A supramolecular complex, a preparation method and application thereof

Abstract

The application provides a supramolecular complex and a preparation method and application thereof, and belongs to the field of biological medicines. The supramolecular complex is prepared from raw materials in the following mass ratio: the mass ratio of puerarin to collagen or a complex thereof is 1:(0.5-20), or the mass ratio of an antibacterial peptide, puerarin and collagen or a complex thereof is 1:5:(2.5-100); the supramolecular complex can form a hydrogel. The supramolecular complex and the hydrogel thereof have significantly improved permeability, can effectively penetrate deep tissue and cells of gingival epithelium, deliver drugs into inner layers of tissue and cells, realize efficient clearance of intracellular pathogens, and break through the problem of insufficient clearance of intracellular bacteria in the existing periodontal drug delivery system. Meanwhile, the supramolecular complex has excellent antibacterial and anti-inflammatory properties and bone formation promoting ability, can effectively treat periodontitis, and provides a new integrated solution for periodontitis treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a supramolecular complex, its preparation method, and its application. Background Technology

[0002] Periodontitis is a chronic inflammation of the periodontal tissues caused by microorganisms, leading to the destruction and resorption of periodontal supporting tissues. *Porphyromonas gingivalis* (Pg) is the main pathogen of periodontitis, causing chronic damage to periodontal tissues through its local colonization and the secretion of endotoxins, gingival proteases, and other virulence factors. Pg can also invade and survive within host cells, effectively evading the body's immune response and altering host cell metabolism. Furthermore, it can reside in epithelial cells and immune cells, continuously releasing toxins and inflammatory factors, weakening osteoblast function, activating osteoclast activity, leading to alveolar bone resorption and soft tissue destruction. Macrophages, as important immune cells, have their immunomodulatory capacity and antibacterial activity greatly impaired when invaded by Pg. For a long time, antibiotics have played an indispensable role in combating bacterial infections; however, due to limited cell permeability, most antibiotics cannot effectively eliminate intracellular bacterial infections. At the same time, antibiotic resistance caused by antibiotic overuse reduces the effectiveness of some antibiotics, rendering them ineffective against bacterial infections.

[0003] With the development of research on biomaterials and natural drugs, antimicrobial peptides (such as polylysine, mussel oligopeptides, and nisin) have received widespread attention in the treatment of periodontitis due to their biocompatibility and antibacterial properties. For example, polylysine has strong antibacterial properties against free bacteria and biofilms, exerting its antibacterial effect by disrupting bacterial cell membranes and interfering with metabolic processes. Its cationic properties allow it to bind to negatively charged bacterial surfaces and biofilm components, enhancing its antibacterial effect; however, it has not yet shown highly effective antagonistic effects against intracellular bacteria. In recent years, studies have found that plant-derived polyphenols can effectively reduce inflammation, kill bacteria, and promote tissue repair. For example, kudzu root extract, an isoflavone compound extracted from the roots of the legume Pueraria lobata or Pueraria truncata, has clear pharmacological effects such as anti-inflammatory, antioxidant, osteoclast inhibition, and tissue repair promotion. In the field of oral diseases, puerarin can reduce periodontal tissue inflammation by downregulating the expression of inflammatory factors, and at the same time, it helps regenerate damaged periodontal tissues through anti-oxidative stress and tissue repair promotion. However, most plant extracts (such as puerarin) are highly hydrophobic and have low solubility in water, which limits their application. At the same time, polyphenolic antibacterial drugs have limited effectiveness against intracellular bacteria when used alone.

[0004] Therefore, current antimicrobial peptides and polyphenolic antimicrobial drugs have weak intracellular penetration capabilities, making it difficult for them to effectively cross the host cell membrane and reach the intracellular bacterial colonization site. Their efficiency in recognizing, binding to, and killing intracellular bacteria is low, achieving only limited extracellular antimicrobial effects. They cannot eradicate pathogenic bacteria hidden within the cell at the source, failing to meet the clinical needs for radical treatment of periodontitis. How to effectively inhibit the growth of intracellular bacteria is a significant challenge currently facing the treatment of periodontitis. Summary of the Invention

[0005] The purpose of this invention is to provide a supramolecular complex, its preparation method, and its application.

[0006] The present invention provides a supramolecular complex, which is prepared from puerarin and collagen or their complex as raw materials, wherein the mass ratio of puerarin to collagen or their complex is 1:(0.5~20).

