Antibacterial repair conjugated polymer nanomaterial and preparation method and application thereof

Abstract

The application discloses a preparation method of a conjugated polymer nanomaterial for antibacterial repair, and belongs to the technical field of medical materials.The preparation method comprises the following steps: preparing a conjugated polymer solution to obtain a BSA@SP II compound; dissolving dipalmitoyl phosphatidylcholine and DSPE-mPEG in an organic solvent, ultrasonic dispersion, film evaporation, water resuspension of the film, ultrasonic dispersion to form a homogeneous solution; adding the BSA@SP II compound and superoxide dismutase into the solution, heating and stirring, and then cooling; and ultrafiltration purification to obtain SPNS nanoparticles.The application further provides the conjugated polymer nanomaterial prepared by using the above method.The application further provides the use of the above nanomaterial in the preparation of a medicine for treating periodontitis.The multifunctional conjugated polymer nanomaterial solves the problems of single function, insufficient curative effect and low biological safety in the existing periodontitis treatment by integrating antibacterial, photothermal treatment and biofilm inhibition functions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical materials, and relates to a conjugated polymer nanomaterial, in particular to an antibacterial repair conjugated polymer nanomaterial and application thereof in periodontitis treatment, and is especially suitable for antibacterial and periodontal tissue regeneration repair of periodontitis. BACKGROUND

[0002] Periodontitis is a chronic infectious disease caused by Porphyromonas gingivalis and other pathogens, and its pathological characteristics include periodontal tissue destruction, inflammatory factor storm and bone defect. Traditional treatment methods (such as antibiotics and surgical debridement) have limitations such as high bacterial drug resistance, short local drug retention time and insufficient ability to promote tissue repair.

[0003] In recent years, nanomaterials have shown potential in the fields of antibacterial and tissue repair due to their high drug loading, controllable release and functional designability. However, most existing nanomaterials only have a single function (such as antibacterial or photothermal therapy), making it difficult to achieve the dual goals of efficient antibacterial and bone regeneration. In addition, although photothermal therapy can enhance antibacterial effect, it has insufficient osteogenic differentiation regulation ability, and some nanomaterials have biological safety risks due to surface charge or hemolytic problems.

[0004] At present, there is no nanomaterial that has photothermal antibacterial, osteoinductive, biofilm inhibitory and good biocompatibility for periodontitis treatment. The present application provides an innovative solution for minimally invasive treatment of periodontitis through multi-component synergistic design. SUMMARY

[0005] In view of the above technical problems in the prior art, the present application provides an antibacterial repair conjugated polymer nanomaterial, a preparation method and application thereof, which solve the technical problem of poor effect of existing drugs on the treatment of periodontitis.

[0006] The present application provides a preparation method of an antibacterial repair conjugated polymer nanomaterial, comprising the following steps:

[0007] 1) Conjugated polymer solution preparation:

[0008] a) Dissolve the conjugated polymer SP II in a first organic solvent, and ultrasonically treat until completely dissolved to obtain a conjugated polymer SP II solution; the structural formula of the conjugated polymer SP II is as follows,

[0009] n = 5-20;

[0010] b) Dissolve bovine serum albumin in water, ultrasonically disperse, and then add dropwise to the conjugated polymer SP II solution, and ultrasonically treat to form a BSA@SP II complex.

[0011] 2) Thin film preparation and reorganization: dipalmitoyl phosphatidylcholine and DSPE-mPEG (distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000) are dissolved in a second organic solvent, ultrasonic dispersion and evaporation into a film, resuspended in water, ultrasonic dispersion to form a homogeneous solution;

[0012] 3) Complex and purification: BSA@SP II complex and superoxide dismutase are added to the solution of step 2), heated at 60-70℃ for 0.5-2 hours, and stirred until cooled;

[0013] 4) Ultrafiltration purification, remove the free components, obtain the conjugated polymer nanomaterial (SPNS nanoparticles) for antibacterial repair.

[0014] Further, in step 3), CaCl2 is added, and stirring is continued for 8-16 hours, so that loaded on the surface of nanoparticles by electrostatic interaction, and SPNS Ca (conjugated polymer nanomaterial containing calcium ions).

