Nano composite material loaded with quorum sensing inhibitor as well as preparation method and application of nano composite material

Abstract

The invention discloses a quorum sensing inhibitor loaded nano composite material and a preparation method and application thereof, and belongs to the field of biomedical materials.The preparation method comprises the steps that MnO2 nano particles are dispersed in a polyallylamine hydrochloride aqueous solution, and a MnO2 / PAH compound is obtained; dispersing the MnO2 / PAH compound in a polyacrylic acid aqueous solution for secondary modification; the modified nano particles are mixed with a MgCl2 solution, ultrasonic treatment and aging treatment are carried out, and a MnO2-Mg compound is obtained; platinum nanoparticles grow in situ and are loaded on the surface of the MnO2-Mg compound by adopting a chemical reduction method to obtain MnO2-Mg / Pt; and loading the BBF on the MnO2-Mg / Pt to obtain the MnO2-Mg / Pt-coated BBF nano composite material. The invention aims to enhance the chemical kinetics efficiency and cooperate with the biomembrane inhibition effect to realize efficient treatment of periodontitis.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, in particular to a nanocomposite material loaded with a quorum sensing inhibitor and a preparation method and application thereof. Background Art

[0002] Periodontitis is a chronic inflammatory disease driven by biofilm. Periodontal inflammation not only causes local tissue damage, manifested as clinical symptoms such as gingival congestion and swelling, bleeding, loose teeth and even tooth loss, but is also closely related to many systemic diseases. Studies have confirmed that this type of inflammatory state can significantly increase the risk of chronic diseases such as cardiovascular and cerebrovascular dysfunction and glucose metabolism imbalance. Pathological studies have shown that the total area of ​​the inner wall of the patient's periodontal pocket can reach 50-72cm 2 The pathological characteristics of periodontitis constitute the anatomical basis for microbial invasion of the circulatory system. Oral pathogens and their active metabolites can enter the blood through this pathological channel, diffuse to various target organs through systemic circulation, induce systemic inflammatory response, and then promote the accelerated development of pathological processes such as atherosclerosis. Therefore, more and more scholars are paying attention to the efficient treatment strategies for periodontitis.

[0003] Pathogenic biofilm plays a vital role in the occurrence and development of periodontitis. As an initiator, it attaches to the tooth surface and triggers an inflammatory response. At the same time, its structural characteristics enable it to protect bacteria from host immune and drug attacks, enhancing bacterial resistance. The bacteria and their metabolites in the biofilm will continuously stimulate the periodontal tissue, release inflammatory factors, directly destroy the periodontal tissue and interfere with the normal metabolism of bone tissue, leading to alveolar bone resorption and tooth loosening. In addition, pathogenic biofilm also promotes the development of bacterial resistance, increases the difficulty of periodontitis treatment, and is a key factor in the occurrence, development and difficulty in curing periodontitis.

[0004] Chemodynamic therapy (CDT) is an emerging antibacterial treatment strategy. Its core principle is to produce reactive oxygen species (ROS) at the lesion site through the catalytic reaction of metal ions, thereby achieving the killing of pathogens. Manganese oxide nanozymes have the advantages of low cost, simple preparation, and low biological toxicity. In addition, due to its characteristics of multiple valence states and multiple structural forms, as well as rapid transfer of electrons and oxygen, it is considered to be a CDT catalyst with application potential. Manganese oxide nanozymes can mimic peroxidase activity under physiological conditions and efficiently catalyze the conversion of hydrogen peroxide into highly toxic hydroxyl radicals in the bacterial microenvironment. This endogenous ROS burst mechanism breaks through the dependence of traditional photodynamic therapy on external light excitation and achieves a non-invasive and sustained antimicrobial effect. In addition, compared with traditional antibiotics, manganese oxide nanozymes have unique advantages: because its antibacterial mechanism is through the oxidation of ROS rather than through specific molecular targets, it does not lead to the development of bacterial resistance. Manganese oxide nanozymes can also promote tissue repair and regeneration, especially showing good application prospects in the treatment of refractory wounds caused by drug-resistant biofilm infections.

[0005] However, despite the great potential of manganese dioxide nanozymes in the field of antibacterial, their practical application still faces multiple challenges. The catalytic activity of nanozymes is affected by many factors, such as hydrogen peroxide concentration, pH value, etc. The complex physiological environment in the body often makes it difficult to achieve the optimal conditions for nanozyme catalysis, which in turn affects its antibacterial effect and is difficult to meet clinical needs. From the perspective of mechanism of action, manganese dioxide nanozymes mainly release manganese ions (Mn 2+ ) reacts with hydrogen peroxide to produce highly oxidative reactive oxygen species such as hydroxyl radicals and superoxide anions. However, in a simulated physiological environment, Mn 2+ The release concentration is usually below 0.5 μM, which is far below the optimal concentration (5-10 μM) required for the traditional Fenton reaction, resulting in low ROS generation efficiency. This limitation stems from the stability of the manganese dioxide nanocrystal structure: its compact lattice structure hinders the Mn 2+, while surface modification or size regulation can partially improve the ion release behavior, it may cause new problems such as nanomaterial agglomeration or decreased biocompatibility. In addition, the catalytic activity of manganese dioxide nanozymes shows significant environmental sensitivity, and its antibacterial efficacy is dynamically regulated by multiple environmental parameters. Among them, the influence of hydrogen peroxide concentration is particularly critical, especially in the bacterial biofilm microenvironment, the concentration gradient of endogenous hydrogen peroxide plays a decisive role in the catalytic pathway and antibacterial effect of nanozymes. Studies have shown that when the concentration of hydrogen peroxide is high (such as the concentration of hydrogen peroxide accumulated locally in the biofilm ≥100μM), its effectiveness as a substrate for Fenton-like reactions is significantly improved, promoting the cleavage of Mn-O bonds. 2+ The nanozyme dissolves and drives the generation of highly active hydroxyl radicals, which can enhance the antibacterial efficiency by 2-3 times. However, under low hydrogen peroxide concentration (<50 μM), the catalytic pathway of the nanozyme switches to O 2 The main production of hydrogen peroxide is the increase in the proportion of singlet oxygen or superoxide anions with weaker oxidative ability, which significantly limits the antibacterial activity. It is worth noting that the spatiotemporal heterogeneity of hydrogen peroxide in bacterial biofilms may further limit the catalytic efficiency of nanozymes: antioxidant enzymes (such as catalase) and reductive metabolites in the biofilm matrix will quickly remove hydrogen peroxide, forming a local "catalytic substrate-deficient zone", thereby weakening the ability to continuously generate highly active hydroxyl radicals.

