Periodontitis targeting sustained-release gel based on epithelial training immunity and preparation method thereof

Abstract

The application discloses a periodontitis targeted sustained-release gel based on epithelial training immunity and a preparation method thereof, relates to the technical field of biological medicine, and the preparation process is as follows: poloxamer is modified and then dissolved in anhydrous acetonitrile; RGD peptide is dissolved in a PBS buffer solution, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are added for activation to obtain an activated RGD peptide solution; the activated RGD peptide solution is added into the modified poloxamer solution, and reaction is carried out 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 to obtain the periodontitis targeted sustained-release gel. The gel provided in the application can specifically target gingival epithelial cells, reduce inflammatory reactions, promote periodontal tissue regeneration, has good biocompatibility and sustained-release performance, and can effectively control periodontitis and prevent recurrence.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a periodontitis-targeted sustained-release gel based on epithelial training immunity and its preparation method. Background Technology

[0002] Periodontitis is a common inflammatory disease of the oral cavity with a complex pathogenesis involving multiple factors such as microbial infection and host immune dysregulation. 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 cannot effectively address the problem of periodontitis recurrence. While these methods can relieve symptoms in the short term, they cannot cure periodontitis in the long run and may lead to the development of drug-resistant bacterial strains and further damage to periodontal tissues.

[0003] In recent years, the concept of immune training has provided a new approach to the treatment of periodontitis. Immune training refers to using specific immune stimuli to induce long-term immune memory in immune cells, thereby enhancing the body's resistance to reinfection. This immune memory not only improves the antibacterial ability of immune cells but also regulates the inflammatory response of non-immune cells, reducing excessive inflammation and tissue damage. Impaired barrier function of gingival epithelial cells is a key pathological basis for periodontitis. Epithelial immune training plays a crucial role in maintaining the dynamic balance between barrier tissue damage and repair; maladaptation can lead to recurrent infections in 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 effectively treat and control periodontitis by specifically targeting gingival epithelial cells. Summary of the Invention

[0005] To provide a periodontitis-targeted sustained-release gel based on trained immunity, which specifically targets gingival epithelial cells, enhances their antibacterial ability, regulates their trained immunity, and reshapes the dynamic balance between epithelial barrier damage and repair, thereby achieving effective treatment and long-term control of periodontitis, this application provides a periodontitis-targeted sustained-release gel based on epithelial trained immunity and its preparation method.

[0006] The preparation method of the periodontitis-targeted sustained-release gel based on epithelial training immunity provided in this application adopts the following technical solution:

[0007] The preparation method of periodontitis-targeted sustained-release gel based on epithelial training immunity includes the following steps:

[0008] S1. Modify poloxamer to obtain modified poloxamer; dissolve the modified poloxamer in anhydrous acetonitrile to obtain a modified poloxamer solution;

[0009] S2. Dissolve the RGD peptide in PBS buffer to obtain an RGD peptide solution; then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and activate at room temperature for 30-50 min to obtain an activated RGD peptide solution.

[0010] S3. Add the activated RGD peptide solution dropwise to the modified poloxamer solution and mix. React at room temperature for 12-24 h. Then remove unreacted reagents by dialysis or ultrafiltration and freeze-dry to obtain RGD peptide-modified poloxamer.

[0011] S4. Dissolve the RGD peptide-modified poloxamer in pure water to obtain a gel matrix solution; dissolve β-glucan and TLR2 agonist in pure water respectively to obtain β-glucan solution and TLR2 agonist solution;

[0012] S5. After mixing the gel matrix solution, β-glucan solution and TLR2 agonist solution, a periodontitis-targeted sustained-release gel based on epithelial training immunity is obtained.

[0013] Preferably, the poloxamer is poloxamer 407; and the β-glucan is yeast β-glucan.

[0014] Preferably, the method for preparing the modified poloxamer includes the following steps:

[0015] T1. Poloxamer 407 was added to acetone at 4-6℃ and swollen and dissolved for 2-3 hours to form a solution with a mass concentration of 4-5 g / mL. Then, it was slowly added to cold hexane to precipitate the solution, centrifuged, and then vacuum dried at 25-37℃ to obtain purified poloxamer 407.

