Tallotannic acid / Cu2 + / minocycline compound and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a tannic acid / Cu < 2 + > / minocycline compound and application thereof. The preparation method of the compound comprises the following steps: complexing tannic acid (TA) and Cu < 2 + >, and loading minocycline (MH) by taking the complex as a carrier. The TA-Cu / MH prepared by the preparation method disclosed by the invention has multiple effects of resisting bacteria, resisting oxidation, resisting inflammation and the like, and also has good safety and a certain effect of promoting tissue regeneration. The research provides an experimental basis for application of a multifunctional drug in periodontitis treatment, and is beneficial to promoting precise drug treatment of periodontitis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a tannic acid / Cu 2+ / Minocycline complex and its use. Background Art

[0002] Periodontitis is a chronic inflammatory disease that can cause destruction of periodontal supporting tissues. Its pathological process begins with the progressive spread of gingival inflammation to the periodontal ligament and alveolar bone tissue, triggering irreversible bone resorption and ultimately leading to tooth loss. Periodontal disease is a disease involving multiple pathogenic factors and promoting factors. The main goal of periodontal treatment is to eliminate inflammation while promoting the recovery and regeneration of periodontal tissues, thereby restoring the physiological function of periodontal tissues. At present, the clinical treatment strategy for periodontitis is centered on the elimination of pathogenic microorganisms. Studies have confirmed that subgingival mechanical debridement combined with drug therapy can enable patients to obtain better clinical benefits by inhibiting the release of pro-inflammatory mediators. However, this treatment method still has disadvantages. First, the current drug treatment mainly relies on antibiotics, but antibiotics are usually associated with drug resistance and are prone to cause dysbiosis. Minocycline hydrochloride (MH), the first-line clinical drug for periodontitis, undergoes characteristic evolution of the oral flora 6 days after local application, with an increase in tetracycline-resistant bacteria. Among them, the drug-resistant phenotype of Streptococcus (such as Streptococcus mitis and Streptococcus sanguinis) is positively correlated with the risk of cardiovascular disease. Moreover, the oral biofilm microenvironment accelerates the horizontal transfer of drug-resistant genes (such as cyclization / conjugation transfer), promoting the evolution and spread of drug-resistant strains.

[0003] In order to circumvent the development of subgingival bacterial resistance caused by traditional antibiotics and achieve multi-target regulation such as anti-inflammatory / antioxidant / osteogenic, the present invention aims to provide a drug for treating periodontitis that retains the antibacterial effect of antibiotics while improving their tendency to develop drug resistance, and has multi-target therapeutic effects of anti-inflammatory, antioxidant, and osteogenic effects. Summary of the Invention

[0004] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to avoid the development of subgingival bacterial resistance caused by traditional antibiotics and achieve multi-target regulation such as anti-inflammatory / antioxidant / osteogenic effects.

[0005] To achieve the above objectives, the present invention provides a composite having multi-target therapeutic effects of anti-inflammatory, anti-oxidation, and osteogenesis, the preparation of which comprises the following steps: Tannic acid (TA) and Cu 2+ The complex is then used as a carrier to load minocycline (MH).

[0006] Preferably, tannic acid (TA) is mixed with Cu 2+The complexation was carried out by thoroughly mixing tannic acid (TA) and CuSO4·5H2O in deionized water.

[0007] Preferably, the thorough mixing is achieved by magnetic stirring for 12 h to 36 h.

[0008] Preferably, the step of loading minocycline is to add MH to the complex to react to obtain a reaction solution, and then collect the precipitate by centrifugation to obtain a complex.

[0009] Preferably, the preparation of the complex further comprises the steps of washing and drying the complex precipitate.

[0010] Preferably, the final concentration of TA is 2 mg / ml.

[0011] Preferably, the final concentration of CuSO4·5H2O is 0.05-0.6 mg / ml.

[0012] Preferably, the final concentration of MH is 0.2-1.2 mg / ml.

[0013] Preferably, the final concentration of CuSO4·5H2O is 0.05-1 mg / ml, 0.1-0.2 mg / ml, 0.2-0.3 mg / ml, 0.3-0.4 mg / ml, 0.4-0.5 mg / ml and / or 0.5-0.6 mg / ml, 0.6 mg / ml.

[0014] Preferably, the final concentration of MH is 0.2-0.4 mg / ml, 0.4-0.6 mg / ml, 0.6-0.8 mg / ml, 0.8-1 mg / ml and / or 1-1.2 mg / ml.

[0015] Preferably, the final concentration of CuSO4·5H2O is 0.5 mg / ml.

[0016] Preferably, the final concentration of MH is 0.6 mg / ml.

[0017] Preferably, the centrifugal speed is 5000-30000 rpm, and the centrifugal time is 10-30 min.

[0018] Preferably, the centrifugal speed is 20000 rpm, and the centrifugal time is 15 min.

[0019] In a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition comprising the complex of the present invention and a pharmaceutically acceptable carrier.

[0020] In another preferred embodiment of the present invention, the present invention provides use of the complex of the present invention and / or the pharmaceutical composition of the present invention in the preparation of a medicament for treating periodontitis.

[0021] This study innovatively designed and constructed a multifunctional complex, TA-Cu / MH, for the treatment of periodontitis. A series of experiments demonstrated that TA-Cu / MH exhibited multiple antibacterial, antioxidant, and anti-inflammatory properties, along with good safety and a moderate tissue regeneration effect. This study provides an experimental basis for the application of multifunctional drugs in the treatment of periodontitis and will help promote precision medicine treatments for periodontitis. Future studies could further explore the mechanism of action of TA-Cu / MH and its application in advanced animal models to promote its clinical translation.

