Nanocomposites Loaded with Quorum Sensing Inhibitors, Preparation Methods Thereof, and Applications

Abstract

The present invention discloses a nano-composite material loaded with a quorum sensing inhibitor, its preparation method and application, belonging to the field of biomedical materials. Among them, the preparation method includes: dispersing MnO2 nanoparticles in an aqueous solution of polyallylamine hydrochloride to obtain a MnO2 / PAH complex; dispersing the MnO2 / PAH complex in an aqueous solution of polyacrylic acid for secondary modification; mixing the modified nanoparticles with a MgCl2 solution, and performing ultrasonic treatment and aging treatment to obtain a MnO2-Mg complex; using a chemical reduction method to in-situ grow platinum nanoparticles and load them on the surface of the MnO2-Mg complex to obtain MnO2-Mg / Pt; loading BBF on MnO2-Mg / Pt to obtain a MnO2-Mg / Pt@BBF nano-composite material. The purpose of the present invention is to enhance the chemodynamic efficiency, synergistically inhibit the biofilm effect, and achieve efficient treatment of periodontitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly to a nano-composite material loaded with a quorum sensing inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] Periodontitis is a chronic inflammatory disease driven by biofilms. Periodontal inflammatory lesions not only cause local tissue damage, manifested as clinical symptoms such as gingival congestive swelling, bleeding, tooth loosening and even tooth loss, but also show a close correlation with many systemic diseases. Research has confirmed that such inflammatory states can significantly increase the incidence risk of chronic diseases such as cardiovascular and cerebrovascular dysfunction and glucose metabolism imbalance. Pathological studies have shown that the total inner wall area of the periodontal pockets of patients can reach 50 - 72 cm 2 The pathological characteristics of which constitute the anatomical basis for microorganisms to invade the circulatory system. Oral pathogenic bacteria and their active metabolic components can enter the blood through this pathological channel, spread to various target organs through the systemic circulation, induce a systemic inflammatory response, and further promote the accelerated development of pathological processes such as atherosclerosis. Therefore, more and more scholars are concerned about the efficient treatment strategies for periodontitis.

[0003] Pathogenic biofilms play a crucial role in the occurrence and development of periodontitis. As an initiating factor, 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 immunity and drug attacks, enhancing bacterial drug resistance. Bacteria and their metabolites in the biofilm continuously stimulate the periodontal tissue, release inflammatory factors, directly damage the periodontal tissue and interfere with the normal metabolism of bone tissue, resulting in alveolar bone resorption and tooth loosening. In addition, pathogenic biofilms also promote the development of bacterial drug resistance, increasing the difficulty of treating periodontitis, and are the key factors for the occurrence, development and intractability of periodontitis.

