Periodontitis targeted sustained-release gel based on epithelium training immunization and preparation method thereof

Abstract

The invention discloses periodontitis targeted sustained-release gel based on epithelial training immunization and a preparation method thereof, and relates to the technical field of biological medicine, and the preparation method comprises the following steps: modifying poloxamer, and dissolving the modified poloxamer in anhydrous acetonitrile; the preparation method comprises the following steps: dissolving RGD peptide in a PBS (Phosphate Buffer Solution), adding N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and activating to obtain an activated RGD peptide solution; adding the activated RGD peptide solution into the modified poloxamer solution, and reacting at room temperature to obtain RGD peptide modified poloxamer; the RGD peptide modified poloxamer solution is mixed with a beta-glucan solution and a TLR2 agonist solution, and the periodontitis targeted slow-release gel is obtained. The gel provided by the invention specifically targets gingival epithelial cells, reduces inflammatory response and promotes periodontal tissue regeneration, has good biocompatibility and slow release performance, and can effectively control periodontitis and prevent relapse.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to a periodontitis-targeted sustained-release gel based on epithelial trained immunity and a preparation method thereof. Background Art

[0002] Periodontitis is a common oral inflammatory disease with a complex pathogenesis involving multiple factors, including microbial infection and dysregulated host immune responses. The goals of periodontitis treatment are to control inflammation, promote periodontal tissue regeneration, and maintain oral health. However, traditional treatments (such as mechanical scaling and antibiotic therapy) have limitations and are ineffective in addressing periodontitis recurrence. While these methods can alleviate symptoms in the short term, they are unable to eradicate periodontitis in the long term and may lead to the development of drug-resistant strains and further damage to periodontal tissues.

[0003] In recent years, the concept of trained immunity has provided new insights into the treatment of periodontitis. Trained immunity refers to the process of enabling immune cells to acquire long-term immune memory through specific immune stimulation, thereby enhancing the body's resistance to reinfection. This immune memory not only improves the antibacterial capacity of immune cells, but also regulates the inflammatory response of non-immune cells, reducing tissue damage caused by excessive inflammation. Impaired barrier function of gingival epithelial cells is a key pathological basis for the development of periodontitis. Epithelial trained immunity plays a vital role in maintaining a dynamic balance between barrier tissue damage and repair; maladaptation can cause recurrent infection of periodontal tissues and increase susceptibility to chronic inflammation.

[0004] Therefore, it is of great significance to provide a periodontitis-targeted sustained-release gel based on trained immunity, which can achieve effective treatment and long-term control of periodontitis by specifically targeting gingival epithelial cells. Summary of the Invention

[0005] In order to provide a periodontitis-targeted sustained-release gel based on trained immunity, by specifically targeting gingival epithelial cells, enhancing their antibacterial ability, and regulating their trained immunity, reshape the dynamic balance of epithelial barrier damage and repair, and achieve effective treatment and long-term control of periodontitis, the present application provides a periodontitis-targeted sustained-release gel based on epithelial trained immunity and a preparation method thereof.

[0006] The preparation method of the periodontitis-targeted sustained-release gel based on epithelial trained immunity provided in this application adopts the following technical solutions: The preparation method of the periodontitis-targeted sustained-release gel based on epithelial trained immunity comprises the following steps: S1. The poloxamer is modified to obtain a modified poloxamer; the modified poloxamer is dissolved in anhydrous acetonitrile to obtain a modified poloxamer solution; S2. The RGD peptide was dissolved in PBS buffer to obtain an RGD peptide solution; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were then added and activated at room temperature for 30-50 minutes to obtain an activated RGD peptide solution; S3. The activated RGD peptide solution was added dropwise to the modified poloxamer solution and mixed, and the mixture was reacted at room temperature for 12-24 hours. The unreacted reagent was then removed by dialysis or ultrafiltration, and the RGD peptide-modified poloxamer was obtained after freeze-drying. S4. dissolving the RGD peptide-modified poloxamer in pure water to obtain a gel matrix solution; dissolving β-glucan and a TLR2 agonist in pure water to obtain a β-glucan solution and a TLR2 agonist solution, respectively; S5. After mixing the gel matrix solution, β-glucan solution and TLR2 agonist solution, a periodontitis-targeted sustained-release gel based on epithelial trained immunity is obtained.

[0007] Preferably, the poloxamer is poloxamer 407; and the β-glucan is yeast β-glucan.