[0022] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of particle size change of six groups of products within 30 days; Figure 2 TA-Cu / MH was quantified using UV spectrophotometry and inductively coupled plasma emission spectrometry. (A) is the UV spectra of TA and MH, (B) is the UV spectra of TA-Cu / MH in sulfuric acid at pH 2, (C) is the standard curve of TA at 278 nm and the standard curve of MH at 278 nm and 351 nm, and (D) is the amount of TA and MH obtained by UV quantification, and the amount of Cu measured by ICP. 2+ the amount; Figure 3 The SEM (A) and TEM (B) images of TA-Cu (left) and TA-Cu / MH (right) are shown. Figure 4 The EDS images of TA-Cu / MH are shown in Figure 2. (A) shows the EDS mapping of TA-Cu and the semi-quantitative analysis of the element ratios in TA-Cu. (B) shows the EDS mapping of TA-Cu and MH and the semi-quantitative analysis of the element ratios in TA-Cu / MH. Figure 5 It is the full XPS spectrum of TA, TA-Cu, and TA-Cu / MH; Figure 6 The X-ray photoelectron spectroscopy results of Cu / MH, TA, and TA-Cu, (A) is the O element peak of each group, (B) is the N element peak of each group, and (C) is the Cu element peak of each group; Figure 7are the Fourier transform infrared spectra results of TA-Cu / MH and TA and MH; Figure 8 The antibacterial results of TA-Cu / MH, (A) is the MBC measured by the coating plate method, (B) is the antibacterial OD value of gradient concentration; Figure 9 TA-Cu / MH inhibits bacterial adhesion - anti Pg and Aa Adhesion electron micrograph; Figure 10 The inhibitory effect of TA-Cu / MH on biofilm formation is shown in Figure 1. (A) is the CLSM image of the biofilm slide after live and dead staining. (B) is the crystal violet staining image and the semi-quantitative analysis of the biofilm after dissolution. Figure 11 TA-Cu / MH destroys bacterial cell membranes Pg and Aa Electron micrograph of cell membrane; Figure 12 TA-Cu / MH destroys the formed biofilm. (A) is the CLSM image of the biofilm slide after live and dead staining. (B) is the photograph of the biofilm slide after crystal violet staining and semi-quantitative analysis after dissolution. Figure 13 Is a single Cu 2+ Figure 1 shows the synergistic antibacterial effect of MH with MH; Figure 14 is the biocompatibility result diagram of TA-Cu / MH gradient concentration; Figure 15 This is the result diagram of the effect of TA-Cu / MH on the proliferation of BMSCs and L929 cells; Figure 16 This is the result diagram of the effect of the composite TA-Cu / MH on the morphology of BMSCs; Figure 17 Figure 1 is a diagram of the free radical scavenging effect of the complex TA-Cu / MH in vitro, (A) is the DPPH free radical scavenging effect of the complex, (B) is the ABTS cation free radical scavenging effect of the complex, and (C) is the FRAP total antioxidant capacity evaluation of the complex; Figure 18 Figure 1 is the antioxidant effect diagram of the TA-Cu / MH complex. (A) is the image of ROS levels in BMSCs cells after treatment with different drugs. (B) is the image of ROS levels in L929 cells after treatment with different drugs. (C) is the statistical analysis of the intracellular fluorescence intensity levels. Figure 19 Flow cytometry was used to detect the ROS levels in RAW264.7 cells after pretreatment with different drugs; Figure 20is a graph of the anti-inflammatory effect of the complex TA-Cu / MH, showing the gene expression levels of inflammatory factors (IL-1β, IL-6, TNF-α) in macrophages and the secretion levels of cytokine proteins (IL-1β, IL-6, TNF-α) in the supernatant of macrophages; Figure 21 Figure 1 is a diagram of the osteogenesis-promoting effect of the TA-Cu / MH composite. (A) is a photograph of BMSCs cells stained with ALP and Alizarin Red, and (B) is the ALP specific activity and calcium nodule quantification. Figure 22 Figure 1 shows the results of the TA-Cu / MH complex inhibiting alveolar bone resorption in rats with periodontitis. (A) is an intraoral image of a rat model with periodontitis. (B) is a schematic diagram of the CEJ-ABC distance. (C) is a Micro-CT image of the alveolar bone around the maxillary second molar four weeks after treatment: the black background is a sagittal Micro-CT image; the blue background is a three-dimensional Micro-CT reconstruction. (D) is a quantitative analysis of CEJ-ABC. (E) is a Micro-CT analysis of the BV / TV, Tb.Sp, Tb.N, and Tb.Th values ​​of rats in each group. Figure 23 are the H&E and Masson staining images of the periodontal tissues of rats in each group after four weeks of treatment; Figure 24 is the PCR result of gingival tissue homogenate of rats in each group after four weeks of treatment; Figure 25 is the ROS level in the periodontal tissue of rats in each group after four weeks of treatment; Figure 26 These are the H&E staining images of the heart, liver, spleen, lung and kidney of rats in each group after four weeks of treatment. DETAILED DESCRIPTION

[0024] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0025] Example 1 Construction and characterization of the composite (TA-Cu / MH) 1. Materials and Methods Experimental materials and equipment

[0026] 2. Experimental Methods Synthesis of the TA-Cu complex: The self-assembling TA-Cu complex was synthesized using a liquid-phase synthesis method at room temperature. TA (calculated to a final concentration of 2 mg / ml) was completely dissolved in deionized water and thoroughly mixed with CuSO₄·5H₂O (dissolved in deionized water) to a final concentration of 0.5 mg / ml. The mixture was allowed to react on a magnetic stirrer for 12 hours, followed by centrifugation at 20,000 rpm for 15 minutes to obtain a precipitate. The precipitate was then washed three times with deionized water and dried in a vacuum dryer to obtain a composite powder.

[0027] Synthesis of the TA-Cu / MH complex: TA-Cu / MH was synthesized using a liquid-phase synthesis method at room temperature. TA (calculated to a final concentration of 2 mg / ml) was completely dissolved in deionized water and thoroughly mixed with CuSO₄·5H₂O (dissolved in deionized water) to a final concentration of 0.5 mg / ml. The mixture was allowed to react on a magnetic stirrer for 12 hours, followed by the addition of MH at a concentration of 0.6 mg / ml. After 4 hours of reaction, the reaction solution was collected and centrifuged at 20,000 rpm for 15 minutes to obtain a precipitate. The precipitate was washed three times with deionized water and dried in a vacuum dryer to obtain a composite powder.

[0028] Example 2 Investigation of the distribution ratio of each component of the composite TA-Cu / MH During the preparation process, TA was set at 2 mg / ml, and the ratio of copper sulfate pentahydrate and MH was adjusted to make the concentration gradient of CuSO4·5H2O 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, and 0.6 mg / ml, and the concentration gradient of MH 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, and 1.2 mg / ml. After obtaining the precipitate, the yield of each group was measured, and the yield was calculated and the six groups with higher yields were screened out; The six groups of complexes selected above were prepared in the same manner, and the complexes were diluted to the same concentration and the particle size changes of each group were measured within one month. The above six groups of complex precipitates were dissolved in sulfuric acid solution with pH = 2. A portion of the dissolved liquid was diluted and used for ICP-OES detection to measure the Cu content of each group of solutions. 2+Concentration; dilute a portion and measure the absorbance of the complex at 278 nm and 351 nm using a UV spectrophotometer. Simultaneously, a standard curve for TA at 278 nm and for MH at 278 nm and 351 nm is generated by serially diluting TA and MH. The resulting wavelength at 351 nm is the MH content in the complex. The absorbance at 278 nm minus the absorbance at 351 nm is the absorbance after dilution of the TA content in the complex. This is used to calculate the content of each component in the complex, the MH entrapment efficiency, and the drug loading rate.

[0029] MH embedding rate (%) = (MH content in TA-Cu / MH / total MH dosage) × 100% MH drug loading (%) = (MH amount contained in TA-Cu / MH / total mass of TA-Cu / MH) × 100% From the above operations, a group with higher yield, more stable particle size, higher MH encapsulation rate and drug loading rate was selected for subsequent characterization and biological evaluation.

[0030] The liquid phase synthesis method was used to prepare the complex by systematically adjusting the concentration ratio of copper sulfate pentahydrate (CuSO4·5H2O) and minocycline (MH). The complex precipitate was obtained after centrifugation and drying. The calculated yields of each group are shown in Table 1.

[0031] In order to reduce production costs and increase the possibility of clinical transformation of the complex, six high-yield combinations of Cu0.4MH0.6, Cu0.4MH0.8, Cu0.4MH1, Cu0.5MH0.6, Cu0.5MH0.8, and Cu0.5MH1 were selected for subsequent optimization of the complex ratio.

[0032] Table 1 Yield of TA-Cu / MH after concentration adjustment

[0033] To study different Cu 2+ The effect of the ratio of MH on the long-term stability of the complex was investigated. The six complex systems screened were prepared at 1 mg / mL and diluted 10 times. The particle size changes were monitored by dynamic light scattering at 1, 5, 10, 15, 20, and 30 days. Figure 1 Shown: Cu 0.4 The average particle size of the three groups was 77-102 nm in the initial stage (1 day); as the storage time increased, the particle size increased significantly, reaching 434-770 nm at 30 days (an increase of 463-654%), indicating that the complex had undergone significant aggregation. 0.5The initial particle size of the three groups was similar to that of the low concentration group (79-95nm); within 30 days, the particle size only increased to 87-210nm (increase rate 1-121%), showing good colloidal stability. 2+ Concentration is a key factor in regulating the long-term stability of the complex, and 0.5 mg / ml Cu 2+ The group maintained a nanoscale dispersion state (<200 nm) within 30 days, meeting the requirements of biomedical materials for colloidal stability and providing a pharmaceutical basis for subsequent in vitro and in vivo experiments.

