Conjugated polymer nano-material for antibacterial repair as well as preparation method and application of conjugated polymer nano-material

Abstract

A preparation method of a conjugated polymer nano material for antibacterial repair comprises the following steps: preparing a conjugated polymer solution to obtain a BSA (at) SP II compound; the preparation method comprises the following steps: dissolving dipalmitoyl phosphatidylcholine and DSPE-mPEG in an organic solvent, carrying out ultrasonic dispersion, evaporating to form a film, adding water to resuspend the film, and carrying out ultrasonic treatment to completely disperse the film so as to form a homogeneous solution; adding a BSA (at) SP II compound and superoxide dismutase into the solution, heating and stirring, and then cooling; and performing ultrafiltration and purification to obtain the SPNS nano-particles. The invention also provides the conjugated polymer nano material prepared by the method. The invention also provides an application of the nano material in preparation of a medicine for treating periodontitis. According to the multifunctional conjugated polymer nano-material disclosed by the invention, the problems of single function, insufficient curative effect and low biological safety in existing periodontitis treatment are solved by integrating antibacterial, photo-thermal treatment and biological membrane inhibition functions.

Description

Technical Field

[0001] This invention belongs to the field of medical materials science and relates to a conjugated polymer nanomaterial, specifically an antibacterial and restorative conjugated polymer nanomaterial and its application in the treatment of periodontitis, especially suitable for antibacterial and periodontal tissue regeneration and repair in periodontitis. Background Technology

[0002] Periodontitis is a chronic infectious disease caused by pathogens such as Porphyromonas gingivalis. Its pathological features include periodontal tissue destruction, cytokine storm, and bone defects. Traditional treatments (such as antibiotics and surgical debridement) have limitations such as high bacterial resistance, short local drug retention time, and insufficient ability to promote tissue repair.

[0003] In recent years, nanomaterials have shown promise in the fields of antibacterial and tissue repair due to their high drug loading capacity, controlled release, and functional designability. However, most existing nanomaterials only possess a single function (such as antibacterial or photothermal therapy), making it difficult to synergistically achieve the dual goals of highly effective antibacterial treatment and bone regeneration. Furthermore, while photothermal therapy can enhance antibacterial effects, its ability to regulate osteogenic differentiation is insufficient, and some nanomaterials pose biosafety risks due to surface charge or hemolytic issues.

[0004] Currently, there are no nanomaterials that combine photothermal antibacterial properties, osteogenic induction, biofilm inhibition, and good biocompatibility for the treatment of periodontitis. This invention provides an innovative solution for minimally invasive treatment of periodontitis through multi-component synergistic design. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides an antibacterial and restorative conjugated polymer nanomaterial, its preparation method, and its application. This antibacterial and restorative conjugated polymer nanomaterial, its preparation method, and its application aim to solve the technical problem of poor efficacy of existing drugs in treating periodontitis.

[0006] This invention provides a method for preparing conjugated polymer nanomaterials for antibacterial repair, comprising the following steps: 1) Preparation of conjugated polymer solution: a) Dissolve the conjugated polymer SPⅡ in a first organic solvent, and sonicate until completely dissolved to obtain a conjugated polymer SPⅡ solution; the structural formula of the conjugated polymer SPⅡ is shown below. n=5~20; b) Dissolve bovine serum albumin in water, disperse it by sonication, and then add it dropwise to the above-mentioned conjugated polymer SPⅡ solution. Sonicate the solution to form a BSA@SPⅡ complex. 2) Thin film preparation and recombination: Dipalmitoylphosphatidylcholine and DSPE-mPEG (distearylphosphatidylethanolamine-polyethylene glycol 2000) were dissolved in a second organic solvent, ultrasonically dispersed, evaporated to form a film, water was added to resuspend the film, and ultrasonically dispersed it completely to form a homogeneous solution. 3) Complexation and purification: Add the BSA@SPⅡ complex and superoxide dismutase to the solution in step 2) above, heat at 60~70℃ for 0.5~2 hours, and stir until cooled; 4) Ultrafiltration purification to remove unencapsulated free components, yielding antibacterial repair conjugated polymer nanomaterials (SPNS nanoparticles).