[0004] Chemodynamic therapy (CDT) is an emerging antibacterial therapy strategy. Its core principle is to generate reactive oxygen species (ROS) at the lesion site through the catalytic reaction of metal ions, thereby achieving the killing of pathogens. Manganese oxide nanozyme has the advantages of low cost, simple preparation, and low biological toxicity. Moreover, due to its multiple valence states, multiple structural forms, and characteristics such as rapid electron and oxygen transfer, it is considered a CDT catalyst with application potential. Manganese oxide nanozyme can simulate peroxidase activity under physiological conditions and efficiently catalyze the conversion of hydrogen peroxide in the bacterial microenvironment into highly toxic hydroxyl radicals. This endogenous ROS burst mechanism breaks through the dependence on external light excitation in traditional photodynamic therapy and realizes a non-invasive and continuous antimicrobial effect. In addition, compared with traditional antibiotics, manganese oxide nanozyme has unique advantages: because its antibacterial mechanism is through the oxidation of ROS rather than through specific molecular targets, it will not lead to the generation of bacterial drug resistance. Manganese oxide nanozyme 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 oxide nanozyme in the antibacterial field, its practical application still faces multiple challenges. The catalytic activity of nanozyme is affected by various factors, such as hydrogen peroxide concentration, pH value, etc. And the complex physiological environment in vivo is often difficult to reach the optimal conditions for nanozyme catalysis, thereby affecting its antibacterial effect and being difficult to meet clinical needs. From the perspective of the mechanism of action, manganese dioxide nanozyme mainly generates reactive oxygen species such as strongly oxidizing hydroxyl radicals and superoxide anions through the release of manganese ions (Mn 2+ ), which reacts with hydrogen peroxide in a Fenton-like reaction. However, in a simulated physiological environment, the release concentration of Mn 2+ is usually lower than 0.5 μM, far lower than the optimal concentration (5 - 10 μM) required for traditional Fenton reaction, resulting in low ROS generation efficiency. This limitation stems from the stability of the manganese dioxide nanocrystal structure: its tight lattice structure hinders the release of Mn 2+for effective dissolution. Although surface modification or size regulation can partially improve the ion release behavior, it may cause new problems such as nanoparticle aggregation or decreased biocompatibility. In addition, the catalytic activity of manganese dioxide nanozymes shows significant environmental sensitivity, and their antibacterial efficacy is dynamically regulated by multiple environmental parameters. Among them, the influence of hydrogen peroxide concentration is particularly crucial. 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. Research shows that when the hydrogen peroxide concentration is high (such as the hydrogen peroxide concentration locally accumulated in the biofilm ≥ 100 μM), its effectiveness as a substrate for the Fenton-like reaction is significantly improved, promoting Mn 2+ dissolution and driving the generation of highly reactive hydroxyl radicals, and the antibacterial efficiency can be enhanced by 2-3 times. However, under the condition of low hydrogen peroxide concentration (<50 μM), the catalytic pathway of nanozymes turns to mainly generate O2, resulting in an increased proportion of singlet oxygen or superoxide anion with weak oxidation ability, and the antibacterial activity is significantly limited. It should be noted that the spatiotemporal heterogeneity of hydrogen peroxide in bacterial biofilms may further limit the catalytic efficiency of nanozymes: antioxidant enzymes (such as catalase) and reducing metabolites in the biofilm matrix will rapidly scavenge hydrogen peroxide, forming a local "catalytic substrate depletion area", thus weakening the continuous generation ability of highly reactive hydroxyl radicals.

[0006] In summary, manganese oxide nanozymes show the potential to efficiently remove drug-resistant bacterial biofilms through ROS-mediated chemodynamic effects, and their advantages lie in the multi-functional synergy of non-invasive catalysis, anti-drug resistance, and tissue repair promotion. However, the Mn 2+ release kinetics is limited (such as surface passivation, lattice stability) and the high sensitivity of catalytic activity to the microenvironment result in the ROS generation efficiency being difficult to meet the requirements of ideal antibacterial. Summary of the Invention

[0007] The purpose of the present invention is to provide a preparation method of a nano-composite material loaded with a quorum sensing inhibitor to solve the problems proposed in the above background technology.

[0008] To achieve the above purpose, the embodiments of the present invention provide the following technical solutions: A preparation method of a nano-composite material loaded with a quorum sensing inhibitor, comprising the following steps:

[0009] Disperse MnO2 nanoparticles in an aqueous solution of polyallylamine hydrochloride for surface modification, and then perform centrifugation to collect the product to obtain the MnO2 / PAH complex;

[0010] Disperse the MnO2 / PAH complex in an aqueous solution of polyacrylic acid for secondary modification, and then perform centrifugation to collect the product to obtain the modified nanoparticles;

[0011] Mix the modified nanoparticles with the MgCl₂ solution, and perform ultrasonic treatment and aging treatment, followed by centrifugation to collect the product, obtaining the MnO₂-Mg composite;

[0012] Use the chemical reduction method to in-situ grow and load platinum nanoparticles on the surface of the MnO₂-Mg composite to obtain the MnO₂-Mg / Pt nanocomposite;

[0013] Select a surface immobilization strategy based on azide-nitrene chemistry to load (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone on the MnO₂-Mg / Pt nanocomposite to obtain the MnO₂-Mg / Pt@BBF nanocomposite.