[0034] Subsequently, the UV spectrophotometer was used to quantify TA and MH. First, the UV spectra of TA and MH after dissolving in sulfuric acid with pH = 2 were measured ( Figure 2 A), it can be seen that MH has an absorption peak at a wavelength of 351nm, while TA has no absorption at this wavelength; TA has an absorption peak at a wavelength of 278nm, at which time the absorption value of MH exists; therefore, TA and MH are dissolved in sulfuric acid with a pH of 2 and diluted according to a concentration gradient, and the standard curve of TA at 278nm and the standard curve of MH at 351nm and 278nm are measured ( Figure 2 C). At the same time, the complex was dissolved in concentrated sulfuric acid at pH = 2, and its UV spectrum was measured after dilution according to the concentration gradient. Figure 2 B. At the same time, we prepared each complex into a 1 mg / ml solution according to the yield, dissolved it in concentrated sulfuric acid at pH = 2, and measured the absorbance of each complex at 278 nm and 351 nm.

[0035] Substitute the measured absorbance value into Figure 2 The amount of TA and MH in each group was calculated from the standard curve of each group, and the amount of Cu in each group was measured by ICP-OES. 2+ The concentration of each component is as follows Figure 2 As shown in Figure D, with the increase of copper ion concentration in the reaction solution, the drug loading rate of the complex to TA decreased, while the drug loading rate to MH increased; at the same time, with the increase of MH concentration, the concentration of TA changed little, and Cu 2+ The concentration began to decrease.

[0036] The encapsulation efficiency of MH in each group was calculated based on the amount of each component (Table 2). 0.5 MH 0.6 The encapsulation efficiency of the complex reaches its maximum when , and the drug loading rate is also relatively high at this time.

[0037] Table 2 MH encapsulation efficiency

[0038] Example 3 Structural Characterization The structures of MH and its complexes (TA-Cu, TA-Cu / MH) were characterized by Fourier transform infrared spectroscopy (ATR mode). An appropriate amount of sample was mixed with potassium bromide and ground, and then pressed into pellets. The test wavenumber range was set to 500-4000 cm -1 , the spectral resolution remains at 1cm -1 . The surface of the material was subjected to elemental analysis by X-ray photoelectron spectrometer: 100eV was used to complete a wide-range full spectrum scan of 0-1400eV, and the surface element distribution data was recorded; 30eV was selected to perform high-resolution scanning of each element, and the obtained spectrum was analyzed by peak fitting using Avantage analysis software to determine the element valence information; all spectral lines were calibrated with the C1s characteristic peak of 284.8eV as the benchmark. In terms of morphological characterization, the freeze-dried composite material was dispersed in deionized water, and the suspension was dropped on a silicon wafer. After complete drying, it was sprayed with gold for 30 seconds, and the surface micromorphology of the sample was observed using a scanning electron microscope; another drop of the dispersion was added to a copper mesh, and the morphology of the composite was collected by transmission electron microscopy, and the elemental composition and distribution of the selected area were quantitatively determined by combining energy spectrum analysis technology.

[0039] The result is as follows: like Figure 3 As shown in B, the appearance of TA-Cu / MH is a lot of dispersed particles, most of which are round or nearly round. The particle diameter is about 50 to 100 nanometers. Figure 3 The TA-Cu complex in A is distributed relatively evenly, but some particles are interconnected to form chains or small aggregates. This may be due to the adhesion of polyphenols, which makes the complex present a network cross-linked morphology.

[0040] like Figure 4 As shown, the atomic number contrast (Z-contrast) of TA-Cu / MH particles is significantly lower than that of TA-Cu, which is consistent with the local enrichment of Cu element in EDS Mapping ( Figure 4 A), further verified the effect of MH modification on Cu 2+ Regulatory effect of coordination structure. EDS Mapping shows that the four elements C, N, O, and Cu in the TA-Cu / MH complex are uniformly dispersed in space, indicating that the components are combined through chemical reactions. Semi-quantitative analysis of the selected area by EDS point scanning shows that the C / N atomic ratio increases from 66.83% and 0.04% in TA-Cu to 77.44% and 17.67% in TA-Cu / MH, indicating that the introduction of amino groups (-NH2) in MH changes the surface chemical environment of the complex.

[0041] In summary, it can be confirmed that the elements in the TA-Cu / MH composite are evenly distributed and successfully combined.

[0042] like Figure 5 As shown: TA-Cu shows a Cu 2p characteristic peak at a binding energy of 932.6 eV compared to TA, confirming that Cu 2+ It was successfully loaded on the TA surface through coordination. In addition to the C 1s (284.8 eV), O 1s (531.5 eV) and Cu 2p characteristic peaks in the TA-Cu / MH spectrum, an obvious N 1s characteristic peak was presented at 399.2 eV. This result was consistent with the XPS response characteristics of -NH2 in the MH molecular structure, confirming that the MH molecule was effectively loaded in the complex system through chemical reaction.

[0043] The successful grafting of TA and MH was further verified by the elemental quantitative analysis data in Table 3: the atomic percentage of N element in the TA-Cu / MH system (3.49%) was significantly higher than that in TA-Cu (not detected), which was consistent with the conclusion of EDS characterization. 2+ The atomic proportion of TA-Cu decreased from 1.61% to 0.80%, which can be attributed to the interaction between the amino groups in MH molecules and the TA molecules on Cu. 2+ The competitive coordination effect of the organic ligands results in some metal binding sites being occupied by organic ligands.

[0044] Table 3 Element composition and proportion of each group

[0045] Figure 6 is the XPS high-resolution spectrum, in the O 1s spectrum ( Figure 6 In Figure A), the TA-Cu and TA-Cu / MH complexes showed a significant leftward shift in binding energy compared to TA (ΔE≈0.3 eV), suggesting that the electron cloud density in the complex changed, which may be related to the charge transfer between the components. Further peak fitting showed that TA-Cu and TA-Cu / MH had a characteristic peak at 531.4 eV, which was attributed to the formation of Cu-O coordination bonds, directly confirming that copper ions were successfully grafted onto the TA skeleton through chemical coordination. N 1s spectrum ( Figure 6 In Figure B), the TA-Cu / MH complex exhibits peaks at 399.6 eV and 402.6 eV, corresponding to neutral amino groups and protonated amino groups, respectively, which coincide with the NH stretching vibration peak (~1560 cm-1) in the Fourier transform infrared spectrum. -1 ) shows the same enhancement trend, indicating that MH may be loaded onto TA-Cu through intermolecular forces. Figure 6 C) It can be found that the main peak of Cu 2p in TA-Cu / MH complex is located at 935.1 eV, accompanied by typical satellite peaks (~944.2 eV), confirming that Cu 2+ The stable existence of Cu +This may be due to the effect of TA making Cu 2+ was reduced, and compared with TA-Cu, the Cu ＋ The relative content decreased by about 9.7% (calculated by the satellite peak area ratio), which may be attributed to the positively charged amino group (–NH3 + The electrostatic interaction between Cu and TA phenolic hydroxyl groups (–OH) partially inhibited the antioxidant capacity of TA, resulting in the 2+ Local restoration.