[0007] Furthermore, in step 3), CaCl2 is added, and stirring is continued for 8-16 hours to allow it to... SPNS was obtained by electrostatic loading onto the surface of nanoparticles and then ultrafiltration. Ca (Conjugated polymer nanomaterials containing calcium ions).

[0008] Furthermore, the concentration of the SPⅡ solution is 0.2~0.5 mg / ml; the concentration of bovine serum albumin is 10~30 mg / ml; the mass ratio of SPⅡ to bovine serum albumin is 0.25:(10~30); and the first organic solvent is tetrahydrofuran.

[0009] Furthermore, the concentration of the dipalmitoylphosphatidylcholine solution is 2-8 mg / ml; the concentration of DSPE-mPEG is 2-8 mg / ml; the mass ratio of dipalmitoylphosphatidylcholine to DSPE-mPEG is 1:(1-3); and the second organic solvent is chloroform.

[0010] Furthermore, the volume ratio of BSA@SPⅡ complex, superoxide dismutase (SOD), and solution in step 2) is 1:1:10.

[0011] Furthermore, the BSA@SPⅡ complex, superoxide dismutase (SOD), and CaCl₂... 2、 The material ratio of the solution in step 2) is 1 ml: 1 ml: 50 mg: 10 ml.

[0012] The present invention also provides conjugated polymer nanomaterials prepared by the above method.

[0013] The present invention also provides the use of the above-mentioned nanomaterials in the preparation of medicaments for the treatment of periodontitis.

[0014] Specifically, DSPE-mPEG (distearylphosphatidylethanolamine-polyethylene glycol 2000) is a commercially available material purchased from Shanghai Punosun Biotechnology Co., Ltd.

[0015] The present invention also provides a conjugated polymer, the structural formula of which is shown below. n=5~20.

[0016] Furthermore, the chemical equation for its preparation is shown below. .

[0017] Specifically, the method for preparing a conjugated polymer includes the following steps: 1) Benzobisthiadiazole, 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethyltinyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, The tris(o-tolyl)phosphine was dissolved in a third organic solvent and then transferred to a Schlenk flask, followed by at least two freeze-vacuum-thaw cycles to degas it. 2) Under nitrogen protection, the mixture from step 1) was placed in an oil bath and heated to carry out Stille polymerization. After the reaction was completed, the precipitate was collected by centrifugation. The precipitate was washed to remove impurities and then dried under vacuum overnight to obtain the conjugated polymer.

[0018] This invention aims to provide a multifunctional conjugated polymer nanomaterial (SPNS series) that integrates antibacterial, photothermal therapy, bone differentiation promotion and biofilm inhibition functions to solve the problems of single function, insufficient efficacy and low biosafety in the current treatment of periodontitis.

[0019] The multifunctional conjugated polymer nanomaterials (SPNS series) developed in this invention are based on the photoelectric properties of the conjugated polymer SPⅡ, and are loaded with superoxide dismutase (SOD). By incorporating active ingredients and modifying DSPE-mPEG to improve biocompatibility, the following technological breakthroughs have been achieved: Broad-spectrum antibacterial and photothermal synergy: through It releases substances that disrupt the bacterial membrane structure, and combined with the photothermal effect triggered by a 1064 nm laser, it significantly improves antibacterial efficiency and reduces drug resistance; Bone repair and osteogenic differentiation regulation: Loaded SOD can clear excess ROS, directionally induce osteogenic differentiation of bone marrow mesenchymal stem cells (high expression of genes such as ALP and Runx2), and accelerate the repair of periodontal bone defects. Biofilm-targeted inhibition: By disrupting the biofilm structure of Porphyromonas gingivalis, it reduces the levels of inflammatory factors such as IL-1β and TNF-α, thereby improving the periodontal microenvironment; Biocompatibility optimization: DSPE-mPEG modification significantly reduces the hemolysis rate of the material (<5%), and in vivo experiments have confirmed that it is non-toxic to organs such as the heart and spleen.

[0020] Compared with existing technologies, the technical effects of this invention are positive and significant. Through the design and optimization of multifunctional conjugated polymer nanomaterials, this invention overcomes the limitations of traditional periodontitis treatment, demonstrating application potential in antibacterial efficiency, bone repair capacity, and biocompatibility. It provides a highly efficient, low-toxicity, and comprehensive innovative tool for the treatment of oral diseases. Attached Figure Description

[0021] Figure 1 shows the physicochemical properties of the materials; where a represents SPNS and SPN. Ca SPNS Ca The electron microscope images are shown in Figure 1. b represents the average particle size of the nanomaterial, c represents the potential diagram of three different materials, d represents the SOD release diagram of two different materials, e represents the particle size stability diagram of three different materials, and f represents the polydispersity index (PDI).