[0006] In summary, manganese oxide nanozymes show the potential to effectively remove drug-resistant bacterial biofilms through ROS-mediated chemokinetic effects, and their advantages lie in the multifunctional synergy of non-invasive catalysis, anti-drug resistance, and promotion of tissue repair. 2+ Limited release kinetics (such as surface passivation and lattice stability) and high sensitivity of catalytic activity to the microenvironment make it difficult for ROS generation efficiency to meet ideal antibacterial requirements. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing a nanocomposite material loaded with a quorum sensing inhibitor, so as to solve the problems mentioned in the above background technology.

[0008] To achieve the above object, the present invention provides the following technical solution: a method for preparing a nanocomposite material loaded with a quorum sensing inhibitor, comprising the following steps: MnO 2 The nanoparticles were dispersed in a polyallylamine hydrochloride aqueous solution for surface modification, followed by centrifugation to collect the product and obtain MnO 2 / PAH complex; MnO 2 The / PAH complex is dispersed in a polyacrylic acid aqueous solution for secondary modification, followed by centrifugation to collect the product and obtain modified nanoparticles; The modified nanoparticles were mixed with MgCl 2 The solutions were mixed, subjected to ultrasonic treatment and aging treatment, and then centrifuged to collect the product to obtain MnO 2 -Mg complex; In situ growth of platinum nanoparticles and loading on MnO by chemical reduction 2 -Mg complex surface, obtaining MnO 2 -Mg / Pt nanocomposites; A surface immobilization strategy based on azide-nitrene chemistry was selected to load (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone onto MnO 2 -Mg / Pt nanocomposite to obtain MnO 2 -Mg / Pt@BBF nanocomposites.

[0009] Preferably, the MnO 2 The method for preparing nanoparticles comprises the following steps: KMnO 4 The solution is mixed with oleic acid, and reacted by magnetic stirring in a constant temperature water bath; after the reaction system is naturally cooled to room temperature, the brown-black precipitate is collected by centrifugation; and then the residual reactant is removed to obtain the MnO 2 Nanoparticles.

[0010] Preferably, the concentration of the polyallylamine hydrochloride aqueous solution is 0.5-1.5 mg / mL; the concentration of the polyacrylic acid aqueous solution is 0.2-0.8 mg / mL.

[0011] Preferably, the MgCl 2 The concentration of the solution is 0.1-0.3 mol / L.

[0012] Preferably, the platinum nanoparticles are grown in situ and loaded on MnO by chemical reduction. 2 -Mg complex surface, obtaining MnO 2 -Mg / Pt nanocomposite material, specifically comprising: Dissolving polyvinyl pyrrolidone and sodium citrate in ultrapure water to obtain a mixed solution; The mixed solution was transferred to a 75-85 °C constant temperature water bath, mechanically stirred, and chloroplatinic acid aqueous solution was slowly added dropwise at a rate of 0.3-0.7 mL / min to react; Ascorbic acid aqueous solution was added dropwise to the reaction system, followed by the addition of pre-dispersed MnO 2 -Mg complex aqueous solution to react, so that platinum nanoparticles grow in situ and are loaded on MnO 2-Mg complex surface, and then separated and purified to obtain MnO 2 -Mg / Pt nanocomposite materials.

[0013] Preferably, the concentration of the chloroplatinic acid aqueous solution is 5-15 mmol / L; the concentration of the ascorbic acid aqueous solution is 0.05-0.15 mol / L; 2 The concentration of the aqueous solution of the -Mg complex is 0.2-0.6 mg / mL.

[0014] Preferably, a surface immobilization strategy based on azide-nitrene chemistry is selected to load (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone on MnO 2 -Mg / Pt nanocomposite to obtain MnO 2 The steps of manufacturing the Mg / Pt@BBF nanocomposite material specifically include: MnO 2 -1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution was added to the aqueous dispersion of the Mg / Pt nanocomposite material, and the mixture was activated by shaking in a constant temperature shaker at 3-5°C; The activated dispersion was centrifuged, the supernatant was discarded, and then the mixture was washed repeatedly; Under light-proof conditions, the washed composite nanomaterials were redispersed in a Tris-HCl buffer, and then a 4-azidoaniline hydrochloride solution was added, and the mixture was stirred and reacted at 20-30°C; Acetone was added to the reaction system and mixed evenly, and then the mixture was allowed to stand to allow the solvent to evaporate naturally. Subsequently, the reaction system was irradiated under ultraviolet light to induce the conversion of the azide group into a highly active nitrene intermediate and capture the (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone molecule, which was then separated and purified to obtain MnO 2 -Mg / Pt@BBF nanocomposites.