[0046] like Figure 7 As shown in the figure, in the 3500-3000 cm-1 region, OH coordination bonds can be seen in TA, and NH coordination bonds exist in MH, but the OH or NH stretching vibrations in TA-Cu / MH are weakened or changed, which may be due to hydrogen bonds or Cu 2+ The coordination effect of -1 There is a C=O coordination bond on TA in the region, but the intensity and position of the stretching vibration of the C=O coordination bond on TA-Cu / MH are changed, indicating that the carboxyl group may be 2+ Coordination forms a metal complex. 1000-500cm -1 The TA-Cu / MH region shows multiple absorption peaks, which may be due to the formation of Cu-O or Cu-N coordination bonds, further proving that Cu 2+ Coordination reaction with TA and MH. Therefore, we infer that the possible reaction for the formation of the complex is: the hydroxyl (OH) in TA and the carboxyl or amino group in MH may react with Cu 2+ A coordination reaction occurs to form Cu-O (-1000 cm) or Cu-N (500-800 cm) complexes. This coordination will cause the vibration modes of hydroxyl, carboxyl and amine groups to change, thus showing different absorption peaks in the infrared spectrum of TA-Cu / MH. 2+ As a central metal, after coordinating with these groups, the stability and structure of the molecule will change, thus exhibiting different spectral characteristics.

[0047] In summary, Cu 2+ The spectral characteristics of the reaction product with TA and MH (TA-Cu / MH) show significant changes in the vibration modes of carbonyl, hydroxyl, amino groups and Cu-O, Cu-N bonds compared with its components, indicating the formation of the complex TA-Cu / MH.

[0048] Example 4 Evaluation of biological properties of the composite (TA-Cu / MH) 1. Antibacterial properties of the composite (TA-Cu / MH) Determination of the minimum inhibitory concentration and minimum bactericidal concentration of the composite TA-Cu / MH: Staphylococcus aureus Sa ), Escherichia coli, E.coil ), Actinobacillus actinomycetemcomitans, Aa )、 Pg The suspension was diluted to a concentration of 1×10 6 CFU / mL. 100 μL of bacterial solution was added to each well of a 96-well plate. A positive control group was added with only bacterial solution and culture medium, an experimental group was added with bacterial solution and TA-Cu / MH solution, and a negative control group was added with only culture medium. Three replicates were set up for each group. TA-Cu / MH solutions with a final concentration gradient of 0.4 μg / ml, 0.6 μg / ml, 0.8 μg / ml, 1 μg / ml, 5 μg / ml, and 10 μg / ml were prepared. After filtration and sterilization, each concentration solution was added to a 96-well plate and incubated in an anaerobic incubator at 37 °C for 24 hours. Subsequently, the absorbance of each well was measured at a wavelength of 660 nm using a spectrophotometer. The minimum drug concentration when the inhibition rate reached 90% was the minimum inhibitory concentration, and the results were observed with the naked eye.

[0049] The complex TA-Cu / MH inhibits bacterial adhesion and biofilm formation: Pg, Aa The inhibition of bacterial adhesion of each group was investigated separately. The silicon wafer was placed in a 24-well plate, and 100μ of bacterial solution diluted 100 times by shaking overnight was added. TA-Cu, MH, and TA-Cu / MH solutions of the same concentration were prepared. After filtration and sterilization, they were added to the well plate. A positive control group was set up with only bacterial solution and culture medium, an experimental group was set up with bacterial solution and drugs, and a negative control group was set up with only culture medium. Three replicates were set up for each group. The incubation was continued for 24-48 hours until a biofilm was formed on the silicon wafer of the positive control group, and the bacterial solution was slowly aspirated with an insulin needle. Each group of silicon wafers was evaluated using a scanning electron microscope. The silicon wafers were fixed with 2.5% glutaraldehyde at 4°C for more than 4 hours, dehydrated with a gradient of low to high concentration anhydrous ethanol (30%, 50%, 70%, 80%, 90%, 100%), critical point dried, and gold sprayed. The inhibition of TA-Cu, MH, and TA-Cu / MH was observed using a scanning electron microscope. Pg and Aa The role of adhesion; use Pg To explore the inhibition of biofilm formation in each group, PgAfter biofilm formation on cell slides, the slides were washed with saline and incubated with a live / dead stain for 30 minutes. Biofilm formation was observed using a laser confocal microscope (excitation / emission wavelengths Ex / Em = 488 / 525 nm). The slides were fixed in 2.5% glutaraldehyde at 4°C for at least 4 hours, rinsed with sterile deionized water, and dried for 20 minutes. The slides were then stained in a 1% crystal violet solution for 20 minutes. The slides were rinsed once again with sterile deionized water, dried, and photographed. The slides were then placed in a well plate, and 100 μL of glacial acetic acid was added to each well for decolorization. Finally, the absorbance at OD 590 nm was measured using a microplate reader to assess the inhibitory effect on biofilm formation.

[0050] The complex TA-Cu / MH damages the cell membrane of pathogens and destroys the formed biofilm: Pg, Aa To investigate the impact of each group on bacterial cell membrane disruption, silicon wafers were placed in 24-well plates and 100μL of a 100-fold diluted bacterial solution, shaken overnight, was added. A positive control group received only the bacterial solution and culture medium; an experimental group received the bacterial solution, followed by the drug after biofilm formation; and a negative control group received only culture medium. The experimental groups included TA-Cu, MH, and TA-Cu / MH, with three replicates per group. Incubation continued for 24-48 hours, until a biofilm formed on each well of the wafer. At this point, the experimental drug was filter-sterilized and added to the plate. After incubating the formed biofilm with the drug for 4 hours, the bacterial solution was slowly aspirated using an insulin needle. Each set of silicon wafers was evaluated using a scanning electron microscope. The wafers were fixed in 2.5% glutaraldehyde at 4°C for more than 4 hours, dehydrated in a gradient of low to high concentrations of anhydrous ethanol (30%, 50%, 70%, 80%, 90%, and 100%), critical point dried, and sprayed with gold. The damage effects of TA-Cu, MH, and TA-Cu / MH on bacterial cell membranes were observed using a scanning electron microscope. The same experimental procedures were used. Pg To investigate the biofilm destruction status of each group.

[0051] 2. Synergistic antibacterial effect of components in the TA-Cu / MH complex The Staphylococcus aureus suspension was diluted and 100 μL of 1×10 6 CFU / mL bacterial solution. A positive control group was set up with only bacterial solution and culture medium added, an experimental group was set up with bacterial solution and drug added, and a negative control group was set up with only culture medium added. The drugs were MH, copper sulfate pentahydrate solution and their mixed solutions of different concentrations. After filtration and sterilization, they were added to a 96-well plate and incubated in a 37°C constant temperature box for 24 hours. Subsequently, the bacterial solution in the well plate was spread on the agar plate to observe the formation of colonies, so as to confirm the MBC of MH, copper sulfate pentahydrate and their mixed solution to determine whether the two have a synergistic antibacterial effect.

[0052] 3. Biocompatibility of the TA-Cu / MH composite Detection of the effect of the complex TA-Cu / MH on the proliferation of BMSCs and L929 cells: The two cells were cultured at a rate of 5×10 4 Cells were seeded at a density of 100 μg / well in 24-well culture plates. Preliminary experiments established four concentration gradients of the complex solution at 5, 10, 15, and 25 μg / mL for toxicity screening. After determining the dosage of the complex, the final experiment was performed using the same seeding density. The TA-Cu, MH, TA-Cu / MH, and blank control groups were set up at the same concentrations, with three replicates per group. Cells were cultured in a cell culture incubator at 37°C, 5% CO₂, and 95% humidity for 1, 3, and 5 days, respectively. After removing the culture medium, 200 μl of CCK-8 reagent was added to each well and incubated for 4 hours. The absorbance at 450 nm was measured using a microplate reader. Cells were then fixed with 4% paraformaldehyde, stained with rhodamine 123 for 10 minutes, and rinsed with PBS. Cell number was then recorded using an upright fluorescence microscope.