[0014] Preferably, the preparation method of the MnO₂ nanoparticles comprises the following steps:

[0015] Mix the KMnO₄ solution with oleic acid and magnetically stir the reaction in a constant temperature water bath; after the reaction system naturally cools to room temperature, perform centrifugation to collect the brownish-black precipitate; then remove the residual reactants to obtain the MnO₂ nanoparticles.

[0016] 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.

[0017] Preferably, the concentration of the MgCl₂ solution is 0.1 - 0.3 mol / L.

[0018] Preferably, the step of using the chemical reduction method to in-situ grow and load platinum nanoparticles on the surface of the MnO₂-Mg composite to obtain the MnO₂-Mg / Pt nanocomposite specifically comprises:

[0019] Dissolve polyvinylpyrrolidone and sodium citrate together in ultrapure water to obtain a mixed solution;

[0020] Transfer the above mixed solution to a constant temperature water bath at 75 - 85 °C, maintain mechanical stirring, and slowly dropwise add an aqueous solution of chloroplatinic acid at a rate of 0.3 - 0.7 mL / min for reaction;

[0021] Dropwise add an aqueous solution of ascorbic acid to the reaction system, and then immediately add the pre-dispersed aqueous solution of the MnO₂-Mg composite for reaction to in-situ grow and load platinum nanoparticles on the surface of the MnO₂-Mg composite, and then obtain the MnO₂-Mg / Pt nanocomposite through separation and purification.

[0022] 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; the concentration of the MnO2-Mg composite aqueous solution is 0.2 - 0.6 mg / mL.

[0023] Preferably, a surface immobilization strategy based on azide-nitrene chemistry is selected. The steps of loading (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone on the MnO2-Mg / Pt nanocomposite to obtain the MnO2-Mg / Pt@BBF nanocomposite specifically include:

[0024] Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to the aqueous dispersion of the MnO2-Mg / Pt nanocomposite and perform oscillating activation in a constant temperature shaker at 3 - 5 °C.

[0025] Centrifuge the activated dispersion, discard the supernatant, and then perform repeated washing.

[0026] Under light-shielded conditions, redisperse the washed composite nanomaterial in Tris-HCl buffer, and then add 4-azidoaniline hydrochloride solution and stir at 20 - 30 °C for reaction.

[0027] Add acetone to the reaction system, mix evenly and let it stand to allow the solvent to evaporate naturally. Subsequently, perform irradiation treatment under an ultraviolet lamp to induce the conversion of azide groups into highly reactive nitrene intermediates and capture (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone molecules, and then obtain the MnO2-Mg / Pt@BBF nanocomposite through separation and purification.

[0028] Preferably, the concentration of the aqueous dispersion of the MnO2-Mg / Pt nanocomposite is 1 - 3 mg / mL; the final concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1 - 3 mmol / L; the final concentration of the 4-azidoaniline hydrochloride solution is 0.5 - 1.5 mmol / L.

[0029] Another object of the embodiment of the present invention is to provide a nanocomposite prepared by the above preparation method.

[0030] Another object of the embodiment of the present invention is to provide an application of the above nanocomposite in the preparation of periodontitis treatment drugs and / or antibacterial drugs.

[0031] A preparation method of a nanocomposite loaded with a quorum sensing inhibitor provided by the present invention designs a nanomaterial of atom-doped manganese oxide loaded with a small molecule inhibitor, introduces Pt Nps, and through strong metal interaction, makes Mn2+ Increased content; introducing Mg 2+ Enhance 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 purpose of the present invention is to enhance the chemodynamic efficiency, synergistically inhibit the biofilm effect, and achieve efficient treatment of periodontitis. Brief Description of the Drawings

[0032] Figure 1 TEM images of each material prepared in the examples of the present invention; in the figure, (A) is the TEM image of MnO2; (B) is the TEM image of MnO2-Mg; (C) is the TEM image of MnO2-Mg / Pt (the upper right inset is the HR-TEM of NPs); (D) is the TEM image of MnO2-Mg / Pt@BBF;