[0008] Preferably, the preparation method of the modified poloxamer comprises the following steps: T1. Poloxamer 407 was added to acetone at 4-6°C and allowed to swell and dissolve for 2-3 hours to form a solution with a mass concentration of 4-5 g / mL. The solution was then slowly added to cold hexane for precipitation, followed by centrifugation and vacuum drying at 25-37°C to obtain purified poloxamer 407. T2. Purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03-0.04 g / mL purified poloxamer 407 solution; carbonyldiimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018-0.022 g / mL carbonyldiimidazole solution; the purified poloxamer 407 solution was replaced with nitrogen several times, and then 40-60 mL of the carbonyldiimidazole solution was dropwise added to 40-60 mL of the purified poloxamer 407 solution under nitrogen protection. The reaction was stirred at room temperature for 4-6 hours; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was then dispersed in diethyl ether and purified by precipitation and filtration, and the process was repeated multiple times. After vacuum drying, poloxamer 407-CDI was obtained as a white powder. T3. Dissolve poloxamer 407-CDI in anhydrous acetonitrile, add diethylenetriamine dropwise at room temperature with magnetic stirring, and react under nitrogen for 12-24 hours. Then, remove unreacted diethylenetriamine by rotary distillation to obtain a concentrated solution, which is freeze-dried. The dried crude product is then dispersed in diethyl ether and purified by precipitation and filtration. This purification process is repeated multiple times, and the modified poloxamer is obtained after vacuum drying.

[0009] Preferably, the mass ratio of poloxamer 407-CDI to diethylenetriamine in T3 is 1:7-8.

[0010] Preferably, the concentration of the modified poloxamer solution in S1 is 4-8%; the concentration of the RGD peptide solution in S2 is 0.5-1.5 mg / mL.

[0011] Preferably, the amount of N-hydroxysuccinimide added in S2 is 2.3-4.6 mg / mL and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 3.8-7.6 mg / mL.

[0012] Preferably, the volume ratio of the activated RGD peptide solution to the modified poloxamer solution in S3 is 1:10-15.

[0013] Preferably, the concentration of the gel matrix solution in S4 is 3-5%; the concentration of the β-glucan solution is 0.2-0.6%; and the concentration of the TLR2 agonist solution is 2-5 μg / mL.

[0014] Preferably, the volume ratio of the gel matrix solution, β-glucan solution and TLR2 agonist solution in S5 is 8-9:0.5-0.9:0.05-0.1.

[0015] The periodontitis-targeted sustained-release gel based on epithelial trained immunity provided in this application adopts the following technical solutions: A periodontitis-targeted sustained-release gel based on epithelial trained immunity is prepared according to the preparation method of the periodontitis-targeted sustained-release gel based on epithelial trained immunity.

[0016] In summary, this application includes at least one of the following beneficial technical effects: 1. This application uses a modified form of poloxamer 407 as the thermosensitive hydrogel matrix. Poloxamer 407 is a temperature-sensitive polymer that is liquid at low temperatures and transforms into a gel upon entering the oral cavity as the temperature rises. This enables localized sustained release of drugs, increasing their retention time and therapeutic efficacy in periodontal tissues. The gel surface is modified with an arginine-glycine-aspartic acid (RGD) peptide that specifically targets KRT14+ epithelial cells. KRT14 is a marker molecule on the surface of epithelial cells, and the RGD peptide specifically binds to integrin receptors on the surface of epithelial cells. This allows for targeted delivery of the gel to gingival epithelial cells, increasing drug concentration in target cells, enhancing therapeutic efficacy, and minimizing effects on non-target cells.

[0017] 2. This application adds β-glucan as an immunomodulator. β-glucan is a natural polysaccharide with immunomodulatory effects. It can activate immune functions such as epithelial cells and enhance their phagocytic ability and antibacterial activity. It also adds TLR2 agonist, which can specifically activate Toll-like receptor 2 (TLR2) on the surface of epithelial cells, further enhance the immune response of epithelial cells, intervene in their memory acquired through immune training, reshape the balance between epithelial barrier damage and repair, and thus promote periodontal tissue repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of alveolar bone resorption in group 1 of periodontitis mice before and after treatment in Example 1 of the present application.

[0019] Figure 2 This is a localization map of Claudin-1 labeled using immunofluorescence staining in the gingival epithelial cells of periodontitis mice in Example 1 of the present application.

[0020] Figure 3 This is a diagram showing the expression and localization of IL-1β in gingival sections of mice with periodontitis in Example 1 of the present application, using immunohistochemical staining.

[0021] Figure 4 This is a diagram showing the expression and localization of E-cadherin in gingival sections of mice with periodontitis in Example 1 of the present application, using immunohistochemical staining. DETAILED DESCRIPTION

[0022] The present application will be further described in detail below with reference to the embodiments and accompanying drawings.

[0023] The chemical reagents used in the examples and comparative examples provided by the present invention are all commercially available products.