[0053] Detection of the effect of the complex TA-Cu / MH on the morphology of BMSCs: After pre-scratching cells in a 24-well plate, P2 BMSCs were plated at 5×10 4 Cells were seeded on slides at a density of 1 / well and TA-Cu / MH was added at an amount determined by biocompatibility. Control groups were treated with the same concentrations of TA-Cu and MH, as well as a blank group, with three replicates per group. After culturing for 1, 3, and 5 days in a cell culture incubator at 37°C, 5% CO2, and 95% humidity, the remaining liquid in the well plate was aspirated, the cells were washed once with PBS, fixed with 4% paraformaldehyde, washed three times with PBS, permeabilized with 0.5% Triton X-100 solution for 15 minutes, blocked with 5% BSA for 30 minutes, and then stained with rhodamine-phalloidin and DAPI for cytoskeletal actin and nuclei, respectively. The cells were incubated at room temperature in the dark for 30 minutes. Finally, the cell morphology of each group at the corresponding detection time point was observed under an inverted fluorescence microscope.

[0054] 4. Antioxidant effect of the TA-Cu / MH complex DPPH radical scavenging rate: A DPPH radical scavenging experimental system was used. Based on pre-experimental data, gradient concentration sample solutions (including TA-Cu and MH control groups) were prepared using ethanol as the solvent. 1 mL of the sample to be tested was mixed with an equal volume of 0.1 mmol / L DPPH ethanol solution in each group. After reacting in the dark for 30 minutes, the absorbance at 517 nm (A1) was measured. In the blank group, ethanol was used to replace the DPPH solution, and in the background group, distilled water was used to replace the sample. The absorbance values ​​(A0) and (A) were simultaneously measured (DPPH radical scavenging rate (%) = (1-(A1-A0) / A) × 100%). ABTS cation radical scavenging rate: First, mix 7 mmol / L ABTS solution with an equal volume of 2.45 mmol / L potassium persulfate solution to prepare a stock solution. Let it react in a dark environment for 12–16 hours before use. Calibrate the stock solution concentration spectrophotometrically and adjust the absorbance at 734 nm to 0.7 ± 0.02 with deionized water to obtain a standard working solution. Dilute TA-Cu / MH and the control group TA-Cu and MH according to the sample preparation process for the DPPH radical scavenging rate assay. Vortex mix 1 mL of the test solution with 3 mL of the ABTS working solution. After reacting in the dark for 6 minutes, measure the absorbance (A1). Use deionized water as the blank control (A0). Calculate the ABTS radical scavenging rate of each group (ABTS radical scavenging rate (%) = (A-A1) / A × 100%). FRAP total antioxidant capacity assay: Prepare a buffer solution by mixing 0.364 g of anhydrous sodium acetate with 3.2 mL of glacial acetic acid. Adjust the pH to 3.6 with 1 M HCl and dilute to 200 mL. Weigh 0.078 g of TPTZ powder and dilute to 25 mL with 40 mM hydrochloric acid. Separately, weigh 2.78 g of FeCl₃·6H₂O and dilute to 50 mL with RO water. Immediately before use, mix the sodium acetate buffer, TPTZ solution, and FeCl₃ solution in a 10:1:1 volume ratio to prepare the FRAP working solution. For the experiment, the gradient concentration test solution was mixed with the FRAP working solution according to the appropriate ratio, vortexed, and reacted for 5 minutes. The absorbance (A1) was measured at 593 nm. The blank control was replaced by an equal volume of RO water instead of the sample solution, and the measured value was recorded as (A). The positive control was replaced by a standard antioxidant solution instead of the sample solution, and the measured value was recorded as (A2). Based on this, the FRAP free radical scavenging activity of the complex and each control group was calculated (FRAP free radical scavenging rate (%) = (A1-A) / (A2-A) × 100%).

[0055] In vitro ROS scavenging effect of the complex TA-Cu / MH Fluorescence microscopy was used to observe the clearance of ROS by cells: BMSCs and L929 cells were cultured at a rate of 2.0×10 4 Cells were seeded at a density of 100 μg / well in a 24-well plate and cultured overnight in a cell culture incubator at 37°C, 5% CO2, and 95% humidity. The experimental group was replaced with a medium containing the TA-Cu / MH complex, while the control group was treated with a medium containing TA-Cu and MH. The blank group was treated with sterile PBS instead, and the intervention lasted for 12 hours. Both the experimental and control groups were stimulated with a 400 μM H2O2 solution for 2 hours. After establishing the oxidative damage model, the medium was discarded. The cells were replaced with serum-free medium containing the DCFH-DA probe and incubated at 37°C in the dark for 30 minutes. After washing three times with PBS, fluorescence images were acquired using a fluorescence microscope, and fluorescence intensity was quantitatively analyzed using Image J software.

[0056] Determination of intracellular ROS levels by flow cytometry: cells were treated as described above, and serum-free medium was added to resuspend the cells and collected. The cell suspension was filtered through a 40 μm filter and detected by flow cytometry. The FITC channel (excitation wavelength 488 nm, emission wavelength 530 nm) was set to collect at least 1×10 4 The fluorescence intensity of each cell was quantitatively analyzed using FlowJo V10 software.

[0057] 6. Anti-inflammatory effect of the complex TA-Cu / MH qRT-PCR was used to detect the expression of inflammatory factors in the cellular inflammation model. qRT-PCR reactions were prepared using the TB Green™ Premix Ex Taq™ Kit (TaKaRa), and the target genes were amplified using a two-step method. After completion of the reaction, the Ct value of each sample was recorded. Using β-actin rRNA as an internal reference, the difference in gene expression between the TA-Cu / MH complex and its individual components (TA-Cu and MH) relative to the untreated group was calculated using the 2^(-ΔΔCt) method. Here, ΔΔCt = (Ct value of the target gene in the experimental group - Ct value of the internal reference) - (Ct value of the target gene in the negative control group - Ct value of the internal reference). All experiments were performed in triplicate, and the experiments were repeated three times independently.

[0058] ELISA (enzyme-linked immunosorbent assay) assays were used to measure cytokine protein secretion in cell supernatants in a cellular inflammation model. Standard curves for TNF-α, IL-6, and IL-1β were constructed based on the concentrations of the standard substances provided in the kit and their corresponding absorbance values. By comparing the absorbance values ​​of the supernatant with the standard curves, the concentrations of TNF-α, IL-6, and IL-1β were calculated, thereby assessing changes in the expression of these inflammatory mediators under different experimental conditions.

[0059] 7. Osteogenesis effect of the complex P2 BMSCs were cultured at 5×10 4Cells were seeded at a density of 1 / well in a 24-well plate. After 12 hours of attachment, the cells were replaced with osteogenic induction medium (α-MEM basal medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, 10 mM sodium β-glycerophosphate, 0.25 mM ascorbic acid, and 0.1 μM dexamethasone). The medium was changed every 2 days to maintain the induction environment. On day 7 of induction, the cells were fixed with 4% paraformaldehyde and incubated with a BCIP / NBT alkaline phosphatase colorimetric kit for 15 minutes in the dark. The staining was then photographed under a stereomicroscope. The cells were then lysed and total protein was extracted. Protein concentration was determined by the BCA assay, and alkaline phosphatase (ALP) activity per unit protein was measured. Following the same procedure, on day 21 of induction, the cells were fixed and stained with Alizarin Red S for calcium deposits. After photographing, the stained complex was solubilized with 10% cetylpyridinium chloride solution, and the mineralization product was quantified by measuring absorbance at 562 nm using a microplate reader.