[0022] Figure 2 shows the temperature rise curves of the three materials at different concentrations. a) shows the temperature rise curves of SPNS at five different concentrations; b) shows the temperature rise curves of SPN. Ca Temperature curves at 5 different concentrations; c represents SPNS. Ca Temperature rise curves at 5 different concentrations.

[0023] Figure 3 This is a schematic diagram of a single cycle at 100 ppm for different materials.

[0024] Figure 4 This is a schematic diagram of three cycles at 100 ppm for different materials.

[0025] Figure 5 A schematic diagram illustrating the hemolytic activity of the three materials in the biosafety assessment.

[0026] Figure 6 shows the antibacterial performance evaluation of the three materials. a is a schematic diagram of bacterial CFU, and b is a schematic diagram of bacterial viability and inactivation staining.

[0027] Figure 7 This is a schematic diagram of a micro-CT scan used to assess osteogenic performance in mice.

[0028] Figure 8 This is for the in vivo toxicity assessment of the polymer nanomaterials of the present invention. Detailed Implementation Example 1

[0029] This invention provides a method for preparing conjugated polymer nanomaterials for antibacterial repair, comprising the following steps: 1) Preparation of conjugated polymer solution: a) Dissolve 0.25 mg of conjugated polymer SPⅡ (prepared using the method of Example 2) in 1 mL of tetrahydrofuran (THF) and sonicate until completely dissolved to obtain SPⅡ solution.

[0030] b) Dissolve 20 mg of bovine serum albumin (BSA) in 1 mL of water, disperse by sonication, and slowly add it dropwise to the above SPⅡ solution. Continue sonication for 5-10 minutes to form a BSA@SPⅡ complex.

[0031] 2) Thin film preparation and recombination: 20 mg dipalmitoylphosphatidylcholine (DPPC) and 20 mg DSPE-mPEG (distearylphosphatidylethanolamine-polyethylene glycol 2000, Shanghai Pronoson Biotechnology Co., Ltd.) were dissolved in 5 mL of chloroform, ultrasonically dispersed, and then evaporated to form a film at 45℃ and 140 rpm. 10 mL of water was added to resuspend the film, and it was ultrasonicated for 5 minutes to completely disperse it, forming a homogeneous solution.

[0032] 3) Complexation and purification: Add 1 ml of BSA@SPⅡ complex and 1 ml of superoxide dismutase (SOD) to 10 ml of the solution in step 2) above, heat at 65°C for 1 hour, and stir until cooled.

[0033] 4) Ultrafiltration purification to remove unencapsulated free components, yielding SPNS nanoparticles.

[0034] 5) Calcium ion-supported nanomaterials (SPNS) Ca Preparation of SPNS: In the SPNS preparation step, add 0.5 mg CaCl2 and stir continuously for 12 hours. SPNS was obtained by electrostatic loading onto the surface of nanoparticles and then ultrafiltration. Ca .

[0035] This invention also provides a control material (SPN) Ca Preparation method of ) Omit SOD, and follow the same steps as SPNS preparation. Add 0.5 mg CaCl2 and stir continuously for 12 hours. Electrostatic loading was applied to the surface of nanoparticles, followed by ultrafiltration to obtain control nanoparticles (SPN) without functional components. Ca ).

[0036] Physicochemical characterization, particle size and potential analysis: Figure (1a) shows SPNS and SPN Ca SPNS Ca Electron micrographs were obtained, and the average particle size of the nanomaterials was determined using dynamic light scattering (DLS). Figure 1b Polydispersity Index (PDI) Figure 1f ) and Zeta potential ( Figure 1c ), to evaluate its dispersion stability; among which ( Figure 1d () indicates the amount of SOD released under different conditions. Figure 1e The above data shows that the material has uniform dispersion, clear surface charge, and high material stability.