[0015] Preferably, the MnO 2 -The concentration of the aqueous dispersion of the Mg / Pt nanocomposite material is 1-3 mg / mL; the final concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1-3 mmol / L; and the final concentration of the 4-azidoaniline hydrochloride solution is 0.5-1.5 mmol / L.

[0016] Another object of the embodiment of the present invention is to provide a nanocomposite material prepared by the above preparation method.

[0017] Another object of the embodiments of the present invention is to provide a use of the above-mentioned nanocomposite material in the preparation of periodontitis therapeutic drugs and / or antibacterial drugs.

[0018] The invention provides a method for preparing a nanocomposite material loaded with a quorum sensing inhibitor, which designs a nanomaterial of an atom-doped manganese oxide loaded with a small molecule inhibitor, introduces Pt Nps, and makes Mn 2+ Increased content; introduction of Mg 2+ Increase the endogenous hydrogen peroxide content; introduce quorum sensing inhibitor (BBF) to inhibit the formation of biofilm and promote the penetration of OH in the biofilm. The present invention aims to enhance the chemical kinetic efficiency, coordinate the biofilm inhibition effect, and achieve efficient treatment of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 TEM images of various materials prepared in the embodiments of the present invention; in the figure, (A) is MnO 2 TEM image of MnO 2 -TEM image of Mg; (C) is MnO 2 -TEM image of Mg / Pt (the inset in the upper right corner is the HR-TEM of NPs); (D) MnO 2 -TEM image of Mg / Pt@BBF; Figure 2 MnO prepared in the embodiment of the present invention 2 -Mg / Pt mapping diagram; Figure 3 FT-IR images of different materials; Figure 4 This is a comparison chart of Zeta potential of different materials; Figure 5 XPS spectra of different materials; in the figure, (A) MnO 2 -XPS spectra of MgMn 2p; (B) MnO 2 -XPS spectrum of Mn 2p of Mg / Pt@BBF; (C) MnO 2 -XPS spectrum of Pt 4f in Mg / Pt@BBF; (D) MnO 2 -XPS spectrum of Mg / Pt@BBF Mg 1s; Figure 6 Prussian blue agar plate for detection of H 2 O 2 Generate situation result graph; Figure 7 The figure is the result of determining the relative expression of the spxB gene of S. gordonii in the presence of different materials by quantitative reverse transcription polymerase chain reaction (qRT-PCR); Figure 8The results of the hydroxyl radical generation characteristic test are shown in Figure 1. (A) UV-visible spectra of TMB in different material systems; (B) MnO 2 -Mg / Pt@BBF concentration gradient (10-50 μg mL -1 )Regulatory effect on TMB color development reaction; (C) H 2 O 2 Fixed dose nanomaterials (40 μg mL -1 )’s UV-visible spectral characteristics; Fig. 9 The results of the effects of different material interventions on the generation of reactive oxygen species in single and double bacterial species biofilms; in the figure, (A) ROS fluorescence staining image; (B) statistical representation of the fluorescence signal intensity corresponding to a single bacterial species; (C) statistical representation of the fluorescence signal intensity corresponding to double bacterial species (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.); Fig.10 The results of CCK-8 experiments at 24 h and 72 h (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.); Fig.11 This is the result of hemolysis experiment; Fig.12 H&E staining results of major organs (including heart, liver, spleen, lung, and kidney); Fig.13 Comparison of the bacterial content in P.gingivalis single-species biofilm and S.gordonii-P.gingivalis multi-species biofilm evaluated by CFU; in the figure, (A) image of P.gingivalis single-species biofilm; (B) CFU count of P.gingivalis single-species biofilm; (C) image of S.gordonii-P.gingivalis multi-species biofilm; (D) CFU count of S.gordonii-P.gingivalis multi-species biofilm (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.); Fig.14Comparison of bacterial status and membrane thickness between P. gingivalis single-species biofilm and S. gordonii -P. gingivalis multi-species biofilm detected by live / dead staining: (A) Live / dead staining 3D imaging of P. gingivalis single-species biofilm and the live / dead bacterial ratio of bacteria; (B) Live / dead staining 3D imaging of S. gordonii-P. gingivalis mixed-species biofilm and the live / dead bacterial ratio of bacteria; (C) Thickness of P. gingivalis single-species biofilm; (D) Thickness of S. gordonii-P. gingivalis mixed-species biofilm (n=3, *P < 0.05, **P < 0.01, ***P< 0.001. ns, not significant.); Fig.15 SEM images of different bacteria; Fig.16 MTT assay was used to evaluate the metabolic status of bacteria in (A) P. gingivalis single-species biofilm and (B) S. gordonii -P. gingivalis multi-species biofilm (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.); Fig.17 To detect the eDNA content of bacterial biofilms using staining methods and kits: (A) SYTOX staining images of P. gingivalis single-species biofilm and S. gordonii-P. gingivalis multi-species biofilm treated with different nanomaterials. Quantification results of eDNA in (B) P. gingivalis single-species biofilm and (C) S. gordonii-P. gingivalis multi-species biofilm treated with different nanomaterials (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.); Fig.18 Comparison of (A) nucleic acid and (B) protein leakage in P. gingivalis single-species biofilm and S. gordonii-P. gingivalis multi-species biofilm (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.); Fig.19The expression results of P.gingivalis AI-2 are shown in Figure 2 (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.); Fig. 20 The RT-qPCR results of adhesin molecule genes and virulence factor genes in multi-species biofilms (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.); Fig.21 The figures are the experimental results of evaluating the antibacterial effects of different materials by CFU; in the figure, (A) is the antibacterial effect of different materials in rats by CFU; B is the bar graph of CFU (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.); Fig. 22 The results of the three-dimensional modeling experiment of the alveolar bone in the maxillary molar area of ​​rats based on Micro-CT; in the figure, (A) is the three-dimensional modeling of the alveolar bone in the maxillary molar area of ​​rats based on Micro-CT; (B) is the bar graph of the corresponding distance measurement from the cementoenamel junction (CEJ) to the alveolar ridge top (ABC) (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, notsignificant.); Fig.23 H&E staining pathological analysis of periodontal inflammation area: (A) Characterization of inflammatory cell infiltration; (B) Quantitative analysis of immune cell density (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.); Fig.24 Evaluation of collagen status in periodontal inflammation area based on Masson trichrome staining: (A) Collagen fiber morphology image; (B) Quantitative analysis of collagen degradation (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, notsignificant.); Fig.25 Localization and quantitative analysis of IL-6 inflammatory factor in gingival tissue: (A) Immunofluorescence micrograph of gingival tissue showing co-localization of IL-6 (red) and nuclear staining (DAPI, blue); (B) Relative intensity analysis of IL-6 immunofluorescence signal (n= 6, * P < 0.05, ** P< 0.01, *** P < 0.001, ns, not significant.); Fig.26 Localization and quantitative analysis of Arg-1 inflammatory factor in gingival tissue: (A) Immunofluorescence micrographs of gingival tissue showed co-localization of Arg-1 (green) and nuclear staining (DAPI, blue); (B) relative intensity analysis of Arg-1 immunofluorescence signal (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.). DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] In order to improve the antibacterial effect of manganese dioxide enzymes, it is imperative to optimize the preparation method of manganese dioxide nanozymes. For example, manganese dioxide nanozymes (OA-MnO 2 The method can obtain nanoparticles with good dispersion and uniform particle size (about 50 nm); this hollow mesoporous OA-MnO 2 It has good oxidase-like activity and can release manganese ions. It has good antibacterial effects both in vitro and in vivo, and is even better than vancomycin. In addition, the valence ratio of manganese elements on the surface of manganese dioxide can be adjusted by chemical methods (such as increasing Mn 2+ The study found that the presence of platinum nanoparticles (Pt NPs) can induce manganese dioxide (MnO 2 ) surface to form oxygen vacancies (Ovac, OV), which can promote the tetravalent manganese (Mn 4+ ) to divalent manganese (Mn 2+ ), thereby significantly increasing the proportion of divalent manganese in the system.