[0060] 8. Animal Experimentation Establishment of a periodontitis model in SD rats: The animal experiments in this study adhered to the ethical standards set by the Ethics Committee of Anhui Medical University and were approved by the committee. A periodontitis model was first established in SD rats. A periodontal probe was used to separate the gingiva, and a 3-0 ligature was inserted into the interproximal space of the maxillary second molar. The cervical ligature was then secured and coated with Pg-LPS solution. The ligature was checked for loosening every 48 hours for two weeks, and Pg-LPS solution was added. After two weeks, rats were randomly selected and sacrificed, and their maxillae were dissected to confirm the successful establishment of the periodontitis model.

[0061] Forty-five five-week-old male Sprague-Dawley rats weighing approximately 150 g were randomly divided into five groups (n=9 per group): a blank control group (Healthy), a periodontitis group (PD), a periodontitis group treated with MH (MH), a periodontitis group treated with the intermediate product TA-Cu (TA-Cu), and a periodontitis group treated with a TA-Cu / MH complex (TA-Cu / MH). Periodontitis models were established for two weeks according to the periodontitis modeling protocol. The blank group received no treatment. After two weeks, all rats underwent periodontal scaling without removing the ligature. The experimental groups also received medications. Due to the shallow periodontal pockets of the rats, medications were administered every two days. Treatment lasted for four weeks. During this period, the ligatures were inspected for removal, but Pg-LPS was not injected around the pockets. After treatment, the rats were sacrificed and their maxillae were harvested. Maxillary bones from three rats in each group were fixed in 4% paraformaldehyde for subsequent histological analysis. Three more rats were removed for in vivo periodontal tissue ROS level detection, and after the detection, they were fixed and subsequently used for Micro-CT detection. The remaining three rats in each group were sacrificed and the gingiva around the maxillary second molars was immediately stripped (paying attention to the uniform cutting position and tissue quality in each group), and then qRT-PCR testing was performed.

[0062] Micro-CT imaging of the alveolar bone: Bilateral maxillary bone samples from rats were scanned using micro-CT at a resolution of 9 μm. After scanning, the region of interest (ROI) was selected using CT Analyzer software, and three-dimensional reconstruction was performed to observe the alveolar bone condition.

[0063] Histological Analysis: After fixation in 4% paraformaldehyde for 48 hours, the maxillary bones were split midway, trimmed to define the observation area, and decalcified in 10% EDTA at 4°C for 8 weeks. Following dehydration, the bones were routinely embedded in paraffin and sectioned longitudinally at a thickness of 4 μm. The periodontal tissues of the maxillary second molars were primarily observed. The sections were mounted on slides and stained with hematoxylin and eosin (H&E) to assess gingival inflammatory infiltration.

[0064] qRT-PCR: Place the excised tissue in a grinding tube, add enzyme-free grinding beads, and then add 1 ml of Trizol protein extraction solution. Grind in a grinder until the tissue is no longer visible, then add 200 μL of chloroform. Perform qRT-PCR according to the previous steps.

[0065] Antioxidant capacity in animals: Weigh DCFH-DA powder and fully dissolve it in DMSO solution to a final concentration of 2 mg / ml. Store in the dark until ready for use. For rats to be sacrificed after periodontal treatment, anesthetize them with an intraperitoneal injection of 3.0% sodium pentobarbital at a rate of 0.2 mL / 100 g. Subsequently, the DCFH-DA solution is injected into the tail vein of the anesthetized rats. The needle is inserted at an angle and then parallel to the tail at approximately 2 / 3 of the rat's tail. After aspirating blood, the DCFH-DA solution is injected. The rats are sacrificed 2 hours later, and the fluorescence intensity of the periodontal tissue of the maxillary molars of each group of rats is observed and analyzed using a small animal live imaging device.

[0066] Animal biosafety: Organs (heart, liver, spleen, lung, and kidney) from each group of rats were collected and dehydrated using a standardized dehydration procedure. The tissues were then fixed in paraffin. Serial sections of 6-7 μm were prepared using a rotary microtome. After slide mounting and baking, the sections were dehydrated using a gradient of ethanol. Following conventional H&E staining, the sections were mounted in a neutral resin, and histopathological evaluation was performed using a light microscope.

[0067] result: like Figure 8 As shown, TA-Cu / MH has an obvious effect on Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) had an MIC value of 0.6 μg / mL and an MBC value of about 5 μg / mL; while for the periodontitis-related pathogen Aggregatibacter actinomycetemcomitans ( Aa gingivalis ( Pg ) had a MIC value of 0.8 μg / mL and an MBC value of approximately 10 μg / mL. This result indicates that TA-Cu / MH exhibits strong antibacterial and bactericidal activity against both Gram-negative and Gram-positive bacteria, and has a strong bacteriostatic and bactericidal activity against periodontitis-specific pathogens ( Aa and Pg ) also had a significant inhibitory effect.

[0068] like Figure 9 As shown, the blank control group exhibited a typical biofilm structure on the silicon wafer surface, with dense bacterial aggregates cross-linked through the extracellular matrix to form a three-dimensional network structure. Although the TA-Cu and MH groups did not form a complete biofilm structure, high-density bacteria still adhered to the wafer surface, indicating that the TA-Cu and MH groups were unable to effectively block initial bacterial colonization. In contrast, the TA-Cu / MH group had only a very small number of bacteria adhered to the silicon wafer surface compared to the blank control group, indicating that the complex can effectively inhibit bacterial adhesion.

[0069] like Figure 10As shown in A, the biofilm thickness of the TA-Cu and MH groups was significantly reduced compared to the control group. However, the biofilm formation process of the TA-Cu / MH group was completely inhibited, with only scattered isolated bacteria detected on the surface, and no continuous biofilm structure was formed. The results of the crystal violet staining-glacial acetic acid decolorization method ( Figure 10 B) Consistent with the above experimental results: the absorbance value of the TA-Cu / MH group (0.23±0.02) was significantly lower than that of the TA-Cu group (2.65±0.01), the MH group (0.69±0.01) and the control group (2.93±0.03).

[0070] Figure 11 It can be clearly seen that the different treatment groups Pg and Aa Intervention effect of cell membrane structure. A dense biofilm was formed on the silicon wafer in the blank control group. The bacteria had normal morphology, and the bacterial cell membrane was smooth and continuous, with no obvious structural damage observed. In the TA-Cu group, TA-Cu material was observed to adhere to the bacterial surface. Although the biofilm was damaged to a certain extent, a large number of bacteria with complete morphology were still colonized on the surface of the silicon wafer. Due to its excellent antibacterial effect, the MH group had significantly fewer residual bacteria on the surface of the silicon wafer, showing good biofilm removal ability, but no obvious abnormalities in bacterial morphology were observed. In the TA-Cu / MH group, the biofilm structure on the surface of the wafer was obviously destroyed, and the number of residual bacteria was extremely small. The remaining bacteria generally showed irreversible damage morphology such as membrane structure collapse and cytoplasm shrinkage. Some bacteria even leaked cytoplasmic contents.