[0037] Morphological characterization: Transmission electron microscopy (TEM) was used to observe the microstructure and structural integrity of the nanomaterials. Spectroscopic analysis: Ultraviolet-Vis spectroscopy (UV-Vis) was used to detect the characteristic absorption peaks of the conjugated polymer SPⅡ, confirming the material's optical properties. Figure 1a This indicates that the material's microstructure is clear, its size distribution is intuitive, and its structural characteristics are well-defined.

[0038] SOD activity assay: The loading and sustained-release behavior of SOD in nanomaterials were quantitatively analyzed using an SOD activity assay kit. Figure 1d The functional verification experimental steps confirmed that the SOD effective load and activity were well maintained, and that it possessed controllable sustained-release characteristics, meeting the functional requirements. Photothermal conversion performance evaluation, experimental conditions: 1064 nm laser irradiation (power density 1 W / cm²), recording different concentrations of SPNS and SPN. Ca SPNS Ca Temperature changes of the solution (as shown in Figure 2) Figure 2a , Figure 2b , Figure 2c Representing SPNS and SPN respectively Ca SPNS Ca This indicates that the nanoparticles have good photothermal conversion performance and exhibit concentration dependence.

[0039] Stability test: The material was subjected to 5 laser on / off cycles to evaluate the stability of the photothermal conversion efficiency. Figure 3 and Figure 4 The material stability test indicates that the photothermal conversion is very stable.

[0040] Antibacterial performance test, in vitro antibacterial test: Pseudomonas gingivalis suspension ( CFU / mL and SPNS Ca Co-incubate (25 μg / mL), irradiate with laser for 5 minutes, dilute and spread on blood agar plates, anaerobic incubate at 37°C for 16 hours, and count colony forming units (CFU). Figure 6a Bacterial colony counts under two different conditions: no laser illumination and laser illumination. Figure 6b The number of dead bacteria under two different conditions: no laser irradiation and laser irradiation. The above results demonstrate that SPNS... CaIt exhibits excellent antibacterial effects. The material can be efficiently sterilized by laser triggering, significantly reducing the number of viable bacteria, proving that photodynamic synergy can enhance the antibacterial effect.

[0041] In vivo periodontitis treatment experiments, animal model establishment: Periodontitis induced by molar ligation in mice, followed by topical application of SPNS Ca (200 μg / mL), laser irradiation for 5 minutes, 3 times a week, for 2 weeks.

[0042] Treatment efficacy assessment: Histological analysis: H&E staining was used to observe the recovery of gingival papillae, and Masson staining was used to detect collagen deposition.

[0043] Micro-CT scan: Quantitative analysis of alveolar bone volume (BV), bone mineral density (BMD), and bone defect repair. Figure 7 This is a micro-CT image of the alveolar bone in mice. The periodontitis disease model causes some defects in the alveolar bone of mice; SPNS was applied topically. Ca This method can effectively reduce the progression of periodontitis in mice and alleviate the degree of alveolar bone loss.

[0044] Biosafety evaluation, hemolysis test: mouse erythrocytes and SPNS Ca Incubate at room temperature for 2 hours (200 μg / mL), measure absorbance at 540 nm, hemolysis rate must be <5% (e.g., Figure 5 (As shown). The material showed no hemolysis within the tested concentration range, exhibited good blood compatibility, met biomedical material safety standards, and has potential for in vivo application.

[0045] In vivo toxicity assessment: Mouse body weight changes were monitored. After sacrifice, heart, spleen, and kidney tissues were collected for H&E staining to observe organ damage. Figure 8 It is evident that the major organ structures remain intact, with no pathological damage observed, indicating that the material has no significant systemic toxicity and possesses good in vivo safety. Example 2

[0046] The present invention also provides a method for preparing the conjugated polymer SPⅡ of Example 1 above: Benzobisthiadiazole (monomer 1, 25 mg, 0.023 mmol), 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethyltinyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (monomer 2, 21.3 mg, 0.025 mmol), (1.0 mg, 0.001 mmol) and tris(o-tolyl)phosphine (2.0 mg, 0.006 mmol) were dissolved in chlorobenzene (4 mL) and then transferred to a 50 mL Schlenk flask, followed by three freeze-vacuum-thaw cycles to degas the solution.