[0022] The microenvironment-responsive design of manganese dioxide enzymes also greatly enhanced their antibacterial efficiency. 2 O 2 Microenvironment-responsive manganese dioxide nanozymes can regulate local H 2 O 2 The concentration optimizes its catalytic activity and dynamically maintains H2 O 2 The optimal reaction threshold (usually 1-5 mM) of the reaction was set, thereby achieving a simultaneous improvement in ROS generation efficiency and bacterial removal efficiency. This intelligent response mechanism not only overcomes the H 2 O 2 The problem of strong concentration dependence can also be avoided by self-regulating the catalytic process to avoid high concentration H 2 O 2 The oxidative damage to host tissues shows the application advantage of precise antibacterial. In the periodontitis microenvironment, the leader of subgingival plaque, Streptococcus gordonii (S.gordonii), produces hydrogen peroxide (H 2 O 2 ), a process that is highly dependent on the secretion of pyruvate oxidase (SpxB). SpxB can oxidize pyruvate into acetic acid, carbon dioxide, and hydrogen peroxide. 2 O 2 The expression level was significantly increased, but the endogenous H 2 O 2 Acting alone is still not enough to achieve ideal antibacterial efficiency. Studies have found that Mg 2+ By promoting the binding of the SpxB enzyme to its key cofactor thiamine diphosphate (ThDP), it directly enhances its catalytic efficiency. SpxB catalyzes the conversion of pyruvate to acetyl phosphate and generates H 2 O 2 , while Mg 2+ The concentration-dependent binding of H (especially at 1 mM) significantly increased the kinetics of the reaction, resulting in 2 O 2 Increased production. Mg 2+ It can also prolong the functional activity of SpxB by stabilizing its structure or protecting it from protease degradation. 2+ The increase in SpxB synthesis may be indirect through regulating mRNA stability or translation efficiency (such as ribosome binding efficiency). 2+ The metabolic enhancement mechanism mediated by Mg also significantly enhanced the ecological competitiveness of S. gordonii in the oral microbial community. 2+ Induced high H 2 O 2The production of manganese dioxide can not only directly inhibit the growth of pathogenic bacteria such as Streptococcus mutans (S. mutans), but also provide energy advantages for S. gordonii through the acetyl phosphate produced by SpxB. As a global regulatory molecule, acetyl phosphate can activate bacterial stress response pathways (such as the expression of virulence factors), thereby enhancing the adaptability and competitiveness of S. gordonii in the biofilm environment. Based on this, Mg was doped on the surface of manganese dioxide enzyme. 2+ The design strategy can regulate H in the periodontitis microenvironment by catalyzing the SpxB enzyme activity of S. gordonii. 2 O 2 concentration.