[0024] Example 1: S1. Poloxamer is modified to obtain a modified poloxamer; the modified poloxamer is dissolved in anhydrous acetonitrile to obtain a modified poloxamer solution having a concentration of 4%; S2. Dissolve 25 mg of RGD peptide in 50 mL of PBS buffer to obtain an RGD peptide solution; then add 115 mg of N-hydroxysuccinimide and 190 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and activate at room temperature for 30 min to obtain an activated RGD peptide solution; S3. 10 mL of the activated RGD peptide solution obtained in S2 was added dropwise to 100 mL of the 4% modified poloxamer solution obtained in S1, and the mixture was reacted at room temperature for 12 h to obtain the RGD peptide-modified poloxamer; unreacted reagents were removed by dialysis or ultrafiltration, and the mixture was freeze-dried to obtain the RGD peptide-modified poloxamer; S4. The RGD peptide-modified poloxamer obtained in S3 was dissolved in pure water to obtain a 3% gel matrix solution; yeast β-glucan and TLR2 agonist were dissolved in pure water to obtain a 0.2% yeast β-glucan solution and a 2 μg / mL TLR2 agonist solution, respectively; S5. The gel matrix solution, yeast β-glucan solution, and TLR2 agonist solution were mixed at a volume ratio of 8:0.5:0.05 to obtain a periodontitis-targeted sustained-release gel based on epithelial trained immunity. The steps for modifying poloxamer in S1 are as follows: S11. Poloxamer 407 was added to acetone at 4°C and allowed to swell and dissolve for 2 hours to form a solution with a mass concentration of 4 g / mL. The solution was then slowly added to cold hexane for precipitation, followed by centrifugation and vacuum drying at 25°C to obtain purified poloxamer 407. S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03 g / mL purified poloxamer 407 solution; carbonyldiimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018 g / mL carbonyldiimidazole solution; the purified poloxamer 407 solution was replaced with nitrogen three times, and then 40 mL of carbonyldiimidazole solution was dropwise added to 40 mL of the purified poloxamer 407 solution under nitrogen protection, and the reaction was stirred at room temperature for 4 h; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was then dispersed in 50 mL of diethyl ether and purified by precipitation and filtration. The purification was repeated three times, and after vacuum drying at 35°C, poloxamer 407-CDI was obtained as a white powder; S13. Dissolve 2 g of poloxamer 407-CDI in 60 mL of anhydrous acetonitrile. Add 14 g of diethylenetriamine dropwise at room temperature with magnetic stirring. React for 12 h under nitrogen protection. Then, remove unreacted diethylenetriamine by rotary distillation to obtain a concentrated solution, which is freeze-dried. The dried crude product is then dispersed in 50 mL of ether and purified by precipitation and filtration. Repeat this purification process three times. Finally, dry the product in vacuo at 35°C to obtain the modified poloxamer.

[0025] Example 2: S1. Poloxamer is modified to obtain a modified poloxamer; the modified poloxamer is dissolved in anhydrous acetonitrile to obtain a modified poloxamer solution having a concentration of 6%; S2. 50 mg of RGD peptide was dissolved in 50 mL of PBS buffer to obtain an RGD peptide solution; then 172 mg of N-hydroxysuccinimide and 285 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were added and activated at room temperature for 40 min to obtain an activated RGD peptide solution; S3. 10 mL of the activated RGD peptide solution obtained in S2 was added dropwise to 125 mL of the 6% modified poloxamer solution obtained in S1, and the mixture was reacted at room temperature for 18 h to obtain the RGD peptide-modified poloxamer; unreacted reagents were removed by dialysis or ultrafiltration, and the mixture was freeze-dried to obtain the RGD peptide-modified poloxamer; S4. The RGD peptide-modified poloxamer obtained in S3 was dissolved in pure water to obtain a 4% gel matrix solution; yeast β-glucan and TLR2 agonist were dissolved in pure water to obtain a 0.4% yeast β-glucan solution and a 3.5 μg / mL TLR2 agonist solution, respectively; S5. The gel matrix solution, yeast β-glucan solution, and TLR2 agonist solution were mixed at a volume ratio of 8.5:0.7:0.075 to obtain a periodontitis-targeted sustained-release gel based on epithelial trained immunity. The steps for modifying poloxamer in S1 are as follows: S11. Poloxamer 407 was added to acetone at 5°C and allowed to swell and dissolve for 2.5 hours to form a solution with a mass concentration of 4.5 g / mL. The solution was then slowly added to cold hexane for precipitation, followed by centrifugation and vacuum drying at 31°C to obtain purified poloxamer 407. S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.035 g / mL purified poloxamer 407 solution; carbonyldiimidazole was dissolved in anhydrous acetonitrile to obtain a 0.02 g / mL carbonyldiimidazole solution; the purified poloxamer 407 solution was replaced with nitrogen three times, and then 50 mL of carbonyldiimidazole solution was dropwise added to 50 mL of the purified poloxamer 407 solution under nitrogen protection, and the reaction was stirred at room temperature for 5 h; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was then dispersed in 60 mL of ether and purified by precipitation and filtration. The purification was repeated three times, and after vacuum drying at 36°C, poloxamer 407-CDI was obtained as a white powder; S13. Dissolve 2 g of poloxamer 407-CDI in 60 mL of anhydrous acetonitrile. Add 15 g of diethylenetriamine dropwise at room temperature with magnetic stirring. React for 18 h under nitrogen protection. Then, remove unreacted diethylenetriamine by rotary distillation to obtain a concentrated solution, which is freeze-dried. The dried crude product is then dispersed in 60 mL of ether and purified by precipitation and filtration. Repeat this purification process three times. The product is then dried in vacuo at 36°C to obtain the modified poloxamer.