[0071] Figure 12 A shows that the biofilm in the blank control group showed a uniformly distributed fluorescence signal, indicating that the bacteria successfully formed a mature biofilm with a three-dimensional structure. Abnormally strong fluorescence aggregation was observed in the TA-Cu experimental group, and the fluorescence intensity of the MH treatment group was significantly reduced, indicating that the biofilm in this group had obviously disintegrated, but the fluorescence signal of live bacteria could still be observed in the residual structure. In the TA-Cu / MH complex treatment group, only a certain amount of dead bacteria were observed adhering to the surface of the slide, indicating that the TA-Cu / MH complex can not only effectively destroy the biofilm structure, but also completely kill the residual microorganisms. Semi-quantitative analysis of the biofilm was performed by crystal violet staining-glacial acetic acid decolorization method ( Figure 12 B) The results showed that the absorbance value of the TA-Cu group (2.98±0.07) was significantly higher than that of the blank control group (1.52±0.05), consistent with the material residue observed by confocal microscopy. The absorbance value of the TA-Cu / MH group (0.38±0.01) was 29.6% lower than that of the MH group (0.54±0.03) (p<0.01), indicating that the complex has a significant advantage in destroying bacterial structure and biofilm integrity.

[0072] The checkerboard dilution method combined with drug sensitivity test revealed that ( Figure 13 When the concentration of CuSO4·5H2O was reduced to 0.5 mg / mL (equivalent to 1 / 8 of its MIC value), its combination with MH 0.04-0.06 μg / mL (1 / 3-1 / 2 of its MIC value) produced a significant synergistic bactericidal effect. This suggests that the two compounds may have a synergistic antibacterial mechanism, enabling the two groups of compounds to achieve a synergistic antibacterial effect below their respective MICs.

[0073] like Figure 14 As shown in the results, the proliferation activity of both cell lines showed a dose-dependent decline with increasing material concentration. At a concentration of 10 μg / mL, TA-Cu / MH still exhibited good biocompatibility, with cell survival rates remaining within a safe range. However, at a concentration of 15 μg / mL, significant cytotoxicity was observed, with a statistically significant difference in cell proliferation rate compared to the blank control group (P<0.001).

[0074] The results of rhodamine staining ( Figure 15 ) Further findings revealed that cells in the TA-Cu / MH group and the other control groups, TA-Cu and MH, showed healthy cell proliferation, with no significant decrease in fluorescence intensity, consistent with the cell proliferation trend in the blank control group. This confirmed that all control groups and TA-Cu / MH exhibited ideal biocompatibility within the effective antibacterial concentration range.

[0075] like Figure 16 As shown, after one day of culture, image analysis using rhodamine-labeled phalloidin and DAPI staining revealed that cells in all groups exhibited good adhesion and active spreading on the bottom of the culture dish. By the third day of culture, the number of cells in each group increased significantly, indicating active cell proliferation. By the fifth day, the cell density had reached over 90%, with tight junctions formed between cells, demonstrating a typical fusion state. Cell morphology in the TA-Cu / MH group, its intermediate TA-Cu group, and the MH group showed no significant differences compared to the blank control group, indicating that these materials had no significant effect on cell morphology at the experimental concentrations.

[0076] Figure 17 A shows that TA, MH and TA-Cu / MH all have certain DPPH free radical scavenging activity. The ability of TA-Cu / MH to scavenge DPPH free radicals has reached more than 85% at 10 μg / mL, which is close to the scavenging rate of TA (P<0.05); Figure 17 B shows that TA and TA-Cu / MH have ABTS free radical scavenging activity at extremely low concentrations and exhibit concentration gradient dependence. At 0.05 μg / mL, the ABTS cation free radical scavenging rate reaches over 90%, while MH has no ABTS free radical scavenging effect at this concentration. Figure 17C shows that TA, MH and TA-Cu / MH have FRAP total antioxidant capacity at lower concentrations and show gradient dependence. It can be seen that under the same concentration conditions, the antioxidant capacity of the complex is better than TA but worse than MH. The FRAP total antioxidant capacity of the complex reaches more than 90% at 0.5 μg / mL.

[0077] The results showed that while MH possessed some free radical scavenging ability, its overall scavenging effect was significantly lower than that of TA at the same concentration. In contrast, the free radical scavenging activity of the complex was slightly lower than that of TA at the same concentration, but it still exhibited strong antioxidant activity. Therefore, the free radical scavenging ability of the complex was significantly enhanced due to the introduction of TA, indicating that TA plays a key antioxidant role in the complex, thereby significantly enhancing the free radical scavenging effect of the complex. This finding provides important evidence for optimizing the antioxidant properties of the complex.

[0078] like Figure 18 As shown in A and B, H2O2 stimulation significantly increased the level of intracellular ROS, and the control group showed the most significant green fluorescence signal. After treatment with TA-Cu, MH, and TA-Cu / MH, the fluorescence intensity of cells in each group decreased to varying degrees, with the TA-Cu / MH group showing the most significant decrease. Quantitative analysis showed ( Figure 18 C). The TA-Cu / MH group demonstrated significantly better ROS scavenging efficiency than the control group (P < 0.01), with relative fluorescence intensity decreasing by approximately 40.8% and 51.1% compared with the TA-Cu and MH groups, respectively, and by 46.3% and 62.5%. These data suggest that TA-Cu / MH significantly enhances antioxidant activity through a synergistic effect, effectively alleviating oxidative stress-induced damage to oral soft and hard tissue cells.

[0079] like Figure 19 As shown, H2O2 stimulation resulted in a significant increase in the ratio of reactive oxygen species (ROS)-positive cells in RAW264.7 macrophages compared with the normal group (P<0.01), confirming the successful establishment of the oxidative stress model. The TA-Cu / MH complex-treated group demonstrated significant ROS scavenging ability, with the ratio of ROS-positive cells reduced by 32.7% compared with the normal group (P<0.001). Its antioxidant potency was significantly superior to that of the single components TA-Cu (reduction of 25.6%, P<0.01) and MH (reduction of 8%, P<0.05).

[0080] qRT-PCR results showed that compared with the normal control group, the mRNA expression levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the LPS-stimulated model group (Control group) showed a significant upregulation (P < 0.01). This result indicates that RAW264.7 macrophages were successfully polarized to the pro-inflammatory M1 phenotype under LPS induction, validating the reliability of the inflammatory cell model. After drug pretreatment, the mRNA expression of inflammatory factors in the LPS+drug group was significantly reduced (P < 0.05), with the TA-Cu / MH combined treatment group showing a more significant inhibitory effect (P < 0.01). These data suggest that TA-Cu / MH can effectively regulate macrophage polarization and reverse LPS-induced M1 transformation, thereby improving the inflammatory immune microenvironment.

[0081] Experimental data showed that after 24 hours of culture, significant levels of macrophage-derived pro-inflammatory cytokines were detected in the supernatant of the control group (IL-6: 1096.5±69.9 pg / mL; IL-1β: 1073.8±77.9 pg / mL; TNF-α: 1201.1±144.7 pg / mL). Compared with the control group, the expression levels of these inflammatory factors in the MH-treated group were statistically significantly decreased (IL-6 decreased by 45.5%, P<0.05; IL-1β decreased by 22.9%, P<0.05; TNF-α decreased by 36.8%, P<0.05). The TA-Cu group exhibited a more pronounced inhibitory effect, with inhibition rates of 21.92%-27.5% higher than those in the MH group (P<0.01). The TA-Cu / MH group showed the best anti-inflammatory effect, with the secretion levels of IL-6, IL-1β, and TNF-α further reduced by 20.3%, 33.38%, and 11.68%, respectively, compared with the TA-Cu group (P<0.01).