[0023] The multi-species biofilm formed by periodontal pathogens has highly heterogeneous structural characteristics. The extracellular polymeric ddubstances (EPS) matrix in its three-dimensional structure not only limits the penetration and diffusion of nanocatalysts through physical barriers, but also the microenvironment of hypoxia, low pH and high glutathione concentration in the biofilm significantly weakens the catalytic efficiency of Fenton / Fenton-like reactions. More importantly, the rich polysaccharide and protein components in EPS can quickly remove ROS, resulting in a sharp attenuation of the bactericidal effect of CDT in the deep layer of the biofilm. It is worth noting that the structural complexity of the biofilm is not only due to physical and chemical barriers, but also closely related to the quorum sensing regulatory network of bacteria. The quorum sensing system (QS) is a common intercellular information transmission mechanism in the microbial world. Its action is based on the ability of microorganisms to secrete and recognize specific chemical signal factors (such as autoinducers, AI). This mechanism enables microorganisms to monitor the concentration fluctuations of the same or different bacterial communities in the environment in real time. In the QS signal transduction network, the AI-2 type regulatory pathway uses autoinducing molecules as information carriers to achieve cross-species communication, and its signal response intensity is positively correlated with the size of the microbial population. The synthesis of AI-2 is catalyzed by the LuxS protein (S-ribosylhomocysteine ​​degrading enzyme) encoded by the LuxS gene. In the oral periodontal flora, AI-2 not only promotes cross-kingdom communication between different species, but also plays a key regulatory role in the formation of biofilms. For example, P.gingivalis not only uses the AI-2 signaling system to enhance the formation and attachment of biofilms, but the expression of its virulence factors is also regulated by the LuxS gene.

[0024] Bromofuran compounds: (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BBF) can specifically inhibit the catalytic function of LuxS enzyme by covalently modifying its active site (Cys residue), thereby reducing the synthesis of AI-2. This inhibitory effect can significantly weaken the biofilm formation ability of P.gingivalis - experiments show that after BBF treatment, the biofilm biomass is reduced by 72%, and the extracellular DNA and protein content in the matrix are reduced by 58% and 65%, respectively. It is worth noting that a composite coating containing BBF was constructed on the titanium surface, which showed excellent and relatively long-lasting antibacterial activity against P.gingivalis (the inhibition rate remained >85% within 28 days), and the biofilm biomass inhibition rate reached 72%, confirming that BBF has important application value in the field of antibacterial materials. This strategy of blocking the pathogenic behavior of pathogens by targeting QS provides a new way of precise intervention for the treatment of biofilm-related infections such as periodontitis.

[0025] The technical problems to be solved by the embodiments of the present invention are as follows: Problem 1: Improving the efficiency of CDT in producing ·OH at the lesion site is the key to ensuring the antibacterial effect of CDT: manganese oxide can be 2+ With H 2 O 2 The reaction produces ·OH. However, there is also Mn in manganese oxide. 2+ With Mn 4+ How to regulate Mn 2+ With Mn 4+ The proportion of Mn 2+ The content is the key to improving the efficiency of ·OH generation. Pt NPs weaken the surface Mn-O bonds through strong metal interactions, increase the mobility of lattice oxygen, and make Mn 2+ The present invention proposes to introduce Pt Nps to adjust the content of Mn in manganese oxide. 2+ With Mn 4+ In addition, increasing the H 2 O 2 The concentration of ·OH can also effectively increase the efficiency of OH production. Streptococcus Gordonii in biofilm produces hydrogen peroxide (H 2 O 2 In the pyruvate metabolism of Streptococcus gordonii, pyruvate oxidase (SpxB) catalyzes the conversion of pyruvate into acetyl phosphate, while producing H 2 O 2 and O 2 Mg 2+ It is a cofactor for the catalytic activity of SpxB. Supplementation of magnesium will increase the catalytic efficiency of SpxB and thus increase H 2 O 2The present invention proposes to introduce Mg 2+ Increase endogenous H 2 O 2 content.

[0026] Problem 2 to be solved: Destroy the complex structure of biofilm and promote the penetration of ·OH in biofilm: In the oral periodontal bacterial community, AI-2 can promote information communication between oral bacteria and play an important role in the formation of biofilm. (Z-)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BBF) is a brominated furanone. BBF can inhibit the activity of LuxS (S-ribosyl homocysteine ​​lyase) by covalently modifying it, thereby reducing the synthesis of AI-2, and then inhibiting the action of Porphyromonas gingivalis to reduce the formation of biofilm. A composite coating containing a new antibacterial agent BBF was prepared on titanium, and it was found that the composite coating had excellent and relatively long-term antibacterial activity against Porphyromonas gingivalis, proving that BBF also has a good application in the antibacterial aspect of implants. The embodiment of the present invention intends to introduce BBF to destroy the co-aggregation of pathogens, greatly reduce the ability of plaque biofilm formation, and increase the penetration of ·OH in the biofilm.

[0027] Example 1: This example provides a method for preparing a nanocomposite material loaded with a quorum sensing inhibitor, comprising the following steps: S1, 50 mL 0.1 mol / L KMnO 4 The solution was mixed with 5 mL of oleic acid (OA, analytical grade) in a 50 mL three-necked flask and reacted with magnetic stirring in a 60 °C constant temperature water bath for 12 hours. After the reaction system was naturally cooled to room temperature (25 °C), the brown-black precipitate was collected by centrifugation at 6000 rpm for 10 minutes. The product was washed three times with anhydrous ethanol to remove the residual reactants and obtain MnO 2 Nanoparticles.

[0028] S2, 10 mg MnO 2 The nanoparticles were dispersed in 10 mL of polyallylamine hydrochloride (PAH, Mw = 15000) aqueous solution (1 mg / mL) and ultrasonically treated at 300 W for 30 minutes to fully disperse them. Then, the PAH was coated by magnetic stirring at 25 °C for 2 hours. After the reaction, the product was collected by centrifugation at 10000 rpm for 15 minutes and washed three times with ultrapure water (18.2 MΩ·cm) to obtain MnO 2 / PAH complex.