[0007] Furthermore, the mass ratio of puerarin to collagen or a complex thereof is 1:10.

[0008] The present invention also provides a supramolecular complex, characterized in that it is prepared from antimicrobial peptides, puerarin and collagen or a complex thereof as raw materials, wherein the mass ratio of the antimicrobial peptides, puerarin and collagen or a complex thereof is 1:5:(2.5~100).

[0009] Further, the mass ratio of the antimicrobial peptide, puerarin, and collagen or their complex is 1:5:50; the antimicrobial peptide includes at least one of polylysine, hyperbranched polylysine, mussel adhesive protein, nisin, myristoyl hexapeptide-5, and palmitoyl tripeptide-36.

[0010] Furthermore, the collagen includes at least one of animal-derived type I collagen, animal-derived type III collagen, recombinant type I collagen, recombinant type II collagen, recombinant type III collagen, recombinant type IV collagen, recombinant type V collagen, recombinant type 17 collagen, and hydrolyzed collagen.

[0011] This invention provides a method for preparing a supramolecular complex, comprising the following steps: adding puerarin to an aqueous solution of collagen or its complex, homogenizing, obtaining a supramolecular complex solution after the reaction is complete, and obtaining a supramolecular complex solid after drying.

[0012] The present invention also provides a method for preparing a supramolecular complex, comprising the following steps: first, mixing and dissolving an antimicrobial peptide and collagen or their complex in an aqueous solution to obtain a mixed solution; then, adding puerarin to the mixed solution, homogenizing, and obtaining a supramolecular complex solution after the reaction is completed; and finally, drying to obtain a supramolecular complex solid.

[0013] The present invention also provides the use of supramolecular complexes in the preparation of supramolecular complex hydrogels. The preparation method of the supramolecular complex hydrogel includes the following steps: mixing a supramolecular complex solution formed by puerarin and collagen or their complex, or a supramolecular complex solution formed by antimicrobial peptides, puerarin and collagen or their complex, with an aqueous solution of a crosslinking agent, and then self-crosslinking in a mold to obtain a supramolecular complex hydrogel.

[0014] Further, the crosslinking agent includes at least one of EDC / NHS, genipin, glutamin transferase, and glutaraldehyde; the collagen or collagen complex has a mass fraction of 0.2-25 w / v in the hydrogel; and the crosslinking agent has a mass fraction of 15-25 w / v in the hydrogel.

[0015] The present invention also provides a supramolecular complex hydrogel, which is prepared by mixing and reacting a supramolecular complex solution formed by puerarin and collagen or their complex with an aqueous solution of a crosslinking agent.

[0016] Further, the crosslinking agent includes at least one of EDC / NHS, genipin, glutamin transferase, and glutaraldehyde; the mass fraction of the supramolecular complex in the hydrogel is 0.2~25 w / v, and the mass fraction of the crosslinking agent in the hydrogel is 15~25 w / v.

[0017] The present invention also provides a method for preparing a supramolecular complex hydrogel, comprising the following steps: mixing a supramolecular complex solution formed by puerarin and collagen or their complex, or a supramolecular complex solution formed by antimicrobial peptides, puerarin and collagen or their complex, with an aqueous solution of a crosslinking agent, and then self-crosslinking in a mold to obtain a supramolecular complex hydrogel.

[0018] This invention also provides the application of supramolecular complex hydrogels in the preparation of medicaments for treating periodontitis.

[0019] This invention also provides the use of supramolecular complexes in the preparation of medicaments for treating periodontitis.

[0020] The supramolecular complex and its hydrogel of this invention exhibit significantly enhanced permeability, enabling effective penetration into deep gingival epithelial tissues and cells to deliver drugs into the inner tissue layers and cellular interiors, achieving highly efficient clearance of intracellular pathogens. Simultaneously, it possesses excellent antibacterial activity, anti-inflammatory properties, and osteopromoting capacity, effectively treating periodontitis. Compared to existing periodontal drug hydrogel delivery systems, this invention breakthroughly solves the problem of insufficient intracellular bacterial clearance, significantly reducing bone loss and promoting the repair of damaged tissues, making it suitable for precise treatment of periodontal pockets and root bifurcation areas. Furthermore, the complex preparation method of this invention is simple and environmentally friendly, and the materials are inexpensive, providing a new integrated solution for periodontitis treatment with broad application prospects.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 Characterization of PUE / COL and PL-PUE / COL: (A) X-ray diffraction (XRD) spectra of PUE, COL, PUE-COL and PL-PUE-COL; (B) Differential scanning calorimetry (DSC) curves of PUE, COL and PUE / COL.