[0026] Example 3: S1. Poloxamer is modified to obtain a modified poloxamer; the modified poloxamer is dissolved in anhydrous acetonitrile to obtain a modified poloxamer solution having a concentration of 8%; S2. 75 mg of RGD peptide was dissolved in 50 mL of PBS buffer to obtain an RGD peptide solution; 230 mg of N-hydroxysuccinimide and 380 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were then added and activated at room temperature for 50 min to obtain an activated RGD peptide solution; S3. 10 mL of the activated RGD peptide solution obtained in S2 was added dropwise to 150 mL of the 8% modified poloxamer solution obtained in S1, and the mixture was reacted at room temperature for 24 h to obtain the RGD peptide-modified poloxamer. Unreacted reagents were removed by dialysis or ultrafiltration, and the mixture was freeze-dried to obtain the RGD peptide-modified poloxamer. S4. The RGD peptide-modified poloxamer obtained in S3 was dissolved in pure water to obtain a 5% gel matrix solution; yeast β-glucan and TLR2 agonist were dissolved in pure water to obtain a 0.6% yeast β-glucan solution and a 5 μg / mL TLR2 agonist solution, respectively; S5. The gel matrix solution, yeast β-glucan solution, and TLR2 agonist solution were mixed in a volume ratio of 9:0.9:0.1 to obtain a periodontitis-targeted sustained-release gel based on epithelial trained immunity. The steps for modifying poloxamer in S1 are as follows: S11. Poloxamer 407 was added to acetone at 6°C and allowed to swell and dissolve for 3 hours to form a solution with a mass concentration of 5 g / mL. The solution was then slowly added to cold hexane for precipitation, followed by centrifugation and vacuum drying at 37°C to obtain purified poloxamer 407. S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.04 g / mL purified poloxamer 407 solution; carbonyldiimidazole was dissolved in anhydrous acetonitrile to obtain a 0.022 g / mL carbonyldiimidazole solution; the purified poloxamer 407 solution was replaced with nitrogen three times, and then 60 mL of carbonyldiimidazole solution was dropwise added to 60 mL of the purified poloxamer 407 solution under nitrogen protection, and the reaction was stirred at room temperature for 6 h; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was then dispersed in 70 mL of diethyl ether and purified by precipitation and filtration. The purification was repeated three times, and after vacuum drying at 37°C, poloxamer 407-CDI was obtained as a white powder; S13. Dissolve 2 g of poloxamer 407-CDI in 60 mL of anhydrous acetonitrile. Add 16 g of diethylenetriamine dropwise at room temperature with magnetic stirring. React for 24 h under nitrogen protection. Then, remove unreacted diethylenetriamine by rotary distillation to obtain a concentrated solution, which is freeze-dried. The dried crude product is then dispersed in 70 mL of ether and purified by precipitation and filtration. Repeat this purification process three times. Finally, dry the product in vacuo at 37°C to obtain the modified poloxamer.

[0027] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the poloxamer is not modified, and the gel is directly prepared using poloxamer 407. The preparation method is as follows: S1. Poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 4% poloxamer solution; S2. Dissolve 25 mg of RGD peptide in 50 mL of PBS buffer to obtain an RGD peptide solution; then add 115 mg of N-hydroxysuccinimide and 190 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and activate at room temperature for 30 min to obtain an activated RGD peptide solution; S3. 10 mL of the activated RGD peptide solution obtained in S2 was added dropwise to 100 mL of the 4% poloxamer solution obtained in S1, and the mixture was reacted at room temperature for 12 h to obtain the RGD peptide-modified poloxamer; unreacted reagents were removed by dialysis or ultrafiltration, and the mixture was freeze-dried to obtain the RGD peptide-modified poloxamer; S4. The RGD peptide-modified poloxamer obtained in S3 was dissolved in pure water to obtain a 3% gel matrix solution; yeast β-glucan and TLR2 agonist were dissolved in pure water to obtain a 0.2% yeast β-glucan solution and a 2 μg / mL TLR2 agonist solution, respectively; S5. The gel matrix solution, yeast β-glucan solution and TLR2 agonist solution were mixed in a volume ratio of 8:0.5:0.05 to obtain a periodontitis-targeted sustained-release gel based on epithelial trained immunity.