[0047] Subsequently, under nitrogen protection, the mixture was heated in a 100 °C oil bath for Stille polymerization for 2 hours. After the reaction was completed, the resulting mixture was added dropwise to methanol, and the precipitate was collected by centrifugation. The obtained precipitate was washed three times with methanol to remove impurities, and then dried under vacuum overnight to remove methanol to obtain the conjugated polymer SPⅡ.

[0048] 1H1 H NMR (300 MHz, CDCl33): δ 9.4-8.72 (d, 4H), 7.61-7.39 (m, 2H), 4.04-3.65 (m, 4H), 2.93-2.51 (m, 4H), 1.58 (m, 20H), 1.26-1.11 (m, 46H), 0.94-0.85 (m, 30H).

[0049] The reaction equation is described below:

Claims

1. A method for preparing conjugated polymer nanomaterials for antibacterial repair, characterized in that... The steps include: 1) Preparation of conjugated polymer solution: a) Dissolve the conjugated polymer SPⅡ in a first organic solvent, and sonicate until completely dissolved to obtain a conjugated polymer SPⅡ solution; the structural formula of the conjugated polymer SPⅡ is shown below. ，n=5~20； b) Dissolve bovine serum albumin in water, disperse it by sonication, and then add it dropwise to the above-mentioned conjugated polymer SPⅡ solution. Sonicate the solution to form a BSA@SPⅡ complex. 2) Thin film preparation and recombination: Dipalmitoylphosphatidylcholine and distearylphosphatidylethanolamine-polyethylene glycol 2000 were dissolved in a second organic solvent, ultrasonically dispersed, evaporated to form a film, water was added to resuspend the film, and ultrasonically dispersed to form a homogeneous solution. 3) Complexation and purification: Add the BSA@SPⅡ complex and superoxide dismutase to the solution in step 2) above, heat at 60~70℃ for 0.5~2 hours, and stir until cooled; 4) Ultrafiltration purification yields conjugated polymer nanomaterials for antibacterial repair.

2. The method for preparing an antibacterial and repair-grade conjugated polymer nanomaterial according to claim 1, characterized in that: Add CaCl2 to step 3) and continue stirring for 8-16 hours, so that... SPNS was obtained by electrostatic loading onto the surface of nanoparticles and then ultrafiltration. Ca .

3. The method for preparing an antibacterial and repair-grade conjugated polymer nanomaterial according to claim 1, characterized in that: The concentration of the conjugated polymer SPⅡ solution is 0.2~0.5 mg / ml; the concentration of bovine serum albumin is 10~30 mg / ml; the mass ratio of SPⅡ to bovine serum albumin is 0.25:(10~30); and the first organic solvent is tetrahydrofuran.

4. The method for preparing an antibacterial and repair-grade conjugated polymer nanomaterial according to claim 1, characterized in that: The concentration of the dipalmitoylphosphatidylcholine solution is 2-8 mg / ml; the concentration of distearylphosphatidylethanolamine-polyethylene glycol 2000 is 2-8 mg / ml; the mass ratio of dipalmitoylphosphatidylcholine to distearylphosphatidylethanolamine-polyethylene glycol 2000 is 1:(1-3); and the second organic solvent is chloroform.

5. The method for preparing an antibacterial and repair-grade conjugated polymer nanomaterial according to claim 1, characterized in that: The volume ratio of BSA@SPⅡ complex to superoxide dismutase is 1:

1.

6. The conjugated polymer nanomaterials prepared by the method of claim 1.

7. Use of the nanomaterial of claim 6 in the preparation of a medicament for treating periodontitis.

8. A conjugated polymer, characterized in that: Its structural formula is shown below. ，n=5~20。 9. The method for preparing a conjugated polymer according to claim 8, characterized in that: The chemical equation for its preparation is shown below. 。 10. A method for preparing a conjugated polymer according to claim 9, characterized in that... The steps include: 1) Benzobisthiadiazole, 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethyltinyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, The tris(o-tolyl)phosphine was dissolved in a third organic solvent and then transferred to a Schlenk flask for at least two freeze-vacuum-thaw cycles to degas it. 2) Under nitrogen protection, the mixture from step 1) was placed in an oil bath and heated to carry out Stille polymerization. After the reaction was completed, the precipitate was collected by centrifugation. The precipitate was washed to remove impurities and then dried under vacuum overnight to obtain the conjugated polymer.

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