[0029] S3, the above MnO 2The / PAH complex was redispersed in 10 mL of polyacrylic acid (PAA, Mw=1800) aqueous solution (0.5 mg / mL), and after being treated with 400 W ultrasound for 20 min, it was stirred at room temperature for 2 h for secondary modification. Finally, the product was collected by centrifugation at 12000 rpm for 15 min and washed three times with ultrapure water to obtain modified nanoparticles.

[0030] S4. Mix the modified nanoparticles with 10 mL 0.2 mol / L MgCl 2 The solutions were mixed and ultrasonically treated (power 350 W, frequency 40 kHz) in a 40°C water bath for 30 minutes, and then aged at 25°C for 12 hours. Finally, in the material purification stage, centrifugal separation technology (10,000 rpm, 10 min) was used to separate the solid and liquid to obtain the precipitate, which was then washed three times with ultrapure water to remove impurities. The treated material was placed in a vacuum drying device and continuously dehydrated at a constant temperature of 60°C for 6 hours to obtain MnO 2 -Mg complex.

[0031] S5. In situ growth of platinum nanoparticles (Pt NPs) and loading on MnO by chemical reduction 2 -Mg complex surface, the specific steps are as follows: 0.1 mmol polyvinyl pyrrolidone (PVP, Mw = 55000) and 0.2 mmol sodium citrate (purity ≥ 99%) were dissolved in 100 mL ultrapure water and magnetically stirred at 600 rpm at 25°C until completely dissolved (about 20 minutes). The mixed solution was transferred to a constant temperature water bath at 80°C and mechanically stirred at 800 rpm. 5 mL of 10 mM chloroplatinic acid aqueous solution (chloroplatinic acid purchased from Sigma-Aldrich) was slowly added dropwise at a rate of 0.5 mL / min using a microinjection pump for 10 minutes. After the addition was completed, the reaction was stirred for 5 minutes. 10 mL of 0.1 M ascorbic acid (L-ascorbic acid) aqueous solution was added dropwise to the system (drop speed 1 mL / min), followed by the immediate addition of 50 mL of pre-dispersed MnO 2 -Mg complex aqueous solution (concentration 0.4 mg / mL, dispersed by 40 kHz ultrasonic treatment for 30 minutes). Maintain 80°C and 800 rpm stirring for 4 hours to allow Pt NPs to grow in situ and be loaded on MnO 2 After the reaction, the mixture was cooled to room temperature (25°C) and the precipitate was collected by vacuum filtration through a 0.22 μm polyethersulfone filter membrane; it was washed three times with ultrapure water and anhydrous ethanol respectively; and then it was treated by a vacuum dehydration system at 60°C for 12 hours to obtain MnO 2-Mg / Pt nanocomposite materials.

[0032] S6. A surface immobilization strategy based on azide / nitrene chemistry was selected to load the quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BBF) on MnO 2 -Mg / Pt nanocomposite materials, the specific steps are as follows: Carboxyl activation: Add 10 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution to 10 mL of MnO2-Mg / Pt nanocomposite aqueous dispersion (concentration 2 mg / mL, pH = 5.5) to a final concentration of 2 mM, and activate at 150 rpm in a constant temperature shaker at 4 °C for 12 h; Washing and removal of by-products: The activated dispersion was centrifuged at 12,000 rpm for 15 min, the supernatant was discarded, and the suspension was washed three times with ultrapure water to completely remove unreacted EDC and by-products; Azide group modification: In the dark, the washed nanomaterials were redispersed in 20 mL of Tris-HCl buffer at pH 8.8, 5 mM 4-azidoaniline hydrochloride (AZA) solution was added to a final concentration of 1 mM, and the mixture was reacted at 25 °C with gentle stirring for 4 hours. Solvent-assisted assembly and photocrosslinking: Add 5 mL of acetone (purity ≥ 99.5%) to the reaction system, mix well, and let stand for 30 minutes to allow the solvent to evaporate naturally. Then, irradiate under a 365 nm UV lamp (power 15 mW / cm², distance 10 cm) for 2 minutes to induce the conversion of the azide group into a highly active nitrene intermediate and capture the BBF molecule; The product was collected by centrifugation at 12000 rpm for 15 minutes, washed with anhydrous ethanol and ultrapure water for 3 times (20 mL of washing solution each time, ultrasonic-assisted dispersion for 5 minutes), and finally dried in a clean fume hood at 25°C for 4 hours away from light to obtain BBF-functionalized MnO 2 -Mg / Pt@BBF nanocomposites.

[0033] Example 2: This example provides a method for preparing a nanocomposite material loaded with a quorum sensing inhibitor, which differs from Example 1 in that: The concentration of the polyallylamine hydrochloride aqueous solution used in step S2 is 0.5 mg / mL; The concentration of the polyacrylic acid aqueous solution used in step S3 is 0.2 mg / mL; Step S4 uses 0.1 mol / L MgCl 2 Solution; In step S5, the constant temperature water bath is set at 75°C, and 5 mL of 5 mM chloroplatinic acid aqueous solution is slowly added at a rate of 0.3 mL / min using a microinjection pump; the concentration of the ascorbic acid aqueous solution is 0.05 M, and the pre-dispersed MnO 2 -Mg complex aqueous solution concentration was 0.2 mg / mL; The MnO used in step S6 2 The concentration of the aqueous dispersion of the -Mg / Pt nanocomposite material was 1 mg / mL; the final concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution was 1 mmol / L; the final concentration of the 4-azidoaniline hydrochloride solution was 0.5 mmol / L; the reaction was carried out by gentle stirring at 20 °C.