[0024] Figure 2 A~D represent the dispersion states of different concentrations of PUE immediately after mixing in PBS, COLⅠ, COLⅢ, and COL17, and after standing for 5 minutes, respectively.

[0025] Figure 3 The results of the hydrogelability test are as follows: (A) Images of COL, PL / COL, PUE / COL and PL-PUE / COL before and after gelation; (B) Injectability of PL-PUE / COL hydrogel; (C) Physical flexibility and adhesiveness of PL-PUE / COL hydrogel.

[0026] Figure 4The results of the permeability tests of PUE / COL and PL-PUE / COL are as follows: (A) Permeability of PUE in PBS solution, PUE in DMSO solution, PUE / COL hydrogel and PL-PUE / COL hydrogel to gingival epithelium; (B) Intracellular permeability of PUE in PBS solution, PUE in DMSO solution, PUE / COL hydrogel and PL-PUE / COL hydrogel.

[0027] Figure 5 The results of the intracellular bacterial elimination performance test are as follows: (A) Macrophage phagocytic capacity of the control group, PL / COL group, PUE / COL group, and PL-PUE / COL group; (B) Intracellular bacterial elimination capacity of the control group, PL / COL group, PUE / COL group, and PL-PUE / COL group.

[0028] Figure 6 The results of the biocompatibility and antibacterial properties experiments are as follows: (A) Cell live and dead staining images of COL, PL2 / COL, PL4 / COL, PUE / COL, and PL2-PUE / COL hydrogels (calcine-AM labeled live cells, PI labeled dead cells); (B) Bacterial live and dead staining images of COL, PL2 / COL, PL4 / COL, PUE / COL, and PL2-PUE / COL hydrogels (NucGreen labeled live bacteria, EthD-III labeled dead bacteria); (C) Results of the antibacterial plating experiment of COL, PL2 / COL, PL4 / COL, PUE / COL, and PL2-PUE / COL hydrogels; (D) Statistical bar chart of antibacterial efficiency of COL, PL2 / COL, PL4 / COL, PUE / COL, and PL2-PUE / COL hydrogels.

[0029] Figure 7 The results of the anti-inflammatory performance test are as follows: (A) ROS fluorescence staining images of the control group, LPS group, LPS+PL / COL group, LPS+PUE / COL group, and LPS+PL-PUE / COL group; (B) CD206 immunofluorescence staining images of the control group, LPS group, LPS+PL / COL group, LPS+PUE / COL group, and LPS+PL-PUE / COL group; (C) iNOS immunofluorescence staining images of the control group, LPS group, LPS+PL / COL group, LPS+PUE / COL group, and LPS+PL-PUE / COL group.

[0030] Figure 8The results of the osteogenic performance test are as follows: (A) ALP staining images of the control group, PL / COL group, PUE / COL group, and PL-PUE / COL group; (B) Immunofluorescence staining images of ALP-labeled cells of the control group, PL / COL group, PUE / COL group, and PL-PUE / COL group.

[0031] Figure 9 The in vivo application effects in the periodontitis model are as follows: (A) Three-dimensional reconstruction images of periodontal tissues in the control group, periodontitis model group, and PL-PUE / COL group; (B) Micro CT tomographic images of periodontal tissues in the control group, periodontitis model group, and PL-PUE / COL group; (C) Statistical diagrams of bone volume fraction in the mesial and distal regions of the control group, periodontitis model group, and PL-PUE / COL group; (D) Statistical diagrams of the distance between the cementoenamel junction and alveolar bone top (CEJ-ABC) in the mesial and distal regions of the control group, periodontitis model group, and PL-PUE / COL group. Detailed Implementation

[0032] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0033] Example 1: Preparation and characterization of puerarin / collagen (PUE / COL) supramolecular complex, polylysine-puerarin / collagen (PL-PUE / COL) supramolecular complex and their hydrogels.