[0015] Further, the concentration of the SP II solution is 0.2-0.5 mg / ml; the concentration of bovine serum albumin is 10-30 mg / ml; the mass ratio of SP II and bovine serum albumin is 0.25: (10-30); and the first organic solvent is tetrahydrofuran.

[0016] Further, the concentration of the dipalmitoyl phosphatidylcholine solution is 2-8 mg / ml; the concentration of DSPE-mPEG is 2-8 mg / ml; the mass ratio of dipalmitoyl phosphatidylcholine and DSPE-mPEG is 1: (1-3); and the second organic solvent is chloroform.

[0017] Further, the volume ratio of BSA@SP II complex, superoxide dismutase (SOD), and the solution of step 2) is 1:1:10.

[0018] Further, the volume ratio of BSA@SP II complex, superoxide dismutase (SOD), and CaCl 2、 The material ratio of the solution of step 2) is 1 ml: 1 ml: 50 mg: 10 ml.

[0019] The application also provides a conjugated polymer nanomaterial prepared by the above method.

[0020] The application also provides the use of the above nanomaterial in the preparation of a drug for treating periodontitis.

[0021] Specifically, DSPE-mPEG (distearoylphosphatidylethanolamine-polyethylene glycol 2000) is a commercially available material purchased from Shanghai Punsen Biotechnology Co., Ltd.

[0022] The application also provides a conjugated polymer, whose structural formula is shown as follows,

[0023] n = 5-20.

[0024] Further, the chemical equation for its preparation is shown as follows,

[0025] .

[0026] Specifically, the preparation method of the conjugated polymer comprises the following steps:

[0027] 1) dissolving benzodithiazole, 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethylstannyl)thiophene-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, and tris(o-tolyl)phosphine in a third organic solvent, then transferring into a Schlenk flask, and then performing at least two cycles of freezing-vacuumizing-thawing to degas;

[0028] 2) under the protection of nitrogen, heating the mixture of step 1) in an oil bath to perform a Stille polymerization reaction; after the reaction is completed, collecting the precipitate of the obtained mixture by centrifugation, washing the obtained precipitate to remove impurities, and then drying overnight under vacuum to obtain the conjugated polymer.

[0029] The application aims to provide a multifunctional conjugated polymer nanomaterial (SPNS series) which integrates antibacterial, photothermal therapy, osteogenic differentiation promotion and biofilm inhibition functions, and solves the problems of single function, insufficient curative effect and low biological safety in existing periodontitis treatment.

[0030] The multifunctional conjugated polymer nanomaterial (SPNS series) developed by the application, based on the photoelectric properties of conjugated polymer SP II, by loading active ingredients such as superoxide dismutase (SOD), and modifying DSPE-mPEG to improve biocompatibility, realizes the following technical breakthroughs:

[0031] Broad-spectrum antibacterial and photothermal synergy: by releasing to destroy the bacterial membrane structure, combined with the photothermal effect triggered by 1064 nm laser, significantly improving the antibacterial efficiency and reducing drug resistance;

[0032] Promoting bone repair and osteogenic differentiation regulation: loaded SOD can scavenge excess ROS, direct osteogenic differentiation of bone marrow mesenchymal stem cells (high expression of ALP, Runx2, etc.), and accelerate periodontal bone defect repair;

[0033] Biofilm-targeted inhibition: by destroying the biofilm structure of Porphyromonas gingivalis, reducing the levels of inflammatory factors such as IL-1β and TNF-α, and improving the periodontal microenvironment;

[0034] Biological safety optimization: DSPE-mPEG modification significantly reduces the hemolysis rate of the material (<5%), and in vivo experiments confirm that it has no toxicity to organs such as the heart and spleen.

[0035] Compared with the prior art, the technical effect of the present application is positive and obvious. Through the design and optimization of multifunctional conjugated polymer nanomaterials, the present application breaks through the limitations of traditional periodontitis treatment and has application potential in terms of antibacterial efficiency, bone repair ability and biological safety, providing an efficient, low-toxic and comprehensive innovative tool for the field of oral disease treatment. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a physical and chemical property characterization diagram of the material; wherein a is an electron micrograph of SPNS, SPN Ca , and SPNS Ca , b is the average particle size of the nanomaterial, c is an electrical potential diagram of three different materials, d is an SOD release diagram of two different materials, e is a particle size stability diagram of three different materials, and f is a polydispersity index (PDI).