[0028] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that RGD peptide is not used to modify the modified poloxamer. The preparation method is as follows: S1. Modifying poloxamer to obtain modified poloxamer; S2. The modified poloxamer obtained in S1 was dissolved in pure water to obtain a 3% gel matrix solution; yeast β-glucan and TLR2 agonist were dissolved in pure water to obtain a 0.2% yeast β-glucan solution and a 2 μg / mL TLR2 agonist solution, respectively; S3. The gel matrix solution, yeast β-glucan solution, and TLR2 agonist solution were mixed at a volume ratio of 8:0.5:0.05 to obtain a periodontitis-targeted sustained-release gel based on epithelial trained immunity. The steps for modifying poloxamer in S1 are as follows: S11. Poloxamer 407 was added to acetone at 4°C and allowed to swell and dissolve for 2 hours to form a solution with a mass concentration of 4 g / mL. The solution was then slowly added to cold hexane for precipitation, followed by centrifugation and vacuum drying at 25°C to obtain purified poloxamer 407. S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03 g / mL purified poloxamer 407 solution; carbonyldiimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018 g / mL carbonyldiimidazole solution; the purified poloxamer 407 solution was replaced with nitrogen three times, and then 40 mL of carbonyldiimidazole solution was dropwise added to 40 mL of the purified poloxamer 407 solution under nitrogen protection, and the reaction was stirred at room temperature for 4 h; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was then dispersed in 50 mL of diethyl ether and purified by precipitation and filtration. The purification was repeated three times, and after vacuum drying at 35°C, poloxamer 407-CDI was obtained as a white powder; S13. Dissolve 2 g of poloxamer 407-CDI in 60 mL of anhydrous acetonitrile. Add 14 g of diethylenetriamine dropwise at room temperature with magnetic stirring. React for 12 h under nitrogen protection. Then, remove unreacted diethylenetriamine by rotary distillation to obtain a concentrated solution, which is freeze-dried. The dried crude product is then dispersed in 50 mL of ether and purified by precipitation and filtration. Repeat this purification process three times. Finally, dry the product in vacuo at 35°C to obtain the modified poloxamer.

[0029] Performance testing For the experiments, 6-8-week-old SPF-certified C57BL / 6J male mice, weighing an average of 15-20 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Mice were housed in a well-ventilated, temperature-controlled environment (constant temperature 18-22°C), with a humidity of 45-55%, and a 12-hour light-dark cycle. They were fed a standard laboratory mouse chow and sterile drinking water, with bedding and feed changed twice weekly.

[0030] Forty mice were randomly divided into five groups, and a mouse periodontitis model was established using the following method: the mice were anesthetized by intraperitoneal injection of a mixture of 100 mg / kg ketamine and 10 mg / kg xylazine according to body weight. After the anesthetic took effect, the mice were fixed with a rubber band. Under a stereomicroscope, ophthalmic scissors and ophthalmic forceps were used to pass a 6-0 surgical sterile silk thread between the first and second mandibular molars of the mice and tie a knot on the mesial buccal side of the first molar. After modeling, the mice in each group were observed until they woke up. Two weeks after the periodontitis model was established, groups 1-5 were treated with the gels prepared in Examples 1-3 and Comparative Examples 1-2, respectively. The following tests were performed before treatment (day 0) and after treatment (days 7, 14, and 21) as the detection time points: 1. Use qPCR kit to detect the changes in mRNA levels of inflammatory factors IL-6, IL-18, and TNF-α in gingival tissue from day 0 to day 21. The operation method is as follows: 1. Extraction of Total RNA from Gingival Tissue At the corresponding time points, fresh gingival tissue was isolated and ground from the mice in the above groups, using enzyme-free EP tubes, enzyme-free pipette tips, and DEPC water. Under a fume hood, the tubes were washed two to three times with pre-chilled PBS and 1 mL of Trizol lysis buffer was added. Gently pipette several times to ensure complete cell lysis. The lysate was then transferred to a new enzyme-free EP tube, and 200 μL of chloroform was added. The tubes were shaken on a shaker for thorough extraction. After standing for several minutes, the tubes were centrifuged at 13,000 g for 15 minutes at 4°C. The supernatant was aspirated, avoiding the milky white interphase. 400 μL of isopropanol was then added and shaken on a shaker to mix thoroughly. The tubes were allowed to stand at room temperature for several minutes. After centrifugation for 10 minutes, the supernatant was discarded. Freshly prepared 75% ethanol solution was added to the white precipitate, and the mixture was centrifuged for 5 minutes and the supernatant discarded. This was repeated twice. The ethanol was evaporated at room temperature, and 10 μL of DEPC water was added to dissolve the RNA. The purity and concentration of RNA in each group were determined using a NanoDrop spectrophotometer, and the RNA was diluted to the optimal concentration with DEPC water.

