A melatonin-loaded hydrogel, its preparation method and its application

Abstract

This invention provides a melatonin-loaded hydrogel, its preparation method, and its application in periodontal disease. This invention designs a biomultifunctional melatonin-carboxymethyl chitosan / polyoxymethylene dextran hydrogel using the Schiff base reaction, thereby giving the sustained-release system high wet tissue adhesion and self-healing properties. This avoids the impact of saliva and movement on drug residence time and distribution in traditional drug delivery methods, prolonging the action time of the hydrogel and melatonin within the periodontal pocket.

Description

Technical Field

[0001] This invention relates to a melatonin-loaded hydrogel, its preparation method, and its application, belonging to the field of pharmaceutical preparations. Background Technology

[0002] The most widely used and effective treatment for periodontal disease is mechanical plaque removal. However, mechanical periodontal therapy has limitations, and it is often ineffective for some patients. For these patients, drug therapy may play an important auxiliary role in achieving better therapeutic results. Local sustained-release medication has emerged as a novel drug treatment method both domestically and internationally in recent years. Especially for periodontal disease, which is a locally infectious disease, local medication plays a crucial role in its treatment, as it can maintain a certain drug concentration within the periodontal pocket for a prolonged period. However, current topical periodontitis preparations mainly exert their effects through antibiotics. Long-term use of antibiotics can lead to bacterial resistance, and antibiotics often lack the ability to modulate the body's immune system and promote tissue regeneration. Researching and developing novel topical periodontitis preparations, providing safer, more effective, less toxic, easier-to-use, tissue-regenerating, and less likely to induce bacterial resistance formulations, is of great significance for the treatment of periodontitis. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a hydrogel loaded with melatonin, its preparation method, and its applications.

[0004] On one hand, the present invention provides a hydrogel loaded with melatonin, which is obtained by mixing a melatonin ethanol solution with an oxidized dextran solution and then mixing it with a carboxymethyl chitosan (CMCS) solution.

[0005] Furthermore, the melatonin ethanol solution is a 4% melatonin ethanol solution, prepared by dissolving 4g of melatonin in 100ml of ethanol.

[0006] Furthermore, the oxidized dextran solution is a 10% aqueous solution of oxidized dextran (PDA50). The preparation method is to weigh 1g of PDA50, add 10mL of double-distilled water, and let it stand to dissolve.

[0007] Furthermore, oxidized dextran is synthesized from dextran and NaIO4 in a molar ratio of 1:1.

[0008] Furthermore, the CMCS solution is a 10% CMCS aqueous solution, prepared by weighing 1g of CMCS, adding 10mL of double-distilled water, and stirring to dissolve.

[0009] On the other hand, the present invention provides a method for preparing a hydrogel loaded with melatonin, wherein a melatonin ethanol solution is mixed with an oxidized dextran solution and then mixed with a CMCS solution.

[0010] Furthermore, the melatonin ethanol solution is a 40 mg / mL melatonin ethanol solution.

[0011] Furthermore, the oxidized dextran solution is a 10% aqueous solution of oxidized dextran (PDA50). The preparation method is to weigh 1g of PDA50, add 10mL of double-distilled water, and let it stand to dissolve.

[0012] Furthermore, the CMCS solution is a 10% CMCS aqueous solution, prepared by weighing 1g of CMCS, adding 10mL of double-distilled water, and stirring to dissolve.

[0013] On the other hand, the present invention provides the application of a melatonin-loaded hydrogel in the preparation of antibacterial drugs.

[0014] On the other hand, the present invention provides the application of a melatonin-loaded hydrogel in the preparation of periodontal disease drugs.

[0015] Beneficial effects:

[0016] 1. This invention designs a biomultifunctional melatonin-carboxymethyl chitosan / polyoxymethylene dextran hydrogel through the Schiff base reaction, thereby giving the sustained-release system high wet tissue adhesion and self-healing properties. This avoids the impact of saliva and movement on drug residence time and distribution in traditional drug delivery, thus prolonging the action time of the hydrogel and melatonin in the periodontal pocket.