[0037] Figure 2 is a temperature rise curve diagram of three materials at different concentrations. a is the temperature rise curve of SPNS at five different concentrations; b is the temperature rise curve of SPN Ca at five different concentrations; and c is the temperature rise curve of SPNS Ca at five different concentrations.

[0038] Figure 3 is a one-time circulation diagram of different materials at 100 ppm.

[0039] Figure 4 is a three-time circulation diagram of different materials at 100 ppm.

[0040] Figure 5 is a biological safety evaluation of three materials, a hemolytic diagram.

[0041] Figure 6 is an antibacterial performance evaluation of three materials, a is a CFU diagram of bacteria, and b is a live and dead performance staining diagram of bacteria.

[0042] Figure 7 is a micro-CT diagram for detecting the osteogenic performance of mice.

[0043] Figure 8 In-vivo toxicity evaluation of the polymer nanomaterials of the present application. DETAILED DESCRIPTION Example 1

[0044] The present application provides a preparation method of a conjugated polymer nanomaterial for antibacterial repair, comprising the following steps:

[0045] 1) Conjugated polymer solution preparation:

[0046] a) 0.25 mg of conjugated polymer SP II (prepared by the method of Example 2) was dissolved in 1 mL of tetrahydrofuran (THF), and ultrasonic treatment was performed until complete dissolution to obtain an SP II solution.

[0047] b) 20 mg of bovine serum albumin (BSA) was dissolved in 1 mL of water, and after ultrasonic dispersion, it was slowly added dropwise to the above SP II solution, and ultrasonic treatment was continued for 5-10 minutes to form a BSA@SP II complex.

[0048] 2) Thin film preparation and reorganization: 20 mg of dipalmitoyl phosphatidylcholine (DPPC) and 20 mg of DSPE-mPEG (distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, Shanghai Punsen Biotechnology Co., Ltd.) were dissolved in 5 mL of chloroform, and after ultrasonic dispersion, a film was evaporated at 45°C and 140 rpm. Resuspend the film in 10 mL of water, and ultrasonic for 5 minutes to completely disperse it to form a homogeneous solution.

[0049] 3) Complexation and purification: 1 ml of BSA@SP II complex and 1 ml of superoxide dismutase (SOD) were added to 10 ml of the solution of step 2) above, and heated at 65°C for 1 hour, and stirred until cooled.

[0050] 4) Ultrafiltration purification to remove free components not wrapped to obtain SPNS nanoparticles.

[0051] 5) Preparation of calcium ion loaded nanomaterial (SPNS Ca ): In the SPNS preparation step, 0.5 mg of CaCl2 was added, and stirring was continued for 12 hours to allow loading on the surface of the nanoparticles by electrostatic interaction, and after ultrafiltration, SPNS Ca was obtained.

[0052] The present application also provides a preparation method of a control material (SPN Ca ):

[0053] SPNS preparation was omitted, and the remaining steps were consistent with SPNS preparation, 0.5 mg of CaCl2 was added, and stirring was continued for 12 hours to allow Electrostatic loading was applied to the surface of nanoparticles, followed by ultrafiltration to obtain control nanoparticles (SPN) without functional components. Ca ).

[0054] 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 diagram shows the stability of three different material particles. The data above indicates that the material is uniformly dispersed, has a defined surface charge, and exhibits high stability.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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... Ca It 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.

[0060] In vivo periodontitis treatment experiments, animal model establishment:

[0061] 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.

[0062] Treatment efficacy assessment:

[0063] Histological analysis: H&E staining was used to observe the recovery of gingival papillae, and Masson staining was used to detect collagen deposition.

[0064] 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.

[0065] 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.