[0016] T2. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03-0.04 g / mL purified poloxamer 407 solution; carbonyl diimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018-0.022 g / mL carbonyl diimidazole solution; the purified poloxamer 407 solution was purged with nitrogen several times, and then, under nitrogen protection, 40-60 mL of carbonyl diimidazole solution was added dropwise to 40-60 mL of purified poloxamer 407 solution, and the reaction was stirred at room temperature for 4-6 h; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was dispersed in diethyl ether and purified by precipitation and filtration, repeated several times, and then dried under vacuum to obtain a white powder of poloxamer 407-CDI;

[0017] T3. Dissolve poloxamer 407-CDI in anhydrous acetonitrile, add diethylenetriamine dropwise under room temperature and magnetic stirring, react for 12-24 h under nitrogen protection, remove unreacted diethylenetriamine by rotary vacuum distillation, obtain a concentrated solution, freeze dry; then disperse the dried crude product in diethyl ether, purify by precipitation and filtration, repeat the purification process multiple times, and obtain modified poloxamer by vacuum drying.

[0018] Preferably, the mass ratio of poloxamer 407-CDI to diethylenetriamine in T3 is 1:7-8.

[0019] Preferably, the concentration of the modified poloxamer solution in S1 is 4-8%; and the concentration of the RGD peptide solution in S2 is 0.5-1.5 mg / mL.

[0020] Preferably, the amount of N-hydroxysuccinimide 2.3-4.6 mg / mL and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide added in S2 is 3.8-7.6 mg / mL.

[0021] Preferably, the volume ratio of the activated RGD peptide solution to the modified poloxamer solution in S3 is 1:10-15.

[0022] 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.

[0023] 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.

[0024] The periodontitis-targeted sustained-release gel based on epithelial training immunity provided in this application adopts the following technical solution:

[0025] Epithelial-trained immune-targeted sustained-release gel for periodontitis, prepared according to the method described above.

[0026] In summary, this application includes at least one of the following beneficial technical effects:

[0027] 1. This application uses poloxamer 407 modified material as the temperature-sensitive hydrogel matrix. Poloxamer 407 is a temperature-sensitive polymer that is liquid at low temperatures and transforms into a gel state as the temperature rises after entering the oral cavity, enabling local sustained release of the drug and improving the retention time and therapeutic effect of the drug in periodontal tissues. Furthermore, the gel surface is modified with arginine-glycine-aspartic acid (RGD) peptides that specifically target KRT14+ epithelial cells. KRT14 is a marker molecule on the surface of epithelial cells, while RGD peptides can specifically bind to integrin receptors on the surface of epithelial cells, thereby achieving targeted delivery of the gel to gingival epithelial cells, increasing the drug concentration in target cells, enhancing the therapeutic effect, and reducing the impact on non-target cells.

[0028] 2. This application adds β-glucan as an immunomodulator. β-glucan is a natural polysaccharide with immunomodulatory effects, which can activate immune functions such as epithelial cells, enhance their phagocytic capacity and antibacterial activity; and adds a TLR2 agonist, which can specifically activate Toll-like receptor 2 (TLR2) on the surface of epithelial cells, further enhancing the immune response of epithelial cells, intervening in the memory acquired by their trained immunity, reshaping the balance between epithelial barrier damage and repair, thereby promoting periodontal tissue repair. Attached Figure Description

[0029] Figure 1 This is a diagram showing the alveolar bone resorption of mice with periodontitis in Example 1 of this application before and after treatment.

[0030] Figure 2 This is a localization map of Claudin-1-labeled gingival epithelial cells of mice with periodontitis in Example 1 of this application, using immunofluorescence staining.

[0031] Figure 3 This is a diagram showing the expression and localization of IL-1β labeled by immunohistochemical staining in gingival sections of mice with periodontitis in Example 1 of this application.

[0032] Figure 4 This is a diagram showing the expression and localization of E-cadherin labeled by immunohistochemical staining in gingival sections of mice with periodontitis in Example 1 of this application. Detailed Implementation

[0033] The present application will be further described in detail below with reference to the embodiments and accompanying drawings.

[0034] The chemical reagents used in the embodiments and comparative examples provided in this invention are all commercially available products.

[0035] Example 1: S1. Modified poloxamer was obtained by modifying it; the modified poloxamer was dissolved in anhydrous acetonitrile to obtain a 4% modified poloxamer solution.

[0036] 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.