[0017] 2. This invention successfully prepared a hydrogel loaded with MT. The material ratio, experimental procedures, and specific experimental parameters were optimized to a certain extent, providing a guarantee for subsequent experiments and meeting the medication needs of patients with periodontal disease.

[0018] 3. Both CPM1 and CPM2 hydrogels of the present invention have excellent self-healing capabilities, and the self-healing capability of CPM1 is specified in Example 3. During the treatment of periodontitis, the hydrogel will inevitably be subjected to external pressure, so a hydrogel with good self-healing properties can maintain its structural integrity to a certain extent, restore its original function through self-repair, and extend its service life.

[0019] 4. Both CPM1 and CPM2 hydrogels of the present invention have excellent anti-swelling ability, as specified in Example 2. In the treatment of periodontitis, the mechanical properties of hydrogels are strongly affected by the degree of swelling. Resistance to swelling helps the hydrogel maintain its original shape and mechanical properties, and preserves good stability.

[0020] 5. Both CPM1 and CPM2 hydrogels of the present invention have excellent adhesion properties, as specified in Example 4. During the treatment of periodontitis, the hydrogel is inevitably affected by external forces. Good adhesion properties prevent the hydrogel from detaching from the periodontal pocket, and these properties can also seal the periodontal pocket to prevent the reinvasion of periodontal pathogens.

[0021] 6. Both CPM1 and CPM2 hydrogels of this invention exhibit excellent in vitro release properties, as specified in Example 5. During periodontitis, melatonin levels in periodontal tissues decrease. The hydrogels release melatonin to increase the level of melatonin within the periodontal pockets, thereby exerting their antioxidant and immunomodulatory effects.

[0022] 7. Both CPM1 and CPM2 hydrogels of the present invention exhibit excellent antibacterial activity, as specified in Example 6. In the treatment of periodontitis, the excellent antibacterial activity of the hydrogels can resist the invasion of oral pathogens, providing a favorable environment for periodontal tissue repair.

[0023] 8. Both CPM1 and CPM2 hydrogels of the present invention have excellent biocompatibility, as specified in Example 7. In the treatment of periodontitis, the good biocompatibility of the hydrogels can prevent inflammatory reactions in periodontal tissues and promote their repair.

[0024] 9. Both CPM1 and CPM2 hydrogels of this invention have excellent cell migration-promoting effects, as illustrated in Example 8. In the treatment of periodontitis, the cell migration-promoting effect of the hydrogels can help periodontal tissues repair rapidly. Attached Figure Description

[0025] Figure 1 A schematic diagram of CPM synthesis.

[0026] Figure 2 Dex and PDA50's FT-IR and 1 1H NMR characterization. (A) Infrared spectra of Dex and PDA50; (B) 1H NMR spectra of Dex and PDA50.

[0027] Figure 3 Morphology and diameter distribution of hydrogels. (A~C) SEM images of CP, CPM1, and CPM2 hydrogels (200×); (D~F) SEM images of CP, CPM1, and CPM2 hydrogels (500×); (G~I) Pore size statistics of cross-sections of CP, CPM1, and CPM2 hydrogels.

[0028] Figure 4 Infrared absorption spectra of MT, CP, CPM1 and CPM2.

[0029] Figure 5 Swelling rates of different hydrogels in PBS.

[0030] Figure 6 Photo of CPM1 hydrogel self-healing.

[0031] Figure 7 Photographs of hydrogel adhesion. (A-C) Photographs of CPM1 adhesion on pigskin under different torsion states; (D) Photograph of CPM1 adhesion on pigskin surface under water flow impact.

[0032] Figure 8 Images showing the release of different hydrogels in PBS. (A) CPM1; (B) CPM.

[0033] Figure 9 Evaluation of the antibacterial activity of hydrogels. (A) Photographs of bacterial colonies surviving on agar plates after co-culturing with hydrogels; (B) Count of Staphylococcus aureus colonies on hydrogels; (C) Count of Staphylococcus aureus colonies on hydrogels.