[0066] 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

[0067] The present application also provides a preparation method of the conjugated polymer SP II of the above embodiment 1:

[0068] Benzobisthiazole (monomer 1, 25 mg, 0.023 mmol), 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethylstannyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (monomer 2, 21.3 mg, 0.025 mmol), Pd(PPh3)4(1.0 mg, 0.001 mmol) and tri(o-tolyl)phosphine (2.0 mg, 0.006 mmol) were dissolved in chlorobenzene (4 mL) and then transferred into a 50 mL Schlenk flask, followed by 3 cycles of freeze-vacuum-thaw to degas. (1.0 mg, 0.001 mmol) and tri(o-tolyl)phosphine (2.0 mg, 0.006 mmol) were dissolved in chlorobenzene (4 mL) and then transferred into a 50 mL Schlenk flask, followed by 3 cycles of freeze-vacuum-thaw to degas.

[0069] After that, the mixture was heated in an oil bath at 100 °C for 2 hours under nitrogen protection for Stille polymerization. After the reaction was completed, the obtained mixture was added dropwise into methanol, and the precipitate was collected by centrifugation. The obtained precipitate was washed with methanol for 3 times to remove impurities, and then dried overnight under vacuum to remove methanol to obtain the conjugated polymer SP II.

[0070] 1H1H 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).

[0071] The reaction equation is described as follows:

[0072]

Claims

1. A method for preparing conjugated polymer nanomaterials for antibacterial repair, characterized in that... Comprising the following steps: 1) conjugated polymer solution preparation: a) dissolving conjugated polymer SP II in a first organic solvent, ultrasonic treatment until completely dissolved to obtain a conjugated polymer SP II solution; the structural formula of the conjugated polymer SP II is as follows, ，n=5~20； b) dissolving bovine serum albumin in water, ultrasonic dispersion, then adding dropwise to the above conjugated polymer SP II solution, ultrasonic treatment to form a BSA@SP II complex; 2) film preparation and reorganization: dissolving dipalmitoyl phosphatidylcholine and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 in a second organic solvent, ultrasonic dispersion, then evaporating into a film, adding water to resuspend the film, ultrasonic treatment to disperse to form a homogeneous solution; 3) complexing and purification: adding BSA@SP II complex and superoxide dismutase to the solution of step 2) above, heating at 60~70℃ for 0.5~2 hours, stirring until cooling; 4) ultrafiltration purification to obtain a conjugated polymer nanomaterial for antibacterial repair.

2. The method according to claim 1, wherein the method is characterized by: In step 3), CaCl2 is added, and stirring is continued for 8-16 hours, so that Ca2+ is loaded on the surface of the nanoparticles by electrostatic action, and SPNS is obtained after ultrafiltration Ca .

3. The method according to claim 1, wherein the method is characterized by: The concentration of the conjugated polymer SP II solution is 0.2~0.5 mg / ml; the concentration of bovine serum albumin is 10~30 mg / ml; the mass ratio of SP II to bovine serum albumin is 0.25:(10~30); the first organic solvent is tetrahydrofuran.

4. The method according to claim 1, wherein the method is characterized by: The concentration of the dipalmitoyl phosphatidylcholine solution is 2~8 mg / ml; the concentration of the distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is 2~8 mg / ml; the mass ratio of dipalmitoyl phosphatidylcholine to distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is 1:(1~3); the second organic solvent is chloroform.

5. The method according to claim 1, wherein the method is characterized by: The volume ratio of BSA@SP II complex to superoxide dismutase is 1:

1.

6. The conjugated polymer nanomaterial obtained by the method of claim 1.

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

8. A conjugated polymer characterized by: The structural formula is as follows, ，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 as follows, 。 10. A method for preparing a conjugated polymer according to claim 9, characterized in that... Comprising the following steps: 1) dissolving benzobisthiazole, 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethylstannyl)thiophene-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, Pd2(dba)3 and tri(ortho-tolyl)phosphine in a third organic solvent, then transferring to a Schlenk flask, and performing at least two cycles of freezing-vacuumizing-thawing to degas; 2) under nitrogen protection, heating the mixture of step 1) in an oil bath to perform Stille polymerization reaction; after the reaction is completed, the obtained mixture is collected by centrifugation to collect the precipitate, the obtained precipitate is washed to remove impurities, and then dried under vacuum overnight to obtain a conjugated polymer.

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