[0037] S3. Add 10 mL of the activated RGD peptide solution obtained from S2 dropwise to 100 mL of the 4% modified poloxamer solution obtained from S1 and mix. React at room temperature for 12 h to obtain RGD peptide-modified poloxamer. Remove unreacted reagents by dialysis or ultrafiltration, and freeze-dry to obtain RGD peptide-modified poloxamer.

[0038] S4. Dissolve the RGD peptide-modified poloxamer obtained from S3 in pure water to obtain a 3% gel matrix solution; dissolve yeast β-glucan and TLR2 agonist in pure water to obtain a 0.2% yeast β-glucan solution and a 2 μg / mL TLR2 agonist solution, respectively.

[0039] 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 training immunity.

[0040] The steps for modifying poloxamer described in S1 are as follows:

[0041] S11. Poloxamer 407 was added to acetone at 4℃ and swollen and dissolved for 2 hours to form a solution with a mass concentration of 4 g / mL. Then, it was slowly added to cold hexane to precipitate the solution, centrifuged, and then vacuum dried at 25℃ to obtain purified poloxamer 407.

[0042] S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03 g / mL purified poloxamer 407 solution; carbonyl diimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018 g / mL carbonyl diimidazole solution; the purified poloxamer 407 solution was subjected to nitrogen purging three times, and then, under nitrogen protection, 40 mL of carbonyl diimidazole solution was added dropwise to 40 mL of purified poloxamer 407 solution, 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 dispersed in 50 mL of diethyl ether, purified by precipitation and filtration, and the purification was repeated three times. After vacuum drying at 35 °C, a white powder of poloxamer 407-CDI was obtained.

[0043] S13. Dissolve 2g of poloxamer 407-CDI in 60mL of anhydrous acetonitrile. Under room temperature and magnetic stirring, add 14g of diethylenetriamine dropwise. After reacting for 12h under nitrogen protection, remove unreacted diethylenetriamine by rotary vacuum distillation to obtain a concentrated solution, which is then freeze-dried. The dried crude product is then dispersed in 50mL of diethyl ether and purified by precipitation and filtration. This purification process is repeated 3 times. The product is then dried under vacuum at 35℃ to obtain modified poloxamer.

[0044] Example 2: S1. Modified poloxamer was obtained by modifying it; the modified poloxamer was dissolved in anhydrous acetonitrile to obtain a 6% modified poloxamer solution.

[0045] S2. Dissolve 50 mg of RGD peptide in 50 mL of PBS buffer to obtain an RGD peptide solution; then add 172 mg of N-hydroxysuccinimide and 285 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and activate at room temperature for 40 min to obtain an activated RGD peptide solution.

[0046] S3. Add 10 mL of the activated RGD peptide solution obtained from S2 dropwise to 125 mL of the 6% modified poloxamer solution obtained from S1 and mix. React at room temperature for 18 h to obtain RGD peptide-modified poloxamer. Remove unreacted reagents by dialysis or ultrafiltration, and freeze-dry to obtain RGD peptide-modified poloxamer.

[0047] S4. Dissolve the RGD peptide-modified poloxamer obtained from S3 in pure water to obtain a 4% gel matrix solution; dissolve yeast β-glucan and TLR2 agonist in pure water to obtain a 0.4% yeast β-glucan solution and a 3.5 μg / mL TLR2 agonist solution, respectively.

[0048] 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 training immunity.

[0049] The steps for modifying poloxamer described in S1 are as follows:

[0050] S11. Poloxamer 407 was added to acetone at 5℃ and swollen and dissolved for 2.5 h to form a solution with a mass concentration of 4.5 g / mL. Then, it was slowly added to cold hexane to precipitate the solution, centrifuged, and then vacuum dried at 31℃ to obtain purified poloxamer 407.

[0051] S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.035 g / mL purified poloxamer 407 solution; carbonyl diimidazole was dissolved in anhydrous acetonitrile to obtain a 0.02 g / mL carbonyl diimidazole solution; the purified poloxamer 407 solution was subjected to nitrogen purging three times, and then, under nitrogen protection, 50 mL of carbonyl diimidazole solution was added dropwise to 50 mL of purified poloxamer 407 solution, 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 dispersed in 60 mL of diethyl ether, purified by precipitation and filtration, and the purification was repeated three times. After vacuum drying at 36 °C, a white powder of poloxamer 407-CDI was obtained.