[0034] Figure 10 Evaluation of the toxicity of hydrogels to cells. (A) Live / dead fluorescence images of L929 cells after co-culturing with hydrogels of different components for 24 h; (B) Quantitative analysis of the fluorescence images of live / dead cells; (C) Cell proliferation images of L929 cells after incubation with hydrogels for 1, 2 and 3 days.

[0035] Figure 11 Effects of hydrogels on cell migration. (A) Representative images of L929 cell migration in scratch healing assays at different time points. (B) Scratch area treatments between different components at 0, 12, 24, 36, and 48 h. Data represent mean ± SD (n = 3) (*p < 0.05, **p < 0.005). Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions of this disclosure, the technical solutions of this disclosure will be described in detail below with reference to specific embodiments and comparative examples.

[0037] Example 1: Preparation of a multifunctional hydrogel loaded with melatonin

[0038] 1.1 Synthesis of oxidized dextran (PDA)

[0039] Oxidized dextran, named PDA50, was synthesized by reacting dextran and NaIO4 in a 1:1 molar ratio. Experiments verified that the hydrogel obtained from this ratio exhibited higher rapid absorption performance, quickly reaching swelling equilibrium after local application to periodontal pockets and demonstrating rapid stimulus response. Dextran and NaIO4 were dissolved in deionized water and stirred at room temperature in the dark for 24 hours. The solution was then dialyzed with single-distilled water (molecular weight cutoff 8-10 kDa) for 72 hours, during which the dialysate had to be changed frequently. Finally, the original dialyzed solution was freeze-dried to obtain a white solid, which was stored under dry conditions.

[0040] 1.2 Synthesis of melatonin hydrogel (CPM)

[0041] First, a melatonin ethanol solution was prepared. A certain amount of melatonin was weighed and added to anhydrous ethanol to obtain a 40 mg / mL melatonin ethanol solution. Then, 1 g of PDA50 was weighed and added to 10 mL of double-distilled water. The solution was allowed to stand and dissolve to obtain a 10% PDA50 aqueous solution. The melatonin ethanol solution and the 10% PDA50 solution were combined at a volume ratio of 1:1 to obtain a 5% PDA50 solution containing 20 mg / mL melatonin, named Solution 1. 1 g of CMCS was weighed and added to 10 mL of double-distilled water. The solution was stirred and dissolved to obtain a 10% CMCS solution, named Solution 2. Solution 1 and Solution 2 were injected into a hydrogel mold at a volume ratio of 1:1 using a suspension syringe. After standing for about 3 minutes, a melatonin (MT) CMP hydrogel containing 10 mg / mL was obtained, named CPM2. A CMP hydrogel containing 5 mg / mL MT was prepared using the same method and named CPM1. A hydrogel without MT was prepared using the same method and named CP. Figure 1 This is a schematic diagram of CPM synthesis.

[0042] By performing FT-IR analysis on dextran (Dex) and PDA50, and 1 H NMR characterization ( Figure 2 As can be seen, we successfully oxidized Dex to obtain dextran with aldehyde groups. (e.g.) Figure 2 As shown in Figure A, the infrared absorption peaks of Dex and PDA50 are similar, with PDA50 showing a peak at 1730 cm⁻¹. -1 There is a relatively weak vibrational absorption peak at 1730 cm⁻¹, which is because the synthesized oxidized dextran contains a small amount of aldehyde groups. The absorption peak of PDA50 is at 1730 cm⁻¹. -1 It wasn't very obvious. Therefore, we successfully synthesized oxidized dextran. For example... Figure 2 As shown in Figure B, PDA50 produced significant amounts of hemiacetals between 4.4 and 5.8 ppm. FT-IR and 1 ¹H NMR characterization confirmed that Dex was oxidized to PDA with aldehyde groups.