[0033] Figure 2 Mapping diagram of MnO2-Mg / Pt prepared in the examples of the present invention;

[0034] Figure 3 FT-IR images of different materials;

[0035] Figure 4 Zeta potential comparison diagram of different materials;

[0036] Figure 5 XPS spectra of different materials; in the figure, (A) XPS spectrum of Mn 2p of MnO2-Mg; (B) XPS spectrum of Mn 2p of MnO2-Mg / Pt@BBF; (C) XPS spectrum of Pt 4f of MnO2-Mg / Pt@BBF; (D) XPS spectrum of Mg 1s of MnO2-Mg / Pt@BBF;

[0037] Figure 6 Results diagram of detecting H2O2 generation by Prussian blue agar plate;

[0038] Figure 7 Results diagram of measuring the relative expression level of S. gordonii spxB gene in the presence of different materials by quantitative reverse transcription polymerase chain reaction (qRT-PCR);

[0039] Figure 8 Results diagram of detecting the generation characteristics of hydroxyl radicals; in the figure, (A) UV-visible spectra of TMB in different material systems; (B) Regulation effect of concentration gradient change (10 - 50 μg mL -1 ) of MnO2-Mg / Pt@BBF on the TMB color reaction; (C) Regulation of fixed-dose nanomaterials (40 μg mL -1) UV-Vis spectral characteristics;

[0040] Figure 9 Results of the effects of different material interventions on the generation of reactive oxygen species in mono- and dual-species biofilms; In the figure, (A) ROS fluorescence staining images; (B) Statistical characterization of the fluorescence signal intensity of the mono-species; (C) Statistical characterization of the fluorescence signal intensity of the dual-species (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.).

[0041] Figure 10 Results of the CCK-8 assay at 24 h and 72 h (*P < 0.05, **P < 0.01, ***P <0.001, ns, not significant.).

[0042] Figure 11 Results of the hemolysis assay;

[0043] Figure 12 Results of H&E staining of major organs (including the heart, liver, spleen, lung, and kidney);

[0044] Figure 13 Comparison of the bacterial content in P. gingivalis mono-species biofilms and S. gordonii-P. gingivalis multi-species biofilms evaluated by CFU; In the figure, (A) Image of P. gingivalis mono-species biofilm; (B) CFU count of P. gingivalis mono-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.).

[0045] Figure 14Comparison diagrams of bacterial status and biofilm thickness of P. gingivalis monoculture biofilms and S. gordonii - P. gingivalis multi - species biofilms detected by live / dead staining: (A) 3D imaging of live / dead staining and live / dead bacterial ratio of P. gingivalis monoculture biofilms; (B) 3D imaging of live / dead staining and live / dead bacterial ratio of S. gordonii - P. gingivalis mixed - species biofilms; (C) Thickness of P. gingivalis monoculture biofilms; (D) Thickness of S. gordonii - P. gingivalis mixed - species biofilms (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.);

[0046] Figure 15 SEM images of different bacteria;

[0047] Figure 16 Evaluation of bacterial metabolism in (A) P. gingivalis monoculture biofilms and (B) S. gordonii - P. gingivalis multi - species biofilms by MTT assay (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.);

[0048] Figure 17 Detection of eDNA content in bacterial biofilms by staining method and kits: (A) SYTOX staining images of P. gingivalis monoculture biofilms and S. gordonii - P. gingivalis multi - species biofilms after treatment with different nanomaterials. Quantification results of eDNA in (B) P. gingivalis monoculture biofilms and (C) S. gordonii - P. gingivalis multi - species biofilms treated with different nanomaterials (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.);

[0049] Figure 18 Comparison diagrams of (A) nucleic acid and (B) protein leakage in P. gingivalis monoculture biofilms and S. gordonii - P. gingivalis multi - species biofilms (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.);

[0050] Figure 19 Expression results of P. gingivalis AI-2 (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.);