[0052] S13. Dissolve 2g of poloxamer 407-CDI in 60mL of anhydrous acetonitrile. Under room temperature and magnetic stirring, add 15g of diethylenetriamine dropwise. After reacting for 18h under nitrogen protection, remove unreacted diethylenetriamine by rotary vacuum distillation to obtain a concentrated solution, which is then freeze-dried. The dried crude product is then dispersed in 60mL of diethyl ether and purified by precipitation and filtration. This purification process is repeated three times. The product is then dried under vacuum at 36℃ to obtain modified poloxamer.

[0053] Example 3: S1. Modified poloxamer was obtained by modifying it; the modified poloxamer was dissolved in anhydrous acetonitrile to obtain a modified poloxamer solution with a concentration of 8%;

[0054] S2. Dissolve 75 mg of RGD peptide in 50 mL of PBS buffer to obtain an RGD peptide solution; then add 230 mg of N-hydroxysuccinimide and 380 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and activate at room temperature for 50 min to obtain an activated RGD peptide solution.

[0055] S3. Add 10 mL of the activated RGD peptide solution obtained from S2 dropwise to 150 mL of the 8% modified poloxamer solution obtained from S1 and mix. React at room temperature for 24 h to obtain RGD peptide-modified poloxamer. Remove unreacted reagents by dialysis or ultrafiltration, and freeze-dry to obtain RGD peptide-modified poloxamer.

[0056] S4. Dissolve the RGD peptide-modified poloxamer obtained from S3 in pure water to obtain a 5% gel matrix solution; dissolve yeast β-glucan and TLR2 agonist in pure water to obtain a 0.6% yeast β-glucan solution and a 5 μg / mL TLR2 agonist solution, respectively.

[0057] S5. The gel matrix solution, yeast β-glucan solution and TLR2 agonist solution were mixed at a volume ratio of 9:0.9:0.1 to obtain a periodontitis-targeted sustained-release gel based on epithelial training immunity.

[0058] The steps for modifying poloxamer described in S1 are as follows:

[0059] S11. Poloxamer 407 was added to acetone at 6℃ and swollen and dissolved for 3 hours to form a solution with a mass concentration of 5 g / mL. Then, it was slowly added to cold hexane to precipitate the solution, centrifuged, and then vacuum dried at 37℃ to obtain purified poloxamer 407.

[0060] S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.04 g / mL purified poloxamer 407 solution; carbonyl diimidazole was dissolved in anhydrous acetonitrile to obtain a 0.022 g / mL carbonyl diimidazole solution; the purified poloxamer 407 solution was purged with nitrogen three times, and then, under nitrogen protection, 60 mL of carbonyl diimidazole solution was added dropwise to 60 mL of the purified poloxamer 407 solution, 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 dispersed in 70 mL of diethyl ether, purified by precipitation and filtration, and the purification was repeated three times. After vacuum drying at 37 °C, a white powder of poloxamer 407-CDI was obtained.

[0061] S13. Dissolve 2g of poloxamer 407-CDI in 60mL of anhydrous acetonitrile. Under room temperature and magnetic stirring, add 16g of diethylenetriamine dropwise. After reacting for 24h under nitrogen protection, remove unreacted diethylenetriamine by rotary vacuum distillation to obtain a concentrated solution, which is then freeze-dried. The dried crude product is then dispersed in 70mL of diethyl ether and purified by precipitation and filtration. This purification process is repeated three times. The product is then dried under vacuum at 37℃ to obtain modified poloxamer.

[0062] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that poloxamer was not modified; poloxamer 407 was used directly for gel preparation. The preparation method is as follows:

[0063] S1. Dissolve poloxamer 407 in anhydrous acetonitrile to obtain a 4% poloxamer solution;

[0064] 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.

[0065] S3. Add 10 mL of the activated RGD peptide solution obtained from S2 dropwise to 100 mL of the 4% poloxamer solution obtained from S1 and mix. React at room temperature for 12 h to obtain RGD peptide-modified poloxamer. Remove unreacted reagents by dialysis or ultrafiltration, and freeze-dry to obtain RGD peptide-modified poloxamer.

[0066] S4. Dissolve the RGD peptide-modified poloxamer obtained from S3 in pure water to obtain a 3% gel matrix solution; dissolve yeast β-glucan and TLR2 agonist in pure water to obtain a 0.2% yeast β-glucan solution and a 2 μg / mL TLR2 agonist solution, respectively.

[0067] 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 training immunity.

[0068] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that the modified poloxamer was not modified with RGD peptide. The preparation method is as follows:

[0069] S1. Modified poloxamer is obtained by modifying poloxamer.