[0043] Scanning electron microscopy (SEM) revealed that the hydrogel has a porous structure. Figure 3 The aperture size in the CP is uniform. Figure 3 (A)). Quantitative analysis was performed using ImageJ, with a pore size of 170 μm. Figure 3 G) such as Figure 3 As shown in (B) and (C), after loading with MT, the pore size in CPM1 and CPM2 is also uniform. Quantitative analysis using ImagJ showed that the pore size of CPM1 is 166.17 μm. Figure 3 (H)). The pore size ratio is smaller than that of the unloaded MT group, which may be because MT is dissolved in anhydrous ethanol, and the anhydrous ethanol reduces the pore size of the hydrogel during gelation. Figure 3 (C) is a cross-sectional view of the CPM2 group hydrogel. SEM microscopy reveals dense and uniform pores within the hydrogel. After pore size quantification, the pore diameter is 176.93 μm. Figure 3 (I)) Compared with CPM1, CPM2 has a larger diameter, which may be because CPM2 is loaded with more MT, resulting in a larger pore size of the synthesized hydrogel. Figure 3 (D~F) are enlarged SEM images of CP, CPM1 and CPM2, respectively.

[0044] The CPM hydrogel was characterized using Fourier transform infrared spectroscopy. Figure 4 As shown, the infrared characteristic absorption peak of pure MT is at 3300 cm⁻¹. -1 The stretching vibration of NH at 2991 cm -1 The stretching vibration of saturated CH at 1556 cm⁻¹ -1 The CN stretching vibration in the secondary amide in the MT molecule structure is at 1213 cm⁻¹. -1 The stretching vibration of aromatic ether COC occurs at a depth of 3000–3500 cm⁻¹. -1 Between these peaks, the peaks of CPM1 and CPM2 are stronger than that of CP, indicating an increase in NH bonds in CPM. Compared with the infrared absorption peak of CP, the peak at 2991 cm⁻¹ is stronger. -1 At 1212 cm⁻¹, obvious characteristic peaks of saturated CH stretching vibrations were observed in both CPM1 and CPM2. -1 The stretching vibration of aromatic ether COC, CPM1 at 1553 cm⁻¹ -1 The characteristic absorption peak appeared at 1555 cm⁻¹. -1 At CPM2, CN stretching vibrations of secondary amide were observed. Therefore, the characteristic peaks of MT were observed in both CPM1 and CPM2, indicating that MT was completely loaded in the CP hydrogel.

[0045] Example 2: Swelling of CMP hydrogel

[0046] Three hydrogels, CP, CPM1, and CPM2, were prepared, with three replicates for each type. The hydrogels were placed in 12-well plates containing PBS (pH-7) at 37°C. Every 5 minutes, the hydrogels were removed, the PBS solution was blotted dry with absorbent paper, and the mass of the hydrogel was measured until a constant weight was reached. The mass of the hydrogel at each time point was recorded. The swelling ratio was calculated as follows:

[0047]

[0048] The results were obtained through hydrogel swelling experiments, such as Figure 5 As shown, CP, CPM1, and CPM2 hydrogels underwent two swelling processes in PBS solution. The swelling rate reached its maximum after 30 minutes, likely due to the larger pore size and faster water absorption. After 30 minutes, the mass of all three hydrogels began to decrease. Once swelling reached saturation, degradation occurred, leading to a decrease in mass. After 24 hours in PBS, the swelling essentially reached dynamic equilibrium. Within 24 hours in PBS solution, the shape of the three hydrogels remained unchanged after absorbing PBS, maintaining a stable structure.

[0049] Example 3: CMP hydrogel self-healing

[0050] Taking CPM1 hydrogel as an example, this study demonstrates the self-healing properties of CPM1 hydrogel at 37°C. First, CPM1 hydrogel was prepared. Then, CPM1 hydrogel containing red and green "T" shaped letters was prepared. The two hydrogels were cut apart at the junction of the horizontal and vertical axes of the "T" with a blade. The red and green halves were placed together and left to stand in a humid environment at 37°C for 1 hour. The self-adhesion of the hydrogels was observed and photographed.