[0031] 2. Tissue RNA Reverse Transcription According to the instructions of PrimeScriptTM® RT regent Kit with gDNA Eraser RNA reverse transcription kit (Takara, Japan): (1) Elute genomic DNA: Incubate in a PCR instrument at 42°C for 2 minutes. Prepare a 10 μL system with 2 μL of 5g DNA Eraser Buffer, 1 μL of gDNA Eraser, and 1 μg of RNA, and top up with RNase-free H2O.

[0032] (2) RNA reverse transcription: Incubate on a PCR instrument at 37°C for 15 minutes and 85°C for 5 seconds. Prepare 20 μL of the system using 10 μL of the first step reaction product, 1 μL of Enzyme I, 1 μL of Primer Mix, and 4 μL of 5× Buffer II. Make up the remaining volume with RNase-free H2O.

[0033] (3) Dilute the obtained cDNA sample to 100 μL with DEPC water and store it or use it for the next experiment.

[0034] 3. Real-time quantitative reverse transcription polymerase chain reaction Using the cDNA from the previous step as the template for amplification, a reaction system was prepared and added to a dedicated 96-well plate (Biorad, USA), with two replicate wells per sample. A total of 20 μL of the system consisted of: 2.0 μL cDNA sample; 10 μL SYBER Green MasterMix; 6.4 μL DEPC water; and 0.8 μL primers. The qRT-PCR amplification protocol was set up. After the amplification was completed and the plate was disconnected from the plate, quantification was performed using the 2-ΔΔCt method. The results are shown in Table 1.

[0035] The specific test results are as follows: Table 1 Changes in mRNA levels of inflammatory factors IL-6, IL-18, and TNF-α in gingival tissue detected by qPCR

[0036] (P less than 0.001) The test results in Table 1 show that IL-6, IL-18, and TNF-α levels briefly increased at 7 days but subsequently declined. The expression levels of all inflammatory factors were significantly lower than baseline values ​​(day 0) at 14 and 21 days, with IL-6 showing the greatest decrease, indicating that the gel's anti-inflammatory effect is cumulative over time. Furthermore, all data from 14 and 21 days showed highly significant differences (P<0.001), confirming the gel's sustained inhibitory effect on chronic periodontal inflammation.

[0037] 2. Micro-CT was used to detect alveolar bone resorption in mice with periodontitis in Example 1, 0-21 days after gel injection. The operation method was as follows: The mice in the above groups were euthanized at the corresponding time points, and the maxilla was isolated. The maxilla of the mouse with the gingival tissue removed was placed in 75% alcohol for CT analysis. 14 mm sample tubes were placed in sequence, and μCT 80 was installed for scanning with a scanning accuracy of 7 μm and a resolution of 500 proj / 180°. After being removed from the machine, three-dimensional reconstruction and analysis of the mouse teeth and maxilla were performed in SCANCO software. According to the different densities of the reconstructed mandible, single-channel analysis of the crown and other parts was performed using SCANCO Visualizer software (1.1.18.0) to generate a heat map to evaluate bone absorption. The level of bone absorption was quantitatively evaluated by measuring the vertical distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC). The results are shown in Figure 2. Figure 1 As shown, the red shadow represents the alveolar bone absorption area between adjacent teeth, scale bar = 1mm, N = 8.

[0038] The results showed that the area of ​​alveolar bone resorption between adjacent teeth continued to decrease over time, especially after 14 days, when recovery was accelerated.

[0039] 3. Immunofluorescence staining was used to detect the localization and distribution of Claudin-1 in the gingival epithelial cells of the periodontitis mice in Example 1. The operation method is as follows: 1. Decalcification, dehydration, wax impregnation and embedding Mouse maxillary bone specimens with gingival tissue were immersed in 20% EDTA decalcifying solution (pH 7.4) for 5-8 weeks in a shaker at 4°C, with the decalcifying solution changed 1-2 times weekly. After decalcification, the specimens were washed twice in PBS, trimmed, preserving the target area without damage, and placed in an embedding cassette before being placed in an orderly fashion in a dehydrator. After dehydration and transparency, the specimens were immersed in embedding wax for at least 2 hours and then embedded in the desired sectioning orientation.

[0040] 2. Slicing Mount the embedded tissue wax block on a microtome and perform rough trimming at a thickness of 10-20 μm. Once near the tooth and jaw regions, section at a setting of 5 μm. Ensure the axial alignment of the crown and root, then obtain buccal and lingual longitudinal sections. Spread the sections thoroughly and dry the slides in a 65°C oven before storing them at 4°C.

[0041] 3. Dewaxing and Rehydration: Bake the sections at 65°C for 1-2 hours. Dewax in xylene I and II, sequentially for 10 minutes each. Next, dewax in ethanol I, II, 90% ethanol, and 80% ethanol, sequentially for 5-10 minutes each. Wash three times with PBS buffer, 5 minutes each.