[0051] Figure 20 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.);

[0052] Figure 21 Experimental results of evaluating the antibacterial effects of different materials by CFU; In the figure, (A) is the antibacterial effect diagram of different materials in rats evaluated by CFU; B is the bar chart of CFU (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.);

[0053] Figure 22 Experimental results of three-dimensional modeling of alveolar bone in the maxillary molar area of rats based on Micro-CT; In the figure, (A) is the three-dimensional modeling diagram of alveolar bone in the maxillary molar area of rats based on Micro-CT; (B) is the bar chart of the measurement of the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.);

[0054] Figure 23 H&E staining pathological analysis of the 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.);

[0055] Figure 24 Evaluation of the collagen state in the periodontal inflammation area based on Masson trichrome staining: (A) Images of collagen fiber morphology; (B) Quantitative analysis of collagen degradation degree (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.);

[0056] Figure 25Localization 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) Analysis of relative intensity of IL-6 immunofluorescence signal (n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant.);

[0057] Figure 26 Localization and quantitative analysis of Arg-1 inflammatory factor in gingival tissue: (A) Immunofluorescence micrograph of gingival tissue showing co-localization of Arg-1 (green) and nuclear staining (DAPI, blue); (B) Analysis of relative intensity of Arg-1 immunofluorescence signal (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.). Detailed implementation manners

[0058] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0059] In order to improve the antibacterial effect of manganese dioxide enzyme, it is imperative to optimize the preparation method of manganese dioxide nanozyme. For example, manganese dioxide nanozyme (OA-MnO2) is prepared by reducing potassium permanganate with oleic acid nano-template; the method can obtain nanoparticles with good dispersion and uniform particle size (about 50 nm); this hollow mesoporous OA-MnO2 has good oxidase-like activity, can release manganese ions, and can exert good antibacterial effects both in vitro and in vivo, even superior to vancomycin. In addition, the valence ratio of manganese elements on the surface of manganese dioxide can also be adjusted by chemical methods (such as increasing the content of Mn 2+ ), thereby improving its catalytic activity and further enhancing the antibacterial effect. It is found that the presence of platinum nanoparticles (Pt NPs) can induce the formation of oxygen vacancies (Oxygen Vacancy, Ovac, OV) on the surface of manganese dioxide (MnO2), and these oxygen vacancies can promote the reduction of tetravalent manganese (Mn 4+ ) to divalent manganese (Mn 2+ ), thus significantly increasing the proportion of divalent manganese in the system.

[0060] The microenvironment responsive design of manganese dioxide enzymes also greatly enhances their antibacterial efficiency. For example, manganese dioxide nanozymes that respond to the H2O2 microenvironment can optimize their catalytic activity by regulating the local H2O2 concentration, dynamically maintaining the optimal reaction threshold of H2O2 (usually 1-5 mM) at the infection site, thereby achieving a simultaneous improvement in ROS generation efficiency and bacterial removal efficiency. This intelligent response mechanism not only overcomes the problem of excessive dependence on H2O2 concentration in the traditional Fenton reaction, but also avoids oxidative damage to host tissues by high concentrations of H2O2 through self-regulating catalytic processes, showing the application advantages of precise antibacterial. In the periodontitis microenvironment, the leader of subgingival plaque, Streptococcus gordonii (S.gordonii), produces hydrogen peroxide (H2O2) through its metabolic activities, a process that is highly dependent on its secreted pyruvate oxidase (SpxB). SpxB can oxidize pyruvate into acetic acid, carbon dioxide, and hydrogen peroxide. Although the expression level of H2O2 in infected sites was significantly increased compared with healthy sites, endogenous H2O2 alone was still insufficient to achieve ideal antibacterial efficiency. 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 H2O2, while Mg 2+ The concentration-dependent binding of Mg (especially at 1 mM) significantly improved the kinetics of the reaction, leading to increased H2O2 production. 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+ The induced high H2O2 production 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 the H2O2 concentration in the periodontitis microenvironment by catalyzing the SpxB enzyme activity of S. gordonii.