[0082] ALP activity ( Figure 21 ) showed that drug intervention significantly enhanced the expression of ALP, an early osteoblast marker. Quantitative results showed that ALP activity in the TA-Cu group (6.9±0.6 U / mg prot) and the TA-Cu / MH group (6.0±1.2 U / mg prot) increased 6-7 times compared with the blank control group (1.0±0.13 U / mg prot) (P<0.001). However, there was no statistical difference between the MH group (2.5±1.1 U / mg prot) and the control group (P>0.05), consistent with the ALP imaging results. Alizarin red staining and quantitative analysis revealed that calcium nodule density in the TA-Cu and TA-Cu / MH groups was significantly higher than that in the control and MH groups (P<0.001).

[0083] The modeling success (such as Figure 22A) SD rats treated for 2 weeks were sacrificed and the alveolar bone resorption of maxillary second molars was analyzed by Micro-CT. Figure 22 Figure B shows the healthy group with intact alveolar bone structure and no obvious signs of bone resorption. The PD group exhibited typical pathological changes, including extensive periradicular bone loss and furcation lesions. Among the intervention groups, the TA-Cu / MH complex group exhibited significantly lower bone resorption than the PD group. Micro-CT scanning was used to locate the distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC) on the palatal side of the maxillary second molar and quantify bone loss. The CEJ-ABC distance in the PD group was significantly increased by 2.3 times compared with the control group (P < 0.01), confirming successful modeling. After drug intervention, the TA-Cu and MH groups decreased by 19.4% and 24.2%, respectively, compared with the PD group, while the TA-Cu / MH complex group decreased by 37.4% (P < 0.01). The between-group differences were statistically significant (P < 0.05), suggesting that the complex inhibits alveolar bone resorption in rats.

[0084] Using Micro-CT three-dimensional reconstruction technology, we quantitatively analyzed bone parameters in the area between the first and second molars of each group of rats, including bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Quantitative analysis revealed significant bone microarchitectural damage in the PD group compared with the control group: BV / TV decreased by 31.05% (P<0.01), Tb.Th decreased by 75.67% (P<0.05), Tb.N decreased by 52.02% (P<0.05), and Tb.Sp increased by 4.08-fold (P<0.001). These changes confirmed the successful modeling of periodontitis. After drug intervention, the TA-Cu / MH group significantly improved bone microstructure compared with the PD group: BV / TV increased to 0.37±0.03 (P<0.01), Tb.Th increased to 0.27±0.03mm (P<0.01), Tb.N recovered to 4.4±0.5 / mm (P<0.01), and Tb.Sp decreased to 0.12±0.02mm (P<0.001), indicating that the complex has a significant bone protective effect.

[0085] Histological observation showed that ( Figure 23): The gingival papilla structure of the healthy control group was intact, the junctional epithelium formed a stable attachment to the enamel surface, and no pathological changes or inflammatory infiltration were observed. The PD group showed typical pathological characteristics of periodontitis: gingival tissue congestion and edema, the junctional epithelium migrated rootward, the alveolar ridge top was significantly absorbed, and a large number of neutrophil infiltration was seen in the periodontal ligament space, indicating that the model was successfully constructed. After drug intervention, the TA-Cu / MH group was significantly improved compared with the PD group: the migration distance of the junctional epithelium was improved compared with the PD group, and the inflammatory cell infiltration was reduced. In contrast, although there was some improvement in the TA-Cu monotherapy group and the MH monotherapy group, the effect was significantly weaker than that of the complex group. The above results indicate that the TA-Cu / MH group can effectively control the inflammation of the periodontal tissue of SD rats, improve the local microenvironment of the periodontal tissue, and inhibit the absorption of alveolar bone.

[0086] RT-qPCR was used to evaluate the levels of inflammatory factors in the gingiva of the second molars of each group after treatment. Figure 24 ) showed that the levels of inflammatory factors (IL-6, IL-1β, and TNF-α) in the gums of the PD group were 5-10 times higher than those in the Healthy group, while those in the TA-Cu and MH groups were 2-3 times higher than those in the Healthy group. However, the inflammatory factor levels in the TA-Cu / MH complex were only 1-2 times higher than those in the Healthy group, indicating no significant difference. This suggests that TA-Cu / MH can alleviate periodontal inflammation by reducing the levels of inflammatory factors in periodontal gingival tissue.

[0087] Fluorescence imaging results ( Figure 25 ) showed that the fluorescence intensity of gingival tissue in the Healthy group was low, indicating that ROS levels were within the physiological range. No significant fluorescence accumulation was observed in the combined epithelium and periodontal ligament regions, suggesting a lack of significant oxidative stress. The fluorescence intensity of gingival tissue in the PD group was significantly increased (approximately 4.04 times that of the Healthy group, P < 0.01), indicating a significant increase in ROS levels. The fluorescence intensity of the TA-Cu / MH group was 67.8% lower than that of the PD group, indicating that the TA-Cu / MH complex effectively inhibited ROS production. The fluorescence intensities of the TA-Cu and MH groups were between those of the PD and TA-Cu / MH groups, indicating that while the single-agent groups had some antioxidant effect, it was significantly weaker than that of the complex group.

[0088] These results indicate that ROS levels significantly increase during periodontitis pathology, and that the TA-Cu / MH complex effectively reduces ROS levels in gingival tissue through its antioxidant activity, with greater efficacy than either component alone. This finding is consistent with the in vitro antioxidant results and further confirms the potential therapeutic value of the TA-Cu / MH complex in ameliorating the oxidative stress microenvironment in periodontitis.

[0089] HE staining results showed that the organ tissue structures of each experimental group were intact, with no pathological changes: myocardial fibers were neatly arranged, the liver lobule structure was clear, the white pulp and red pulp of the spleen were clearly demarcated, the alveolar structure was intact, and the glomeruli and renal tubules were normal in morphology ( Figure 26 These results indicate that the TA-Cu / MH complex and its single-component drugs showed no significant in vivo toxicity at the experimental doses and administration cycles.

[0090] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A composite having multi-target therapeutic effects of anti-inflammatory, antioxidant, and osteogenesis, the preparation of which comprises the following steps: Tannic acid (TA) and Cu 2+ The complex is then used as a carrier to load minocycline (MH).

2. The composite according to claim 1, characterized in that The tannic acid (TA) and Cu 2+ The complexation step is to thoroughly mix tannic acid (TA) and CuSO4·5H2O in deionized water.

3. The composite according to claim 2, characterized in that The thorough mixing is achieved by magnetic stirring for 12 h to 36 h.

4. The composite according to claim 1, characterized in that The step of loading minocycline is to add MH to the complex to react to obtain a reaction solution, and then collect the precipitate by centrifugation to obtain a complex.

5. The composite according to claim 1, characterized in that The final concentration of TA was 2 mg / ml.

6. The composite according to claim 1, characterized in that The final concentration of CuSO4·5H2O is 0.05-0.6 mg / ml; Optionally, the final concentration of MH is 0.2-1.2 mg / ml.

7. The composite according to claim 1, characterized in that The final concentration of CuSO4·5H2O is 0.05-1 mg / ml, 0.1-0.2 mg / ml, 0.2-0.3 mg / ml, 0.3-0.4 mg / ml, 0.4-0.5 mg / ml and / or 0.5-0.6 mg / ml, 0.6 mg / ml; Optionally, the final concentration of MH is 0.2-0.4 mg / ml, 0.4-0.6 mg / ml, 0.6-0.8 mg / ml, 0.8-1 mg / ml and / or 1-1.2 mg / ml.

8. The composite according to claim 1, characterized in that The final concentration of CuSO4·5H2O is 0.5 mg / ml; Optionally, the final concentration of MH is 0.6 mg / ml.

9. A pharmaceutical composition comprising the complex according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

10. Use of the complex according to any one of claims 1 to 8 and / or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating periodontitis.

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