[0051] CPM hydrogel is prepared by loading MT through a Schiff base reaction of CMCS and PDA50. During periodontitis treatment, the hydrogel is inevitably subjected to external pressure, leading to fracture and a shortened lifespan. CP hydrogel possesses self-healing properties, allowing it to maintain its structural integrity, restore its original function through self-repair, and extend its lifespan. Figure 6 As shown, after the CPM1 hydrogel was cut open for 1 hour, the red or green end of the hydrogel was held with tweezers. The other end of the hydrogel did not fall off, which shows that the hydrogels were bonded together through a self-healing phase, proving the self-healing properties of the hydrogel.

[0052] Example 4: CPM hydrogel adhesion

[0053] Based on previous research, porcine skin was chosen as the substrate for testing the adhesion of hydrogels to human skin surfaces. We used a double-suspension syringe to inject CMCS and MT PDA50 solutions onto the surface of porcine skin, and by changing the shape of the porcine skin, we assessed the adhesion of the hydrogels to the tissue surface. First, a certain amount of red staining agent was added to the MT PDA50 solution. Using two sterile medical syringes, 0.5 mL of each of the CMCS and MT PDA50 solutions were injected onto the surface of the porcine skin, along with the suspension syringe tip. After gelation, the shape of the porcine skin was changed, and different photographs were taken.

[0054] To verify the tissue adhesion properties of hydrogels, using CPM1 hydrogel as an example, CPM1 hydrogel was injected onto the surface of pigskin through a dual-mixing syringe. Through different torturing methods, the hydrogel could firmly adhere to the pigskin surface. Figure 7 (AC)). This indicates that CPM1 hydrogel can adhere to tissues and also has a certain degree of flexibility, allowing it to be rolled up. Under the impact of a rapid water flow from a tap ( Figure 7 (D) The hydrogel on the pigskin surface was impacted by water flow. The CPM1 hydrogel on the pigskin remained firmly adhered to the pigskin. This demonstrates that the hydrogel can still adhere to the skin under tremendous impact.

[0055] Example 5: In vitro release of CPM hydrogel

[0056] The diffusion of melatonin hydrogel in vitro was assessed. To simulate the in vivo environment, the melatonin hydrogel was released in PBS (pH-7 and pH-5) buffer solutions. 0.8 mL of CPM1 and CPM2 hydrogels were synthesized and placed in dialysis bags (molecular weight cutoff 3500 Da). A certain volume of PBS (pH-7 and pH-5) buffer was added to the dialysis bags, and the mixture was shaken in a centrifuge tube containing 20 mL of PBS buffer solution and released at 37°C. At regular intervals, 2 mL of solution was taken out, fresh PBS buffer was added, and the mixture was shaken and released again at 37°C. The UV absorbance of MT in the extracted solution was measured at 278 nm using a UV spectrophotometer until the release in the hydrogel reached equilibrium. The cumulative drug release was calculated using Origin software. Three replicates were prepared for each sample.

[0057] To verify the release of MT loaded with CPM in vivo, the CPM was placed in a simulated periodontal environment, and the MT content in the solution was measured at different time points. Figure 8 As shown, CPM1 and CPM2 were released in PBS pH-5 and pH-7, respectively. Both groups of hydrogels released more than 90% within 24 hours in both PBS environments. CPM1 was released at 98% in PBS pH-5, and MT was almost completely released.

[0058] Example 6: Evaluation of antibacterial activity

[0059] To verify the antibacterial activity of the hydrogels, *Staphylococcus aureus* (a Gram-positive bacterium) and *Escherichia coli* (a Gram-negative bacterium) were selected as model bacteria. Blank and CP were used as control groups. A total of 200 μL of hydrogels, CPM1 and CPM2, were prepared and irradiated with UV light for 30 min in a clean bench. They were then placed in tryptone-soy broth containing *Staphylococcus aureus* or Luria-Bertani medium containing *Escherichia coli* at the same concentration and co-cultured at 37°C for 12 h. The co-cultured bacterial solution was diluted 10-8 times with PBS (pH-7), and 20 μL of the PBS bacterial broth was evenly spread onto agar plates. The plates were then placed in a 37°C bacterial incubator. After 24 h, the number of colonies was observed. Each sample was plated on three plates. The bacterial colony count (CFU) in the agar medium was counted. The results are expressed as follows:

[0060]

[0061] The oral cavity is prone to bacterial growth, and the treatment of periodontitis mainly focuses on eradicating bacteria. We verified the antibacterial ability of the hydrogel Gel-nHA through in vitro antibacterial experiments using CFU counting. Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli were selected. Figure 9 As shown in (A), different hydrogels exhibit antibacterial effects against E. coli and S. aureurs. Compared to the Blank group, CP shows a certain antibacterial effect. When loaded with MT, it can be seen that the bacterial counts of CPM1 and CPM2 significantly decreased after co-culturing with Escherichia coli and Staphylococcus aureus, respectively. Figure 9 As shown in (B), the average colony count of Escherichia coli in the Blank group was 2.06 × 10⁻⁶, obtained by CFU counting. 12 The CFU / mL count was 4.75 × 10⁻⁶ CPM1 and CPM2, respectively. 11 CFU / mL and 3.610 8 CFU / mL. The average colony count of Staphylococcus aureus in the Blank group was 2.835 × 10⁻⁶. 11 The CFU / mL count was 2.787 × 10⁻⁶ CPM1 and CPM2, respectively. 10 CFU / mL and 1.653×10 10 CFU / mL, ( Figure 9(C)). The results above indicate that CP hydrogel has a good antibacterial effect, and the addition of MT to the CP hydrogel does not affect its antibacterial rate. CPM2 significantly inhibits bacterial growth.

[0062] Example 7: Biocompatibility Testing

[0063] 7.1 Sterilization treatment of hydrogel materials

[0064] CMCS and PDA solutions containing MT were irradiated with ultraviolet light in a clean bench. In a fume hood, 10% CMCS and PDA solutions containing 5 mg / mL and 10 mg / mL MT were taken with sterile medical syringes and mixed in a 1:1 volume ratio to form a hydrogel. The hydrogel was then placed in 4 mL of DMEM cell culture medium and incubated at 37°C for 24 h to obtain the melatonin hydrogel extract.

[0065] 7.2 Cell Culture

[0066] L929 mouse fibroblasts were cultured in cell culture dishes containing DMEM cell culture medium, 10% fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 U / mL) in a 37°C CO2 incubator. Cells were passaged when they reached confluence of 90%. For passage, cells were typically digested with 0.05% trypsin / EDTA for 3 min, the trypsin was removed, culture medium was added, cells were pipetted, and the cells were transferred to new sterile culture dishes.

[0067] 7.3 Determination of CCK-8

[0068] L929 cells from cell culture dishes were transferred to 96-well plates at a density of 5 × 10³ cells / well and cultured in a CO2 cell incubator. After 24 h, L929 cells from 24-well plates were co-cultured with hydrogel extract for 24, 48, and 72 h. Then, 10 μL of CCK-8 solution was added to each well and incubated for 1 h. The absorbance (OD) of CCK-8 was measured using a microplate reader. The control and experimental groups were set up in the same manner as for cell adhesion. Each group was run in triplicate.

[0069] 7.4 Cell Viability

[0070] L929 cells were removed from the cell culture incubator, and the DMEM culture medium was aspirated using a pipette. The cells were washed three times with PBS, and then 1 mL of trypsin was added. The cells were digested at 37°C for 3 min. 2 mL of DMEM culture medium was added, and the adherent cells were detached and counted. L929 cells were seeded in 24-well plates at a density of 2 × 10⁴ cells per well and incubated in a cell culture incubator. After 24 h, melatonin hydrogel extraction buffer was added, and the cells were incubated for another 24 h. Then, PI diluted with PBS and Calcein-AM were added, and the cells were incubated for 20 min. Fluorescence microscopy was used to photograph the cells. Live and dead cell counts were performed using ImagJ.