[0042] 4. Antigen Retrieval: Heat retrieval (steam heating method) in 1× sodium citrate antigen retrieval solution for 15 minutes. After cooling the solution to room temperature, remove the sections and wash three times.

[0043] 5. Blocking: During the wash process, prepare a blocking solution containing 0.2% Triton X-100, 10% BSA, and 5% goat serum. Wipe away any moisture from the tissue surrounding the sections to prevent the samples from drying out for extended periods. Add 50 μL of blocking solution per sample and incubate at 37°C for 1-2 hours.

[0044] 6. Primary antibody incubation: Dilute mouse anti-Claudin-1 in blocking solution at a ratio of 1:500, add 50 μL / sample (the same below), and place in a 4°C refrigerator overnight.

[0045] 7. Secondary Antibody Incubation: After rewarming at 37°C for 1 hour, wash three times with PBS buffer, 10 minutes each time. Add fluorescent Alexa Fluor 550 goat anti-mouse secondary antibody diluted 1:100 in PBS dropwise and incubate at 37°C for 1 hour.

[0046] 8. Nuclear staining: DAPI staining for 5 minutes in the dark, wash 3 times. Seal the slides with water-soluble mounting agent Galetin and collect images under a confocal microscope. The results are as follows. Figure 2 Shown, scale bar = 30 μm.

[0047] The results showed that on day 0, Claudin-1 presented a dense, continuous green fluorescence signal in the epithelium; from day 7 to day 21, the fluorescence signal gradually became discontinuous, and the signal intensity in some areas decreased. By day 21, the Claudin-1 signal in the epithelium was significantly localized or weakened.

[0048] IV. Immunofluorescence staining was used to detect the expression and localization of IL-1β in the gingival epithelial cells of the periodontitis mice in Example 1. The operation method is as follows: 1. Baking: Prepare paraffin samples from the collected mouse maxillary specimens with gingival tissue according to the "decalcification, dehydration, wax impregnation, embedding and sectioning" method. Dewax the samples at 65°C for 2 hours, and then gradually hydrate and wash.

[0049] 2. Inactivate endogenous peroxidase: Wipe off excess water on the slices, protect from light, add 3% hydrogen peroxide, incubate for 15-20 minutes, and wash three times with PBS (the same below).

[0050] 3. Blocking: Steam-heat in antigen retrieval solution for 15 minutes. After cooling to room temperature, block at 37°C for 1 hour.

[0051] 4. Primary Antibody Incubation: Wipe away moisture from the blocked tissue sections and add the appropriate primary antibody diluent, diluted according to the instructions, at 50 μL / sample. Incubate overnight at 4°C. In this experiment, the primary antibody used was Rabbit monoclonal anti-IL-1β, diluted 1:200. A negative control should also be included.

[0052] 5. Secondary Antibody Incubation: Remove the slides from the overnight staining box and rewarm at 37°C for 1 hour. Wash three times with PBS, add secondary antibody, and incubate at room temperature for 30 minutes.

[0053] 6. Wash with PBS three times, add SABC, and incubate at 37°C for 30 minutes.

[0054] 7. Wash three times with PBS, add freshly prepared AEC staining solution, and after complete color development within 10-30 minutes, place in distilled water to terminate the reaction.

[0055] 8. Wash 3 times with PBS, add hematoxylin to stain the nucleus for 10 seconds, and rinse under tap water to turn blue.

[0056] 9. Wipe the moisture around the slices, add a drop of Galetin water-soluble mounting medium, seal the slices, store at 4°C, and take pictures under an Olympus microscope. Figure 3 Shown, scale bar = 50 μm, N = 8.

[0057] 5. Immunofluorescence staining was used to detect the expression and localization of E-cadherin in the gingival epithelial cells of the periodontitis mice in Example 1. The operation method is as follows: 1. Baking: Dewax the mouse paraffin sections at 65°C for 2 hours, hydrate them in a gradient manner, and wash them as described above.

[0058] 2. Inactivate endogenous peroxidase: Wipe off excess water on the slices, protect from light, add 3% hydrogen peroxide, incubate for 15-20 minutes, and wash three times with PBS (the same below).

[0059] 3. Blocking: Steam-heat in antigen retrieval solution for 15 minutes. After cooling to room temperature, block at 37°C for 1 hour.

[0060] 4. Primary Antibody Incubation: Wipe away moisture from the blocked tissue sections and add the appropriate primary antibody diluent, diluted according to the instructions, at 50 μL / sample. Incubate overnight at 4°C. In this experiment, the primary antibody used was mouse monoclonal anti-E-cadherin, diluted 1:500. A negative control should also be included.