[0034] 1. Preparation of PUE / COL supramolecular complex and its hydrogel

[0035] (1) Prepare a collagen aqueous solution with a concentration of 20 w / v% (w / v% is the mass percentage concentration, g / ml%);

[0036] (2) Puerarin (PUE) was added to collagen aqueous solution at a mass ratio of 1:10 and then homogenized at 37°C and 200 rpm for 1 h to obtain PUE / COL supramolecular complex solution.

[0037] (3) Prepare a 40 mg / mL transglutaminase (MTG) aqueous solution and homogenize and stir until well mixed;

[0038] (4) Mix the PUE / COL supramolecular complex solution and MTG aqueous solution in equal volumes, stir quickly to mix evenly, inject into the mold, and self-crosslink at 37℃ for 20 min to obtain PUE / COL supramolecular complex hydrogel.

[0039] 2. Preparation of PL-PUE / COL supramolecular complex and its hydrogel

[0040] (1) Polylysine (PL), puerarin, and collagen were first mixed and dissolved in an aqueous solution at a mass ratio of 1:5:50 to obtain a PL / COL solution. Then, puerarin was added to the PL / COL solution and homogenized at 37°C and 200 rpm for 1 h to obtain a PL-PUE / COL supramolecular complex solution. The concentration of collagen in the PL-PUE / COL supramolecular complex solution was 20 w / v%.

[0041] (2) Prepare a 40 mg / mL aqueous solution of transglutaminase (MTG) and homogenize and stir until well mixed;

[0042] (3) Mix the PL-PUE / COL supramolecular complex solution and MTG aqueous solution in equal volumes, stir quickly to mix evenly, inject into the mold, and self-crosslink at 37℃ for 20 min to obtain PL-PUE / COL supramolecular complex hydrogel.

[0043] 3. Characterization of PUE / COL and PL-PUE / COL supramolecular complexes

[0044] The PUE / COL and PL-PUE / COL supramolecular complex solutions were freeze-dried to remove the solvent, and then the PUE / COL and PL-PUE / COL supramolecular complex solids were characterized.

[0045] The formation of the supramolecular system was characterized by XRD and differential scanning calorimetry (DSC).

[0046] like Figure 1 The XRD pattern shown in Figure A indicates that pure PUE exhibits a series of sharp, high-intensity diffraction peaks at 2θ ranging from 10° to 55°, suggesting that pure PUE is a highly crystalline substance. After forming a supramolecular structure with COL and PL, the characteristic crystalline peaks of PUE / COL and PL-PUE / COL disappear or their intensity significantly decreases, forming an amorphous morphology consistent with COL. This indicates a strong non-covalent interaction between PUE and COL. Figure 1 The DSC results of B showed that COL exhibited an endothermic valley at approximately 61°C, corresponding to the denaturation temperature of collagen triple helix unwinding. Pure PUE did not show a significant endothermic valley, while the endothermic valley of the PUE / COL supramolecular complex formed by the two was significantly shifted towards higher temperatures, indicating that the supramolecular interaction between PUE / COL significantly improved the thermal stability of COL.

[0047] Characterization results demonstrate that puerarin and collagen successfully formed a supramolecular structure, and that supramolecular interactions significantly improved the thermal stability of collagen.

[0048] Comparative Example 1: Preparation of COL Hydrogel

[0049] The COL solution from Example 1 was mixed with an equal volume of 40 mg / mL MTG aqueous solution, stirred rapidly until homogeneous, injected into a mold, and self-crosslinked at 37°C for 20 min to obtain COL hydrogel.

[0050] Comparative Example 2: Preparation of PL / COL hydrogel

[0051] The PL / COL solution from Example 1 was mixed with an equal volume of 40 mg / mL MTG aqueous solution, stirred rapidly to mix thoroughly, injected into a mold, and self-crosslinked at 37°C for 20 min to obtain PL / COL hydrogel.

[0052] The collagen used in Example 1 and Comparative Example 1 was type 1 collagen. The concentration of COL in COL, PL / COL, PUE / COL and PL-PUE / COL hydrogels was 10 w / v%, and the concentration of MTG was 20 mg / mL.

[0053] Experiment Example 1: Solubility, Hydrogelability, and Stability Experiments

[0054] 1. Experimental Methods

[0055] (1) ① Dissolve different concentrations of PUE (2, 4, 6, 8, 10 mg / mL) in 100 mg / mL PBS respectively. Figure 2 A) and 100 mg / mL COLⅠ (type 1 collagen)-PBS solution ( Figure 2 B), ② Dissolve different concentrations of PUE (4, 6, 8, 10 mg / mL) in 100 mg / mL COLⅢ (type 3 collagen), Figure 2 C) and 100 mg / mL COL17-PBS (type 17 collagen, Figure 2 D), observe the condition of different collagens after mixing with PUE, and take photos to record the state of the solution immediately after mixing and after standing for 5 minutes.