[0070] S2. Dissolve the modified poloxamer obtained from S1 in pure water to obtain a 3% gel matrix solution; dissolve yeast β-glucan and TLR2 agonist in pure water to obtain a 0.2% yeast β-glucan solution and a 2 μg / mL TLR2 agonist solution, respectively.

[0071] 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 training immunity.

[0072] The steps for modifying poloxamer described in S1 are as follows:

[0073] S11. Poloxamer 407 was added to acetone at 4℃ and swollen and dissolved for 2 hours to form a solution with a mass concentration of 4 g / mL. Then, it was slowly added to cold hexane to precipitate the solution, centrifuged, and then vacuum dried at 25℃ to obtain purified poloxamer 407.

[0074] S12. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03 g / mL purified poloxamer 407 solution; carbonyl diimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018 g / mL carbonyl diimidazole solution; the purified poloxamer 407 solution was subjected to nitrogen purging three times, and then, under nitrogen protection, 40 mL of carbonyl diimidazole solution was added dropwise to 40 mL of purified poloxamer 407 solution, 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 dispersed in 50 mL of diethyl ether, purified by precipitation and filtration, and the purification was repeated three times. After vacuum drying at 35 °C, a white powder of poloxamer 407-CDI was obtained.

[0075] S13. Dissolve 2g of poloxamer 407-CDI in 60mL of anhydrous acetonitrile. Under room temperature and magnetic stirring, add 14g of diethylenetriamine dropwise. After reacting for 12h under nitrogen protection, remove unreacted diethylenetriamine by rotary vacuum distillation to obtain a concentrated solution, which is then freeze-dried. The dried crude product is then dispersed in 50mL of diethyl ether and purified by precipitation and filtration. This purification process is repeated 3 times. The product is then dried under vacuum at 35℃ to obtain modified poloxamer.

[0076] Performance testing

[0077] The experimental animals were 6-8 week old SPF-grade male C57BL / 6J mice, with an average weight of 15-20g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a hospital animal laboratory with a well-ventilated environment, suitable temperature (constant temperature 18-22°C), and humidity maintained at 45-55%, with a 12-hour daily light-dark cycle. They were fed standard laboratory mouse feed and provided with sterile water. Bedding and feed were changed twice a week.

[0078] Forty mice were randomly divided into 5 groups, and a mouse periodontitis model was established using the following method: Mice were anesthetized by intraperitoneal injection of a mixture of 100 mg / kg ketamine and 10 mg / kg toluidine. After the anesthetic took effect, the mice were fixed with rubber bands, and under a stereomicroscope, ophthalmic scissors and forceps were used to thread a 6-0 surgical suture between the first and second mandibular molars of the mice, tying 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 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):

[0079] I. The changes in mRNA levels of inflammatory factors IL-6, IL-18, and TNF-α in gingival tissue from 0 to 21 days were detected using a qPCR kit. The procedure is as follows:

[0080] 1. Extraction of total RNA from gingival tissue

[0081] At the corresponding time points, fresh gingival tissue was isolated from the mice in the above-mentioned groups and ground, using enzyme-free EP tubes, enzyme-free pipette tips, and DEPC water throughout the process. Under a fume hood, the tissue was washed 2-3 times with pre-cooled PBS, and 1 mL of Trizol lysis buffer was added. To ensure complete cell lysis, the tissue was gently pipetted several times. The lysis buffer was then transferred to a new enzyme-free EP tube, and 200 μL of chloroform was added. The tube was shaken to extract the RNA thoroughly. After standing for several minutes, the tube was centrifuged at 13000g for 15 minutes at 4°C. The supernatant was aspirated, taking care not to touch the milky white intermediate layer. 400 μL of isopropanol was added and the tube was shaken to mix. The mixture was 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 tube was centrifuged for 5 minutes. The supernatant was then discarded, and this process 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 measured using a NanoDrop spectrophotometer, and the RNA was diluted with DEPC water to the optimal concentration.

[0082] 2. Tissue RNA reverse transcription reaction

[0083] Follow the instructions for the PrimeScript™® RT regent kit with gDNA Eraser RNA reverse transcription kit (Takara, Japan):

[0084] (1) Elution of genomic DNA: React at 42°C for 2 minutes on a PCR instrument. Prepare a 10 μL system with 2 μL of 5 g DNA Eraser Buffer, 1 μL of g DNA Eraser, and 1 μg of RNA, and make up the difference with RNase-free H2O.