[0061] 5. Secondary Antibody Incubation: Remove the slides from the overnight staining box and rewarm at 37°C for 1 hour. Wash three times with PBS, add secondary antibody, and incubate at room temperature for 30 minutes.

[0062] 6. Wash 3 times with PBS, add SABC, incubate at 37℃ for 30 minutes.

[0063] 7. Wash 3 times with PBS, add AEC solution, color completely within 10-30 minutes, then stop the reaction in distilled water.

[0064] 8. Wash 3 times with PBS, add hematoxylin nuclear dye for 10 seconds, then rinse back to blue under tap water.

[0065] 9. Dry the water, add Galetin water-soluble mounting medium for mounting, then take pictures under an Olympus microscope. The results are shown in Figure 4 , scale = 50 μm, N = 8.

[0066] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, and the modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a periodontitis-targeted sustained-release gel based on epithelial trained immunity, characterized by: The following steps are involved: S1. The poloxamer is modified to obtain a modified poloxamer; the modified poloxamer is dissolved in anhydrous acetonitrile to obtain a modified poloxamer solution; S2. The RGD peptide was dissolved in PBS buffer to obtain an RGD peptide solution; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were then added and activated at room temperature for 30-50 minutes to obtain an activated RGD peptide solution; S3. The activated RGD peptide solution was added dropwise to the modified poloxamer solution and mixed, and the mixture was reacted at room temperature for 12-24 hours. The unreacted reagent was then removed by dialysis or ultrafiltration, and the RGD peptide-modified poloxamer was obtained after freeze-drying. S4. dissolving the RGD peptide-modified poloxamer in pure water to obtain a gel matrix solution; dissolving β-glucan and a TLR2 agonist in pure water to obtain a β-glucan solution and a TLR2 agonist solution, respectively; S5. After mixing the gel matrix solution, β-glucan solution and TLR2 agonist solution, a periodontitis-targeted sustained-release gel based on epithelial trained immunity is obtained.

2. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 1, characterized in that: The poloxamer is poloxamer 407; the β-glucan is yeast β-glucan.

3. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 1, characterized in that: The preparation method of the modified poloxamer comprises the following steps: T1. Poloxamer 407 was added to acetone at 4-6°C and allowed to swell and dissolve for 2-3 hours to form a solution with a mass concentration of 4-5 g / mL. The solution was then slowly added to cold hexane for precipitation, followed by centrifugation and vacuum drying at 25-37°C to obtain purified poloxamer 407. T2. Purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03-0.04 g / mL purified poloxamer 407 solution; carbonyldiimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018-0.022 g / mL carbonyldiimidazole solution; the purified poloxamer 407 solution was replaced with nitrogen several times, and then 40-60 mL of the carbonyldiimidazole solution was dropwise added to 40-60 mL of the purified poloxamer 407 solution under nitrogen protection. The reaction was stirred at room temperature for 4-6 hours; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was then dispersed in diethyl ether and purified by precipitation and filtration, and the process was repeated multiple times. After vacuum drying, poloxamer 407-CDI was obtained as a white powder. T3. Dissolve poloxamer 407-CDI in anhydrous acetonitrile, add diethylenetriamine dropwise at room temperature with magnetic stirring, and react under nitrogen for 12-24 hours. Then, remove unreacted diethylenetriamine by rotary distillation to obtain a concentrated solution, which is freeze-dried. The dried crude product is then dispersed in diethyl ether and purified by precipitation and filtration. This purification process is repeated multiple times, and the modified poloxamer is obtained after vacuum drying.

4. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 3, characterized in that: The mass ratio of poloxamer 407-CDI to diethylenetriamine in T3 is 1:7-8.

5. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 1, characterized in that: The concentration of the modified poloxamer solution in S1 is 4-8%; the concentration of the RGD peptide solution in S2 is 0.5-1.5 mg / mL.

6. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 1, characterized in that: The amount of N-hydroxysuccinimide added in S2 is 2.3-4.6 mg / mL and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 3.8-7.6 mg / mL.

7. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 1, characterized in that: The volume ratio of the activated RGD peptide solution and the modified poloxamer solution described in S3 is 1:10-15.

8. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 1, characterized in that: The concentration of the gel matrix solution in S4 is 3-5%; the concentration of the β-glucan solution is 0.2-0.6%; and the concentration of the TLR2 agonist solution is 2-5 μg / mL.

9. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial trained immunity according to claim 1, characterized in that: The volume ratio of the gel matrix solution, β-glucan solution and TLR2 agonist solution in S5 is 8-9:0.5-0.9:0.05-0.

1.

10. A periodontitis-targeted sustained-release gel based on epithelial trained immunity, characterized by: A periodontitis targeted sustained-release gel based on epithelial trained immunity prepared according to the preparation method of the periodontitis targeted sustained-release gel based on epithelial trained immunity according to any one of claims 1 to 9.

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