[0034] Example 3: This example provides a method for preparing a nanocomposite material loaded with a quorum sensing inhibitor, which differs from Example 1 in that: The concentration of the polyallylamine hydrochloride aqueous solution used in step S2 is 1.5 mg / mL; The concentration of the polyacrylic acid aqueous solution used in step S3 is 0.8 mg / mL; Step S4 uses 0.3 mol / L MgCl 2 Solution; In step S5, the constant temperature water bath is 85°C, and 5 mL of 15 mM chloroplatinic acid aqueous solution is slowly added at a rate of 0.7 mL / min using a microinjection pump; the concentration of the ascorbic acid aqueous solution is 0.15 M, and the pre-dispersed MnO 2 -Mg complex aqueous solution concentration was 0.6 mg / mL; The MnO used in step S6 2 The concentration of the aqueous dispersion of the -Mg / Pt nanocomposite material was 3 mg / mL; the final concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution was 3 mmol / L; the final concentration of the 4-azidoaniline hydrochloride solution was 1.5 mmol / L; the reaction was carried out by gentle stirring at 30 °C.

[0035] Experimental Example: MnO prepared in Example 1 2 Nanoparticles, MnO 2 -Mg complex, MnO 2 -Mg / Pt nanocomposites and MnO 2 -Mg / Pt@BBF nanocomposites were observed by transmission electron microscopy (TEM). Figure 1 shown.

[0036] By comparing the elemental composition of different nanomaterials through mapping, the results are as follows Figure 2 shown.

[0037] The different materials obtained in each step of Example 1 were subjected to FT-IR testing. The results are as follows: Figure 3 shown.

[0038] The different materials prepared in each step of Example 1 were tested for Zeta potential. The results are as follows: Figure 4 shown.

[0039] The different materials prepared in each step of Example 1 were subjected to XPS spectrum test. Figure 5 shown.

[0040] The different materials prepared in each step of Example 1 were subjected to a Prussian blue agar plate test. 2 O 2 The generated results are as follows Figure 6 shown.

[0041] The relative expression of the S. gordonii spxB gene in the presence of the different materials prepared in each step of Example 1 was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results are as follows: Figure 7 shown.

[0042] The hydroxyl radical generation characteristics test experiment was carried out on the different materials prepared in each step of Example 1. The results are as follows: Figure 8 shown.

[0043] The different materials prepared in each step of Example 1 were used to conduct intervention experiments on the generation of active oxygen in single and double bacterial biofilms. The results are as follows: Fig. 9 shown.

[0044] The MnO prepared in Example 1 2 -Mg / Pt@BBF nanocomposites were subjected to CCK-8 experiments. The results of CCK-8 experiments at 24 h and 72 h were shown in Fig.10 shown.

[0045] The MnO prepared in Example 1 2 -Mg / Pt@BBF nanocomposite materials were subjected to hemolysis experiments. Fig.11 shown.

[0046] The antibacterial test was carried out on each material prepared in Example 1: the bacterial content in the P. gingivalis single-species biofilm and the S. gordonii-P. gingivalis multi-species biofilm was evaluated by CFU. The results are as follows: Fig.13 As shown; the bacterial status and film thickness of P.gingivalis single-species biofilm and S.gordonii-P.gingivalis multi-species biofilm were detected by live / dead staining. The results are shown Fig.14 As shown; SEM images of different bacteria are shown Fig.15 The MTT method was used to evaluate the metabolism of bacteria in P.gingivalis single-species biofilm and S.gordonii-P.gingivalis multi-species biofilm. The results are shown in Fig.16 As shown; the eDNA content of bacterial biofilm was detected by staining method and kit, and the results were as follows Fig.17 As shown; the leakage of nucleic acids and proteins in P. gingivalis single-species biofilm and S. gordonii-P. gingivalis multi-species biofilm was evaluated, and the results are shown Fig.18 The expression of P.gingivalis AI-2 is shown in Fig.19 The RT-qPCR results of adhesin molecule genes and virulence factor genes in multi-species biofilms are shown in Fig. 20 shown.

[0047] The MnO prepared in Example 1 2 -Mg / Pt@BBF nanocomposites were used for animal experiments: H&E staining results of the main organs of animals are shown in Fig.12 As shown; the antibacterial effect of different materials in rats was evaluated by CFU, and the results are shown Fig.21 As shown; Micro-CT-based three-dimensional modeling of the alveolar bone in the maxillary molar area of ​​rats. Fig. 22 As shown; the results of H&E staining pathological analysis of the periodontal inflammation area are shown Fig.23 The results of the evaluation of collagen status in the periodontal inflammation area based on Masson trichrome staining are shown in Fig.24 As shown; the localization and quantitative analysis results of IL-6 inflammatory factor in gingival tissue are shown Fig.25 As shown; the localization and quantitative analysis results of Arg-1 inflammatory factor in gingival tissue are shown Fig.26 shown.

[0048] It should be noted that the above experimental methods are common knowledge in the art, and the existing conventional experimental methods, experimental instruments and reagents can be used to conduct the experiment, which will not be described in detail here.

[0049] According to the above experimental results, it can be known that: the embodiment of the present invention designs a MnO loaded with a quorum sensing inhibitor 2 -Mg / Pt nanozymes are designed to enhance chemical kinetic efficiency, synergize biofilm inhibition effects, and achieve efficient treatment of periodontitis. To improve the CDT efficiency of manganese dioxide, first, Mg2+ , enhancing the activity of Streptococcus Gordonii SpxB enzyme. This strategy greatly improves the pyruvate metabolism rate of Streptococcus Gordonii, thereby increasing local H 2 O 2 concentration, providing sufficient substrate for the subsequent Fenton reaction. Subsequently, the Mn 2+ Content——Platinum nanoparticles can induce oxygen vacancies on the surface of manganese dioxide, promoting the 4+ To Mn 2+ The reduction of Mn 2+ The ratio of MnO 2 -Mg / Pt nanosystem significantly improves the efficiency of CDT in generating ROS. However, due to the structural barrier effect of bacterial biofilm, MnO 2 -The hydroxyl radicals (·OH) generated by Mg / Pt nanoparticles are difficult to directly target the core of pathogens. For this reason, the embodiment of the present invention introduces BBF, which can inhibit the synthesis of AI-2 signaling molecules of P.gingivalis by covalently modifying the key site of LuxS enzyme (Cys84 residue), thereby destroying the co-aggregation of pathogens and greatly reducing the ability of bacterial plaque biofilm formation. This innovative design significantly enhances the efficacy of CDT through the triple synergistic mechanism of metabolic regulation, catalytic regulation, and quorum sensing inhibition.