[0071] To verify the cytotoxicity of the materials, L929 mouse fibroblasts were used to perform CCK-8 assays and cell viability tests on CP, CPM1, and CPM2 hydrogels. Figure 10 (A) shows fluorescence images of live and dead cells after co-culturing different hydrogel extracts with cells for 24 hours. Staining live and dead cells with Calcein-AM (green) and PI (red) respectively shows that CP, CPM1, and CPM2 have no toxicity to L929 cells. Image J was used to further quantify the number of live / dead cells on the nanofibers in the above images. Compared with the Blank group, the proportion of live cells in the other three groups showed no significant difference. Figure 10 (B) This indicates that CP, CPM1, and CPM2 are almost non-toxic to L929 cells. Further analysis using CCK-8 assays was performed to assess the proliferation of L929 cells using the three hydrogels. Figure 10 As shown in (C), after co-culturing the three groups of cell extracts with L929 for 1, 2, and 3 days, the number of cells in each group increased with the extension of the culture time.

[0072] Example 8: Cell Migration

[0073] Cells in the culture dish were digested, counted, and seeded into 6-well plates at a density of 2 × 10⁵ cells / mL. After incubation for 24 h, each well was marked with a 200 μL pipette tip to create a 400 μm wide incision. Cells were washed three times with PBS, and serum-free hydrogel extraction buffers of different compositions were added. After incubation for 24 h, DMEM complete medium was added, and cells were incubated for 0, 12, 24, and 48 h. Images were then taken under a fluorescence microscope.

[0074] The effect of CPM hydrogel on cell migration was detected using a cell scratch assay. Metallic acid (MT) possesses numerous benefits, including antioxidant, anti-inflammatory, and immunomodulatory effects, and has been shown to inhibit oral cell viability and angiogenesis. Therefore, the cell migration assay demonstrated the antibacterial and wound-healing effects of MT-loaded in CP hydrogel for the treatment of periodontitis. Figure 11As shown, co-incubation of the hydrogel with L929 cells for 24 hours had almost no effect. After 36 hours of incubation, compared with the control group, cell migration began to occur in the CPM1 and CPM2 groups. The wound healing rate of the CPM1 group reached 45%, and that of the CPM2 group exceeded 50%. After 48 hours, the wound healing rate of the CP group was 32%, lower than the 49% healing rate of the Blank group. However, after loading MT into the CP hydrogel, significant cell migration was observed in the scratched area, with the cell migration rate of the CPM2 group reaching 79%. Therefore, the CPM hydrogel can induce cell migration and promote wound repair while treating periodontitis.

Claims

1. A hydrogel loaded with melatonin, characterized in that, The melatonin ethanol solution was mixed with an oxidized dextran solution, and then mixed with a carboxymethyl chitosan solution to obtain the hydrogel. The melatonin ethanol solution was 40 mg / mL, the oxidized dextran solution was a 10% aqueous solution of oxidized dextran, and the carboxymethyl chitosan solution was a 10% aqueous solution of carboxymethyl chitosan. The volume ratio of the melatonin ethanol solution to the oxidized dextran solution was 1:

1. The volume ratio of the mixture of the melatonin ethanol solution and the oxidized dextran solution to the carboxymethyl chitosan solution was 1:

1. The resulting hydrogel had uniform pore size and a cumulative melatonin release rate of ≥90% within 24 h in PBS solution. The oxidized dextran was synthesized from dextran and NaIO4 in a molar ratio of 1:

1.

2. A method for preparing the melatonin-loaded hydrogel according to claim 1, characterized in that, The melatonin ethanol solution is mixed with the oxidized dextran solution, and then mixed with the carboxymethyl chitosan solution to obtain the product.

3. The method for preparing the melatonin-loaded hydrogel as described in claim 2, characterized in that, The melatonin ethanol solution was a 40 mg / mL melatonin ethanol solution, the oxidized dextran solution was a 10% oxidized dextran aqueous solution, and the carboxymethyl chitosan solution was a 10% carboxymethyl chitosan aqueous solution.

4. The use of the melatonin-loaded hydrogel as described in claim 1 in the preparation of antibacterial drugs.

5. The use of the melatonin-loaded hydrogel as described in claim 1 in the preparation of periodontal disease drugs.

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