[0085] (2) RNA reverse transcription: react at 37°C for 15 minutes and 85°C for 5 seconds on a PCR instrument. Prepare a 20 μL system according to the ratio of 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, and make up the difference with RNase-free H2O.

[0086] (3) Dilute the obtained cDNA sample with DEPC water to 100 μL for storage or use in the next experiment.

[0087] 3. Real-time quantitative reverse transcription polymerase chain reaction

[0088] Using the cDNA from the previous step as the amplification template, the reaction mixture was prepared and added to a specialized 96-well plate (Biorad, USA) for the reaction, with two replicates per sample. The total 20 μL mixture consisted of: 2.0 μL cDNA sample; 10 μL SYBER Green MasterMix; 6.4 μL DEPC water; and 0.8 μL primers. A qRT-PCR amplification program was set. After the reaction, quantification was performed using the 2-ΔΔCt method. The results are shown in Table 1.

[0089] The specific test results are as follows:

[0090] Table 1. Changes in mRNA levels of inflammatory factors IL-6, IL-18, and TNF-α in gingival tissue detected by qPCR.

[0091]

[0092] (P < 0.001)

[0093] As shown in Table 1, IL-6, IL-18, and TNF-α transiently increased at day 7, but subsequently decreased. The expression levels of all inflammatory factors were significantly lower than the baseline (day 0) at days 14 and 21, with IL-6 showing the largest decrease, indicating that the anti-inflammatory effect of the gel is cumulative over time. Furthermore, all data at days 14 and 21 showed highly significant differences (P<0.001), validating the gel's sustained inhibitory effect on chronic periodontal inflammation.

[0094] II. Micro-CT was used to detect alveolar bone resorption in mice with periodontitis in Example 1 group from 0 to 21 days after gel injection. The procedure is as follows:

[0095] The mice in the above groups were euthanized at corresponding time points, and the maxillae were isolated. The maxillae of mice with removed gingival tissue were placed in 75% alcohol for CT analysis. 14 mm sample tubes were sequentially placed in the sample tubes, and the samples were scanned using a μCT 80 scanner with a scanning precision of 7 μm and a resolution of 500 proj / 180°. After scanning, three-dimensional reconstruction analysis of the mouse teeth and maxillae was performed in SCANCO software. Based on the different densities of the reconstructed mandible, single-channel analysis of the crown and other sites was performed using SCANCO Visualizer software (1.1.18.0) to generate heatmaps, which could assess bone resorption. The level of bone resorption was quantitatively assessed by measuring the vertical distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC). The results are as follows: Figure 1 As shown, the red shading represents the alveolar bone resorption area between adjacent teeth. Scale bar = 1 mm, N = 8.

[0096] The results showed that the area of ​​alveolar bone resorption between adjacent teeth continued to decrease over time, especially after 14 days when the recovery accelerated.

[0097] III. The distribution of Claudin-1 in the gingival epithelial cells of the periodontitis mice in Example 1 group was detected using immunofluorescence staining. The procedure is as follows:

[0098] 1. Decalcification, dehydration, paraffin impregnation, and embedding

[0099] Mouse maxillary bone samples with gingival tissue were immersed in 20% EDTA decalcification solution (pH 7.4) for 5-8 weeks at 4°C on a shaker, with the decalcification solution changed 1-2 times per week. After decalcification, the samples were washed twice in PBS, trimmed to preserve the target area without damage, placed in an embedding cassette, and then systematically placed in a dehydrator. The dehydrated and cleared samples were then immersed in embedding paraffin solution for at least 2 hours, followed by embedding according to the desired section orientation.

[0100] 2. Slice

[0101] The embedded tissue blocks were fixed on a microtome and coarsely trimmed to a thickness of 10-20 μm. When approaching the tooth and jawbone areas, sections were prepared at a thickness of 5 μm. The crowns and roots were aligned posteriorly to obtain buccal-lingual longitudinal sections. The sections were fully spread, and the slides were thoroughly dried in a 65°C oven before being collected and stored at 4°C.

[0102] 3. Dewaxing and hydration: Place the sections at 65℃ for 1-2 hours. Then, dewax them sequentially in xylene I and II, 10 minutes each time. Next, immerse them sequentially in anhydrous ethanol I, II, 90% ethanol, and 80% ethanol, 5-10 minutes each. Wash three times with PBS buffer, 5 minutes each time.