[0061] The multi-species biofilms formed by periodontal pathogenic bacteria have highly heterogeneous structural characteristics. The extracellular polymeric substances (EPS) matrix in their three-dimensional architecture not only restricts 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 the Fenton / Fenton-like reaction. More critically, the abundant polysaccharide and protein components in EPS can rapidly scavenge ROS, leading to a sharp decline in the bactericidal effect of CDT in the deep layer of the biofilm. It is worth noting that the structural complexity of the biofilm not only stems from physical and chemical barriers, but is also closely related to the quorum sensing regulatory network of bacteria. The quorum sensing system (QS) is a widespread intercellular information transmission mechanism in the microbial world, and its basis lies in the ability of microorganisms to secrete and recognize specific chemical signaling factors (such as autoinducers, AIs). This mechanism enables microorganisms to monitor the concentration fluctuations of the same or different species of bacteria in the environment in real time. In the QS signal transduction network, the AI-2 type regulatory pathway uses autoinducer molecules as information carriers to achieve cross-species communication, and its signal response intensity is positively correlated with the microbial population size. The synthesis of AI-2 is catalyzed by the LuxS protein (S-ribosylhomocysteine lyase) encoded by the LuxS gene. In the oral periodontal flora, AI-2 not only promotes cross-kingdom communication between different species of bacteria, but also plays a key regulatory role in biofilm formation. For example, P. gingivalis not only uses the AI-2 signal system to enhance biofilm formation and attachment, but the expression of its virulence factors is also regulated by the LuxS gene.

[0062] Brominated furan compounds: (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BBF) can specifically inhibit its catalytic function by covalently modifying the active site (Cys residue) of the LuxS enzyme, thereby reducing the synthesis amount of AI-2. This inhibitory effect can significantly weaken the biofilm formation ability of P. gingivalis - experiments have shown that after treatment with BBF, its biofilm biomass decreases by 72%, and the extracellular DNA and protein contents in the matrix decrease by 58% and 65% respectively. It is worth noting that a composite coating containing BBF was constructed on the titanium surface, and this coating showed excellent and relatively long-lasting antibacterial activity against P. gingivalis (the antibacterial rate remained >85% within 28 days), and the biofilm biomass inhibition rate reached 72%, confirming the important application value of BBF in the field of antibacterial materials. This strategy of targeting QS to block the pathogenic behavior of pathogens provides a new way for the precise intervention in the treatment of biofilm-related infections such as periodontitis.

[0063] The technical problems to be solved in the embodiments of the present invention are as follows:

[0064] Problem 1 to be solved: Improving the efficiency of ·OH generation by CDT at the lesion site is the key to ensuring the antibacterial effect of CDT. Manganese oxide can generate ·OH through the reaction of Mn 2+ with H2O2. However, both Mn 2+ and Mn 4+ exist in manganese oxide. How to regulate the ratio of Mn 2+ to Mn 4+ and increase the content of Mn 2+ is the key to improving the generation efficiency of ·OH. Pt NPs weaken the surface Mn-O bond through strong metal interaction, improve the mobility of lattice oxygen, and increase the content of Mn 2+ . The embodiments of the present invention intend to introduce Pt Nps to adjust the ratio of Mn 2+ to Mn 4+ in manganese oxide. In addition, increasing the concentration of H2O2 in the biofilm microenvironment can also effectively increase the generation efficiency of ·OH. Streptococcus gordonii in the biofilm will produce hydrogen peroxide (H2O2). During the pyruvate metabolism of Streptococcus gordonii, pyruvate oxidase (SpxB) catalyzes the conversion of pyruvate into acetyl phosphate, while generating H2O2 and O2. And Mg 2+ is a cofactor for the catalytic activity of SpxB. Supplementing magnesium will increase the catalytic efficiency of SpxB, and then increase the content of H2O2. The embodiments of the present invention intend to introduce Mg 2+ to increase the endogenous H2O2 content.