[0056] (2) Take 1 mL of COL, PL / COL, PUE / COL and PL-PUE / COL solutions respectively, add 20 mg / mL MTG aqueous solution to them, and take pictures to record the gelation of the system.

[0057] 2. Experimental Results

[0058] like Figure 2 As shown in A~D, PUE is more soluble in COL solution than in PBS solution, indicating that PUE can be effectively solubilized in COL solution; among them, PUE is more soluble in COLⅠ.

[0059] like Figure 3As shown, PUE / COL and PL-PUE / COL have good gelling effects, among which PL-PUE / COL hydrogel has excellent injectability, physical flexibility and adhesion.

[0060] The above experimental results show that PUE can be effectively solubilized in COL solution and forms an injectable hydrogel with localized adhesion properties under the action of cross-linking agent.

[0061] Experimental Example 2: Permeability Tests of PUE / COL and PL-PUE / COL

[0062] 1. Experimental Methods

[0063] PUE was labeled with Cy3 fluorescent dye, and PUE-PBS suspension, PUE-DMSO (10% DMSO) solution, PUE / COL solution and PL-PUE / COL solution with PUE concentration of 10 mg / mL were prepared respectively: (1) 20 μL of the above system was evenly spread on the surface of the gingival epithelium of the mandibular teeth of pigs. After 1 hour, it was fixed and frozen sectioned. The cell nuclei were labeled with DAPI nuclear dye and the sections were observed under a fluorescence microscope; (2) The above system was added to the culture medium to make the final concentration of PUE 20 μg / mL. It was co-cultured with RAW264.7 macrophages at 37°C. After 1 hour, the cells were labeled with DIO membrane dye and Hoechst33342 nuclear dye and the entry of PUE into the cell was observed under a fluorescence microscope.

[0064] 2. Experimental Results

[0065] like Figure 4 As shown in Figure A, although PUE-DMSO solved the solubility problem of PUE-PBS, it still could not effectively penetrate into the gingival epithelium and deep lamina propria. However, after PUE / COL and PL-PUE / COL formed supramolecular structures, they effectively promoted the penetration of PUE into the deep tissues of the gingival epithelium.

[0066] like Figure 4 As shown in Figure B, the results of co-culturing with RAW264.7 macrophages showed that almost no PUE entered the cells in the PUE-PBS and PUE-DMSO groups, while the amount of PUE / COL and PL-PUE / COL entering the cells increased significantly after forming supramolecular structures.

[0067] Experimental results show that PUE / COL and PL-PUE / COL can form supramolecular structures, significantly improving their penetration into deep gingival epithelial tissues and cells, delivering PUE and PL into the inner layers of tissues and cells to achieve the purpose of clearing intracellular bacteria.

[0068] Experimental Example 3: Intracellular bacterial clearance ability of PUE / COL and PL-PUE / COL supramolecular complex hydrogels

[0069] 1. Experimental Methods

[0070] (1) Hydrogels of each group (PL / COL, PUE / COL, PL-PUE / COL hydrogels) were immersed in DMEM medium to prepare hydrogel extracts (1g hydrogel was immersed in 10mL of medium). After incubation at 37℃ for 24h, the extracts were collected. Then, RAW264.7 macrophages were cultured in different hydrogel extracts for 24h, and FITC-labeled polystyrene microspheres with a diameter of 100nm were added. 10 7 Cells / well were cultured for 3 hours. After DiI staining of the cell membrane, the clearance of intracellular bacteria was observed under a fluorescence microscope. Experimental groups: control group, PL-COL group, PUE-COL group, PL-PUE / COL group.

[0071] (2) Take P. gingivalis in the logarithmic midphase, label it with FITC (0.15 mg / mL, 30 min in the dark), and infect RAW264.7 macrophages (MOI=100, 3h). First, use gentamicin (500 μg / mL) and metronidazole (200 μg / mL) to clear extracellular bacteria, and then treat each group with hydrogel extract (same as (1)) for 3h. After DiI staining of the cell membrane, observe the clearance of intracellular bacteria under a fluorescence microscope.