[0103] 4. Antigen retrieval: Perform antigen thermal retrieval (steam heating retrieval method) in 1× sodium citrate antigen retrieval solution for 15 minutes. Then, after the antigen retrieval solution has cooled to room temperature, remove the slides and wash them 3 times.

[0104] 5. Blocking: During the washing process described above, prepare a blocking solution containing 0.2% Triton X-100, 10% BSA, and 5% goat serum. Wipe the tissue around the slides dry to prevent prolonged drying of the samples. Add 50 μL of blocking solution per sample and incubate at 37°C for 1-2 hours.

[0105] 6. Primary antibody incubation: Dilute mouse anti-Claudin-1 with blocking buffer at a ratio of 1:500, add 50 μL / sample (the same below), and incubate overnight at 4°C.

[0106] 7. Secondary antibody incubation: After warming to 37°C for 1 hour, wash 3 times with PBS buffer, 10 minutes each time. Add Alexa Fluor 550 goat anti-mouse secondary antibody diluted 1:100 with PBS, and incubate at 37°C for 1 hour.

[0107] 8. Nucleus staining: Stain with DAPI in the dark for 5 minutes, then wash 3 times. Mount with water-soluble Galetin and acquire images under a confocal microscope. Results are as follows. Figure 2 As shown, the scale bar is 30 μm.

[0108] The results showed that on day 0, Claudin-1 showed a dense and continuous green fluorescent signal in the epithelial layer; from day 7 to day 21, the fluorescent signal gradually became discontinuous, and the signal intensity in some areas decreased. By day 21, the Claudin-1 signal in the epithelial layer was significantly localized or weakened.

[0109] IV. The expression and localization of IL-1β in the gingival epithelial cells of the periodontitis mice in Example 1 group were detected using immunofluorescence staining. The procedure is as follows:

[0110] 1. Baking: The collected mouse maxillary samples with gingival tissue were prepared into paraffin samples according to the method of "decalcification, dehydration, wax impregnation, embedding and sectioning". The samples were baked at 65°C for 2 hours to dewax, then graded hydration and washed.

[0111] 2. Inactivate endogenous peroxidase: Wipe off excess moisture from the slices, protect from light, add 3% hydrogen peroxide, incubate for 15-20 minutes, and wash 3 times with PBS (the same applies below).

[0112] 3. Blocking: Heat with steam in antigen retrieval solution for 15 minutes. After cooling to room temperature, block at 37°C for 1 hour.

[0113] 4. Primary antibody incubation: After blocking, wipe away any moisture around the tissue sections, add the appropriate primary antibody dilution buffer (50 μL / sample) according to the manufacturer's instructions, and incubate overnight at 4°C. The primary antibody used in this experiment was Rabbit monoclonal anti-IL-1β antibody, diluted 1:200. A negative control was also included.

[0114] 5. Secondary antibody incubation: Remove the slide box that has been stained overnight and incubate at 37°C for 1 hour. Wash 3 times with PBS, add secondary antibody, and incubate at room temperature for 30 minutes.

[0115] 6. Wash 3 times with PBS, add SABC, and incubate at 37°C for 30 minutes.

[0116] 7. Wash 3 times with PBS, add freshly prepared AEC staining solution, and after complete color development within 10-30 minutes, place in distilled water to stop the reaction.

[0117] 8. Wash 3 times with PBS, add hematoxylin for nuclear staining for 10 seconds, rinse under tap water to return to blue.

[0118] 9. Wipe the area around the section dry, add Galetin water-soluble mounting medium, mount the section, and store at 4°C. Image the section under an Olympus microscope. Results are as follows: Figure 3 As shown, the scale is 50 μm and N is 8.

[0119] V. The expression and localization of E-cadherin in the gingival epithelial cells of periodontitis mice in Example 1 group were detected using immunofluorescence staining. The procedure is as follows:

[0120] 1. Baking: Using the same method as above, dewax mouse-derived paraffin sections were baked at 65°C for 2 hours, then graded for hydration and washed.

[0121] 2. Inactivate endogenous peroxidase: Wipe off excess moisture from the slices, protect from light, add 3% hydrogen peroxide, incubate for 15-20 minutes, and wash 3 times with PBS (the same applies below).

