogel.

[0245] Inactivated *P. gingivalis* bacterial suspension and HGFs were added to six-well plates, followed by the addition of 1 g of ClO2 slow-release hydrogel at different concentrations (MIC and 1 / 2 MIC). The group containing only HGFs was designated as the negative control, and the group infected with *P. gingivalis* and containing HGFs was designated as the positive control. The plates were incubated in a constant temperature incubator (37℃, 5% CO2) for 24 hours. The liquid was then transferred to centrifuge tubes and centrifuged (1000 rpm, 15 min). The supernatant was collected, labeled, and sealed for each group. Finally, the supernatant was stored at -80℃.

[0246] 5) ELISA method to detect the secretion of MMP-1 and MMP-2 of HGFs.

[0247] 2 Experimental Results

[0248] 2.1 Antibacterial effect of ClO2-loaded oral sustained-release hydrogel

[0249] Table 10 shows the effect of different concentrations of ClO2 on the growth of P. gingivalis suspension. The calculation results show that the absorbance values ​​of each experimental group and the control group at 600 nm, as well as the calculated relative growth inhibition rate of P. gingivalis in each experimental group relative to the control group, are statistically significant compared with the control group (p < 0.05).

[0250] Table 10. OD600 and relative growth inhibition rate of different ClO2 concentration groups.

[0251]

[0252] Based on the experimental results, hydrogels loaded with ClO2 at concentrations of 500 mg / L, 400 mg / L, and 250 mg / L all resulted in a relative growth inhibition rate of over 90% for *P. gingivalis*. Specifically, the 250 mg / L ClO2 hydrogel group inhibited 92.1% of *P. gingivalis* growth, which is the minimum ClO2 concentration (MIC) required to achieve a 90% inhibition rate. When the hydrogel was loaded with 125 mg / L ClO2, the relative growth inhibition rate also reached 53.9%. Furthermore, the relative growth inhibition rate of *P. gingivalis* increased positively with increasing ClO2 concentration.

[0253] 2.2 Effect of ClO2-loaded oral sustained-release hydrogel on biofilm formation of P. gingivalis

[0254] Table 11 shows the inhibition rate of ClO2-loaded oral sustained-release hydrogels on P. gingivalis biofilm. The experimental group loaded with 250 mg / L ClO2 hydrogel inhibited 66.2% of P. gingivalis biofilm formation, the group loaded with 125 mg / L ClO2 hydrogel showed an inhibition rate of 40.1%, while the group loaded with 62.5 mg / L ClO2 hydrogel only inhibited 31.4% of biofilm formation. All differences between the experimental groups and the control group were statistically significant (p < 0.05).

[0255] Table 11 shows the inhibition rate of ClO2 oral sustained-release hydrogel on biofilm formation of P. gingivalis.

[0256]

[0257] 2.3 ClO2-loaded oral sustained-release hydrogel's activity against Kgp hydrolysis

[0258] Lysine gingivopeptide (Kgp) is an important virulence factor secreted by *P. gingivalis*, which can directly participate in the destructive and hydrolytic process of periodontal connective tissue and assist *P. gingivalis* in immune evasion by disrupting the normal functioning of the host complement system. Reducing Kgp hydrolytic activity can significantly decrease the pathogenicity of *P. gingivalis*, reduce periodontal tissue damage, and improve local periodontitis symptoms. Figure 17 The curves showing the change in the hydrolytic activity of kgp protein over time reveal that the higher the concentration of ClO2 carried by the hydrogel, the stronger the inhibitory effect on the hydrolytic activity of P. gingivalis gingivin.

[0259] 2.4 Effects of ClO2-loaded oral sustained-release hydrogel on the secretion of MMP-1 and MMP-2

[0260] MMP-1 and MMP-2 are two matrix metalloproteinases secreted by HGCs, also known as collagenase-1 and collagenase-2. They participate in the hydrolytic denaturation of type I collagen fibers, the main type of collagen in periodontal connective tissue, and the expression levels of MMP-1 and MMP-2 are significantly increased in severe periodontitis. Table 12 shows the expression levels of MMP-1 and MMP-2 in different groups. Group 1 was the pure culture group of HGFs; Group 2 was the HGFs group infected with P. gingivalis; Group 3 was the group simultaneously cultured with 250 mg / L ClO2 hydrogel, P. gingivalis, and HGFs; Group 4 was the group simultaneously cultured with 125 mg / L ClO2 hydrogel, P. gingivalis, and HGFs. The results showed that the ClO2-loaded oral sustained-release hydrogel could effectively reduce the secretion of MMP-1 and MMP-2 by HGFs after P. gingivalis stimulation.

[0261] Table 12 shows the calculated concentrations (ng / mL) of MMP-1 and MMP-2 in each sample group.

[0262]

[0263] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0264] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of ClO2-loaded oral sustained-release hydrogel in the preparation of drugs for relieving and / or treating periodontitis, characterized in that, The preparation of the ClO2-loaded oral sustained-release hydrogel includes: An empty-loaded gel was obtained by using the electrostatic interaction between chitosan quaternary ammonium salt and sodium alginate. The empty gel was placed in a ClO2 solution and allowed to stand to obtain the ClO2-loaded oral sustained-release hydrogel.

2. The application according to claim 1, characterized in that, The preparation of the empty gel includes the following steps: Add the chitosan quaternary ammonium salt solution to the sodium alginate solution, react for 10-12 min, then centrifuge to collect the precipitate to obtain the QCS / Alg polyelectrolyte complex. The QCS / Alg polyelectrolyte composite is placed in a mold and allowed to solidify to obtain the empty gel.

3. The application according to claim 2, characterized in that, The mass ratio of chitosan quaternary ammonium salt to sodium alginate is 1-1.5:

1.

4. The application according to claim 3, characterized in that, The concentration of the ClO2 solution is 100-250 ppm.

5. The application according to claim 4, characterized in that, The settling time is 20-24 hours.

6. The use of the ClO2-loaded oral sustained-release hydrogel as described in claim 1 in the preparation of therapeutic drugs for inflammatory diseases.

7. The application according to claim 6, characterized in that, The inflammatory disease is characterized by elevated levels of the inflammatory factors TNF-α and IL-1β.

8. The application of the ClO2-loaded oral sustained-release hydrogel as described in claim 1 in the preparation of antibacterial agents, characterized in that, The formulation is used to inhibit the growth of Porphyromonas gingivalis, inhibit the formation of Porphyromonas gingivalis biofilm, inhibit the hydrolytic activity of gingival glycosides, or inhibit the secretion of matrix metalloproteinases by gingival fibroblasts.

9. The application according to claim 8, characterized in that, The gingival extract is lysine-based gingival extract.

10. The application according to claim 8, characterized in that, The matrix metalloproteinases are MMP-1 and MMP-2.

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