[0072] 2. Experimental Results

[0073] like Figure 5 As shown in Figure A, the PL-PUE / COL group exhibits stronger phagocytic uptake capacity; as... Figure 5 As shown in Figure B, in RAW264.7 macrophages infected with P. gingivalis, the intracellular bacterial load in the PL-PUE / COL group was significantly lower than that in other groups, confirming that the PL-PUE / COL hydrogel can effectively kill intracellular bacteria.

[0074] Experimental Example 4: Cytotoxicity and antibacterial properties of PUE-COL and PL-PUE-COL supramolecular complex hydrogels

[0075] 1. Experimental Methods

[0076] (1) The effects of the control group, COL group, PL2 / COL group (PL concentration 2 mg / mL), PL4 / COL group (PL concentration 4 mg / mL), PUE / COL group and PL2-PUE / COL group (PL concentration 2 mg / mL) on the activity of RAW264.7 macrophages were detected by using a cell live and dead staining kit: RAW264.7 were directly co-cultured on the surface of each group of hydrogel (48-well plate, 200 μL hydrogel / well) and cultured at 37℃ for 24 h. The green fluorescence emitted by live cells was observed under an inverted fluorescence microscope at an excitation wavelength of 490 nm, and the red fluorescence emitted by dead cells was observed under an excitation wavelength of 545 nm.

[0077] (2) The antibacterial effect of the hydrogel was evaluated by live / dead bacteria staining and plating test: Porphyromonas gingivalis (ATCC 33277, 1×10⁻⁶) was stained with live / dead bacteria and plated. 6 CFU / mL) was co-incubated with the control group, COL group, PL2 / COL group, PL4 / COL group, PUE / COL group, and PL2-PUE / COL group (200 μL / group) at 37℃ for 72 h. After SYTO9 / propidium iodide staining, the ratio of live bacteria (green) to dead bacteria (red) was observed under a fluorescence microscope. In the plating experiment, 10 4 The diluted bacterial suspension was evenly spread on BHI blood agar plates, anaerobically incubated at 37°C for 5 days, photographed, and the antibacterial efficiency was calculated based on the colony count.

[0078] 2. Experimental Results

[0079] like Figure 6 As shown in Figure A, cells exhibited green fluorescence on the hydrogel surfaces of the COL, PL2 / COL, PUE / COL, and PL2-PUE / COL groups, with almost no red fluorescence, showing no significant difference from the control group. However, the PL4 / COL group showed more red fluorescence and less green fluorescence than the other groups, indicating that high concentrations of PL affect cell viability.

[0080] like Figure 6 As shown in B~D, both the PL4 / COL group and the PL2-PUE / COL group have high antibacterial activity, which is significantly higher than that of the PL2 / COL group and the PUE / COL group, indicating that the composite synergy of PL and PUE improves the antibacterial efficiency of the hydrogel.

[0081] Experimental Example 5: Anti-inflammatory ability of PUE-COL and PL-PUE-COL supramolecular complex hydrogels

[0082] 1. Experimental Methods

[0083] (1) Anti-inflammatory experiment: RAW264.7 macrophages were cultured for 24 h and then stimulated with 100 ng / mL Escherichia coli lipopolysaccharide (LPS) for 12 h. Then, hydrogel extract (same as in Experiment 3) was added and cultured for another 24 h: ① The intracellular ROS production was observed using the DCFH fluorescent probe; ② After the samples were fixed, immunofluorescence staining was performed, and the expression of macrophage polarization markers CD206 and iNOS (green fluorescence) and cell nuclei (blue fluorescence) were observed under a fluorescence microscope.

[0084] 2. Experimental Results

[0085] Anti-inflammatory test results showed that, compared with the LPS and PL / COL groups, the PUE-COL group and the PL-PUE / COL group significantly reduced the amount of ROS produced after LPS stimulation. Figure 7 A) and the expression levels of inflammatory markers CD206 and iNOS ( Figure 7 (B~C), especially the PL-PUE / COL group.

[0086] Experimental Example 6: Osteogenesis Capacity of PUE-COL and PL-PUE / COL Supramolecular Complex Hydrogels

[0087] 1. Experimental Methods

[0088] Mouse osteoblast precursor cell line MC3T3-E1 was treated with different hydrogel extracts (same as in Experiment 3). The expression level of alkaline phosphatase (ALP) in osteoblasts was observed by ALP staining and cell immunofluorescence staining (ALP labeling).