[0122] 3. Blocking: Heat with steam in antigen retrieval solution for 15 minutes. After cooling to room temperature, block at 37°C for 1 hour.

[0123] 4. Primary antibody incubation: After blocking, wipe away any moisture around the tissue sections, add the appropriate primary antibody dilution buffer (50 μL / sample) according to the manufacturer's instructions, and incubate overnight at 4°C. The primary antibody used in this experiment was Mouse monoclonal anti-E-cadherin antibody, diluted 1:500. A negative control was also included.

[0124] 5. Secondary antibody incubation: Remove the slide box that has been stained overnight and incubate at 37°C for 1 hour. Wash 3 times with PBS, add secondary antibody, and incubate at room temperature for 30 minutes.

[0125] 6. Wash 3 times with PBS, add SABC, and incubate at 37°C for 30 minutes.

[0126] 7. Wash 3 times with PBS, add freshly prepared AEC staining solution, and after complete color development within 10-30 minutes, place in distilled water to stop the reaction.

[0127] 8. Wash 3 times with PBS, add hematoxylin for nuclear staining for 10 seconds, rinse under tap water to return to blue.

[0128] 9. After drying, add Galetin water-soluble mounting medium and mount the slide. Then, image the slide under an Olympus microscope. Results are as follows: Figure 4 As shown, the scale is 50 μm and N is 8.

[0129] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a periodontitis-targeted sustained-release gel based on epithelial training immunity, characterized in that: Includes the following steps: S1. Modify poloxamer to obtain modified poloxamer; dissolve the modified poloxamer in anhydrous acetonitrile to obtain a modified poloxamer solution; S2. Dissolve the RGD peptide in PBS buffer to obtain an RGD peptide solution; then add N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and activate at room temperature for 30-50 min to obtain an activated RGD peptide solution. S3. Add the activated RGD peptide solution dropwise to the modified poloxamer solution and mix. React at room temperature for 12-24 h. Then remove unreacted reagents by dialysis or ultrafiltration and freeze-dry to obtain RGD peptide-modified poloxamer. S4. Dissolve the RGD peptide-modified poloxamer in pure water to obtain a gel matrix solution; dissolve β-glucan and TLR2 agonist in pure water respectively to obtain β-glucan solution and TLR2 agonist solution; S5. After mixing the gel matrix solution, β-glucan solution and TLR2 agonist solution, a periodontitis-targeted sustained-release gel based on epithelial training immunity is obtained; The poloxamer is poloxamer 407; the β-glucan is yeast β-glucan; The method for preparing the modified poloxamer includes the following steps: T1. Poloxamer 407 was added to acetone at 4-6℃ and swollen and dissolved for 2-3 hours to form a solution with a mass concentration of 4-5 g / mL. Then, it was slowly added to cold hexane to precipitate the solution, centrifuged, and then vacuum dried at 25-37℃ to obtain purified poloxamer 407. T2. The purified poloxamer 407 was dissolved in anhydrous acetonitrile to obtain a 0.03-0.04 g / mL purified poloxamer 407 solution; carbonyl diimidazole was dissolved in anhydrous acetonitrile to obtain a 0.018-0.022 g / mL carbonyl diimidazole solution; the purified poloxamer 407 solution was purged with nitrogen several times, and then, under nitrogen protection, 40-60 mL of carbonyl diimidazole solution was added dropwise to 40-60 mL of purified poloxamer 407 solution, and the reaction was stirred at room temperature for 4-6 h; the reaction solution was then concentrated by rotary evaporation and freeze-dried; the dried crude product was dispersed in diethyl ether and purified by precipitation and filtration, repeated several times, and then dried under vacuum to obtain a white powder of poloxamer 407-CDI; T3. Dissolve poloxamer 407-CDI in anhydrous acetonitrile, add diethylenetriamine dropwise under room temperature and magnetic stirring, react for 12-24 h under nitrogen protection, remove unreacted diethylenetriamine by rotary vacuum distillation, obtain a concentrated solution, freeze dry; then disperse the dried crude product in diethyl ether, purify by precipitation and filtration, repeat the purification process multiple times, and obtain modified poloxamer by vacuum drying.

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

3. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial training 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.

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

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

6. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial training 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.

7. The method for preparing the periodontitis-targeted sustained-release gel based on epithelial training 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.

8. The periodontitis-targeted sustained-release gel based on epithelial training immunity prepared by the method of any one of claims 1-7.

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