[0050] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A method for preparing a nanocomposite material loaded with a quorum sensing inhibitor, characterized in that: The following steps are involved: The MnO2 nanoparticles are dispersed in a polyallylamine hydrochloride aqueous solution for surface modification, and then centrifuged to collect the product to obtain a MnO2 / PAH complex; The MnO2 / PAH complex is dispersed in a polyacrylic acid aqueous solution for secondary modification, followed by centrifugation to collect the product and obtain modified nanoparticles; The modified nanoparticles are mixed with a MgCl2 solution, and subjected to ultrasonic treatment and aging treatment, followed by centrifugation to collect the product to obtain a MnO2-Mg complex; The chemical reduction method is used to make platinum nanoparticles grow in situ and load them on the surface of the MnO2-Mg composite to obtain the MnO2-Mg / Pt nanocomposite material; A surface immobilization strategy based on azide-nitrene chemistry was selected to load (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone on MnO2-Mg / Pt nanocomposites to obtain MnO2-Mg / Pt@BBF nanocomposites.

2. The method for preparing a nanocomposite material loaded with a quorum sensing inhibitor according to claim 1, characterized in that: The preparation method of the MnO2 nanoparticles comprises the following steps: The KMnO4 solution and oleic acid are mixed and reacted with magnetic stirring in a constant temperature water bath; after the reaction system is naturally cooled to room temperature, the brown-black precipitate is collected by centrifugation; and then the residual reactants are removed to obtain the MnO2 nanoparticles.

3. The method for preparing a nanocomposite material loaded with a quorum sensing inhibitor according to claim 1, characterized in that: The concentration of the polyallylamine hydrochloride aqueous solution is 0.5-1.5 mg / mL; the concentration of the polyacrylic acid aqueous solution is 0.2-0.8 mg / mL.

4. The method for preparing a nanocomposite material loaded with a quorum sensing inhibitor according to claim 1, characterized in that: The concentration of the MgCl2 solution is 0.1-0.3 mol / L.

5. The method for preparing a nanocomposite material loaded with a quorum sensing inhibitor according to claim 1, characterized in that: The steps of using a chemical reduction method to in-situ grow platinum nanoparticles and load them on the surface of the MnO2-Mg composite to obtain the MnO2-Mg / Pt nanocomposite material specifically include: Dissolving polyvinyl pyrrolidone and sodium citrate in ultrapure water to obtain a mixed solution; The mixed solution was transferred to a 75-85 °C constant temperature water bath, mechanically stirred, and chloroplatinic acid aqueous solution was slowly added dropwise at a rate of 0.3-0.7 mL / min to react; An ascorbic acid aqueous solution is added dropwise to the reaction system, and then a pre-dispersed MnO2-Mg complex aqueous solution is immediately added to react, so that platinum nanoparticles grow in situ and are loaded on the surface of the MnO2-Mg complex, and then separated and purified to obtain a MnO2-Mg / Pt nanocomposite material.

6. The method for preparing a nanocomposite material loaded with a quorum sensing inhibitor according to claim 5, characterized in that: The concentration of the chloroplatinic acid aqueous solution is 5-15 mmol / L; the concentration of the ascorbic acid aqueous solution is 0.05-0.15 mol / L; and the concentration of the MnO2-Mg complex aqueous solution is 0.2-0.6 mg / mL.

7. The method for preparing a nanocomposite material loaded with a quorum sensing inhibitor according to claim 1, characterized in that: A surface fixation strategy based on azide-nitrene chemistry was selected to load (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone on the MnO2-Mg / Pt nanocomposite to obtain the MnO2-Mg / Pt@BBF nanocomposite, which specifically includes: Adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to the aqueous dispersion of the MnO2-Mg / Pt nanocomposite material, and performing shaking activation in a constant temperature shaker at 3-5°C; The activated dispersion was centrifuged, the supernatant was discarded, and then the mixture was washed repeatedly; Under light-proof conditions, the washed composite nanomaterials were redispersed in a Tris-HCl buffer, and then a 4-azidoaniline hydrochloride solution was added, and the mixture was stirred and reacted at 20-30°C; Acetone was added to the reaction system and mixed evenly, then allowed to stand to allow the solvent to evaporate naturally. Subsequently, the system was irradiated under ultraviolet light to induce the conversion of the azide group into a highly active nitrene intermediate and capture the (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone molecule, which was then separated and purified to obtain the MnO2-Mg / Pt@BBF nanocomposite material.

8. The method for preparing a nanocomposite material loaded with a quorum sensing inhibitor according to claim 7, characterized in that: The concentration of the MnO2-Mg / Pt nanocomposite aqueous dispersion is 1-3 mg / mL; the final concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1-3 mmol / L; and the final concentration of the 4-azidoaniline hydrochloride solution is 0.5-1.5 mmol / L.

9. A nanocomposite material prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the nanocomposite material according to claim 9 in preparing a drug for treating periodontitis and / or an antibacterial drug.

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