[0089] 2. Experimental Results

[0090] like Figure 8 As shown in A, the PL-PUE / COL group produces more ALP; Figure 8 B showed that the PL-PUE / COL group had higher ALP protein expression levels in osteoblasts. The experimental results indicate that the PL-PUE / COL supramolecular complex hydrogel can promote ALP production, increase ALP protein expression levels in osteoblasts, and significantly enhance osteogenic capacity.

[0091] Experiment Example 7: Animal Model Therapeutic Effect Verification Experiment

[0092] 1. Experimental Methods

[0093] Male C57 mice were randomly divided into three groups: control group (no modeling); periodontitis model group (periodontitis model + PBS treatment); and PL-PUE / COL hydrogel group (periodontitis model + PL-PUE / COL hydrogel). Periodontitis model construction: Under anesthesia, 5-0 silk sutures were used to ligate the maxillary second molar (M2) subgingivally to induce periodontal tissue inflammation. After 14 days of treatment with PUE-PL / COL hydrogel (20 μL / treatment), the mouse periodontitis model underwent micro-CT testing.

[0094] 2. Experimental Results

[0095] like Figure 9 As shown, compared with the positive control group, the PUE-PL / COL hydrogel group showed a significant reduction in mesial and distal alveolar bone loss (CEJ-ABC distance) and a significant increase in bone volume fraction (BV / TV). The experimental results indicate that PUE-PL / COL hydrogel can effectively treat periodontitis.

[0096] In summary, this invention successfully prepared supramolecular complexes of puerarin / collagen and polylysine-puerarin / collagen, and their hydrogels. The supramolecular interactions significantly improved the thermal stability of collagen. Specifically, the supramolecular complexes and hydrogels of this invention exhibit significantly enhanced permeability, effectively penetrating deep gingival epithelial tissues and cells, delivering PUE and PL into the inner layers of tissues and cells to eliminate intracellular bacteria. Simultaneously, cell experiments confirmed that PUE / COL and PL-PUE / COL hydrogels can effectively kill intracellular bacteria, with PL-PUE / COL hydrogel exhibiting the best phagocytic uptake capacity and the best intracellular bacteria clearance capacity. Furthermore, PUE / COL and PL-PUE / COL hydrogels possess excellent antibacterial activity, anti-inflammatory properties, and osteogenic properties; PL-PUE / COL hydrogel demonstrated good therapeutic effects in the treatment of periodontitis.

Claims

1. The application of a supramolecular complex in the preparation of a drug for treating periodontitis, wherein the drug for treating periodontitis is a drug for use on the gingiva to fight intracellular bacteria in the gingiva; the supramolecular complex is prepared from antimicrobial peptides, puerarin, and type I collagen as raw materials; the molecular weight ratio of the antimicrobial peptides, puerarin, and type I collagen is 1:5:50; the antimicrobial peptide is polylysine; The preparation method of the supramolecular complex includes the following steps: First, antimicrobial peptides and type I collagen are mixed and dissolved in an aqueous solution to obtain a mixed solution. Then, puerarin is added to the mixed solution, homogenized, and after the reaction is complete, a supramolecular complex solution is obtained. After drying, a supramolecular complex solid is obtained. The concentration of type I collagen in the supramolecular complex solution was 20 w / v.

2. The application of a supramolecular complex hydrogel in the preparation of a drug for treating periodontitis, wherein the drug for treating periodontitis is a drug applied to the gingiva to combat intracellular bacteria of the gingiva; the preparation method of the supramolecular complex hydrogel includes the following steps: First, antimicrobial peptides and type I collagen are mixed and dissolved in an aqueous solution to obtain a mixed solution. Then, puerarin is added to the mixed solution, homogenized, and after the reaction is complete, a supramolecular complex solution is obtained. Subsequently, the supramolecular complex solution is mixed evenly with an aqueous crosslinking agent solution, and self-crosslinked in a mold to obtain a supramolecular complex hydrogel. The mass ratio of the antimicrobial peptide, puerarin, and type I collagen is 1:5:

50. The antimicrobial peptide is polylysine. The concentration of type I collagen in the supramolecular complex hydrogel is 10 w / v.

3. The application according to claim 2, characterized in that, The cross-linking agent is transglutaminase.

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