[0065] Problem 2 to be solved: Destroying the complex structure of the biofilm and promoting the penetration of ·OH in the biofilm. In the oral periodontal bacterial community, AI-2 can promote the information communication between oral bacteria and play an important role in the formation of the biofilm. (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BBF) is a brominated furanone. BBF can inhibit the activity of LuxS (S-ribosylhomocysteine lyase) by covalent modification, thereby reducing the synthesis of AI-2, and then inhibiting the action of Porphyromonas gingivalis to reduce the formation of the biofilm. A composite coating containing the novel 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 good application in antibacterial for implants. The embodiments of the present invention intend to introduce BBF to disrupt the co-aggregation of pathogenic bacteria, greatly reduce the ability of plaque biofilm formation, and increase the penetration of ·OH in the biofilm.

[0066] Example 1: This example provides a preparation method of a nano-composite material loaded with a quorum sensing inhibitor, including the following steps:

[0067] S1. 50 mL of 0.1 mol / L KMnO4 solution and 5 mL of oleic acid (OA, analytical pure) were mixed in a 50 mL three-necked flask and magnetically stirred at 60 °C in a constant temperature water bath for 12 hours. After the reaction system was naturally cooled to room temperature (25 °C), it was centrifuged at 6000 rpm for 10 minutes to collect the brownish-black precipitate. The product was washed three times with absolute ethanol to remove the residual reactants, and then MnO2 nanoparticles were obtained.

[0068] S2. 10 mg of MnO2 nanoparticles were dispersed in 10 mL of an aqueous solution of polyallylamine hydrochloride (PAH, Mw = 15000) (1 mg / mL), and ultrasonically treated at a power of 300 W for 30 minutes to ensure complete dispersion. Subsequently, it was magnetically stirred at 25 °C for 2 hours to coat with PAH. After the reaction, the product was centrifuged at 10000 rpm for 15 minutes to collect the product, and washed three times with ultrapure water (18.2 MΩ·cm) to obtain the MnO2 / PAH composite.

[0069] S3. The above MnO2 / PAH composite was redispersed in 10 mL of an aqueous solution of polyacrylic acid (PAA, Mw = 1800) (0.5 mg / mL), ultrasonically treated at 400 W for 20 minutes, and then continuously stirred at room temperature for 2 hours for secondary modification. Finally, the product was centrifuged at 12000 rpm for 15 minutes to collect the product, and washed three times with ultrapure water to obtain the modified nanoparticles.

[0070] S4. The above modified nanoparticles were mixed with 10 mL of 0.2 mol / L MgCl2 solution, ultrasonically treated in a 40 °C water bath (power 350 W, frequency 40 kHz) for 30 minutes, and then left to age at 25 °C for 12 hours. Finally, in the material purification stage, centrifugation separation technology (10000 rpm, 10 min) was used to achieve solid-liquid separation to obtain the precipitate, and then it was washed three times with ultrapure water to remove impurities. The treated material was placed in a vacuum drying equipment and dehydrated at a constant temperature of 60 °C for 6 hours to obtain the MnO2-Mg composite.

[0071] S5. Platinum nanoparticles (Pt NPs) were in-situ grown and loaded on the surface of the MnO2-Mg composite by chemical reduction method. The specific steps are as follows:

[0072] Dissolve 0.1 mmol of polyvinylpyrrolidone (PVP, Mw = 55000) and 0.2 mmol of sodium citrate (purity ≥ 99%) in 100 mL of ultrapure water, and stir magnetically at 600 rpm at 25 °C until completely dissolved (about 20 minutes). Transfer the above mixed solution to an 80 °C constant temperature water bath and maintain mechanical stirring at 800 rpm. Slowly add 5 mL of 10 mM aqueous chloroplatinic acid solution (chloroplatinic acid purchased from Sigma-Aldrich) dropwise at a rate of 0.5 mL / min using a micro-injection pump for 10 minutes. After the addition is complete, continue stirring and reacting for 5 minutes. Gradually add 10 mL of 0.1 M ascorbic acid (L-ascorbic acid) aqueous solution dropwise to the system (dropwise addition rate 1 mL / min), and then immediately add 50 mL of pre-dispersed aqueous solution of MnO2-Mg composite (concentration 0.4 mg / mL, dispersed by ultrasonic treatment at 40 kHz for 30 minutes). Maintain stirring at 80 °C and 800 rpm and react for 4 hours to in-situ grow and load Pt NPs on the surface of the MnO2-Mg composite. After the reaction is completed, naturally cool the mixture to room temperature (25 °C), collect the precipitate by vacuum filtration through a 0.22 μm polyethersulfone membrane; wash it 3 times with ultrapure water and absolute ethanol respectively; then continuously treat it in a vacuum dehydration system at 60 °C for 12 hours to obtain the MnO2-Mg / Pt nanocomposite.

[0073] S6. Select a surface immobilization strategy based on azide / nitrene chemistry to load the quorum sensing inhibitor (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (BBF) on the MnO2-Mg / Pt nanocomposite. The specific steps are as follows:

[0074] Carboxyl activation: Add a 10 mM solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to 10 mL of the aqueous dispersion of the MnO2-Mg / Pt nanocomposite (concentration 2 mg / mL, pH = 5.5) to a final concentration of 2 mM, and activate it by oscillating at 150 rpm in a constant temperature shaker at 4 °C for 12 hours;

[0075] Washing and removing by-products: Centrifuge the activated dispersion at 12000 rpm for 15 minutes, discard the supernatant, and repeat washing 3 times with ultrapure water to completely remove the unreacted EDC and by-products;

[0076] Azide group modification: Under light protection conditions, the washed nanomaterials were redispersed in 20 mL of Tris-HCl buffer solution with pH = 8.8, and 5 mM 4-azidoaniline hydrochloride (AZA) solution was added to a final concentration of 1 mM. The mixture was gently stirred at 25 °C for 4 hours;

[0077] Solvent-assisted assembly and photocrosslinking: 5 mL of acetone (purity ≥ 99.5%) was added to the reaction system and mixed evenly, then left standing for 30 minutes to allow the solvent to evaporate naturally. Subsequently, it was irradiated under a 365 nm ultraviolet lamp (power 15 mW / cm², distance 10 cm) for 2 minutes to induce the azide group to transform into a highly reactive nitrene intermediate and capture BBF molecules;

[0078] The product was collected by centrifugation at 12000 rpm for 15 minutes, washed 3 times with absolute ethanol and ultrapure water respectively (each washing solution volume was 20 mL, and ultrasonic-assisted dispersion was carried out for 5 minutes), and finally dried in the dark at 25 °C in a clean fume hood for 4 hours to obtain the BBF-functionalized MnO2-Mg / Pt@BBF nanocomposite.

[0079] Example 2: This example provides a method for preparing a nanocomposite loaded with a quorum sensing inhibitor, which is different from Example 1 in that:

[0080] The concentration of the polyallylamine hydrochloride aqueous solution used in step S2 is 0.5 mg / mL;

[0081] The concentration of the polyacrylic acid aqueous solution used in step S3 is 0.2 mg / mL;

[0082] Step S4 uses 0.1 mol / L MgCl2 solution;

[0083] The constant temperature water bath in step S5 is 75 °C, and 5 mL of 5 mM chloroplatinic acid aqueous solution is slowly added dropwise at a rate of 0.3 mL / min using a micro-injection pump; the concentration of the ascorbic acid aqueous solution is 0.05 M, and the concentration of the pre-dispersed MnO2-Mg composite aqueous solution is 0.2 mg / mL;

[0084] The concentration of the MnO2-Mg / Pt nanocomposite aqueous dispersion used in step S6 is 1 mg / mL; the final concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 1 mmol / L; the final concentration of the 4-azidoaniline hydrochloride solution is 0.5 mmol / L; the reaction was gently stirred at 20 °C.

[0085] Example 3: This example provides a method for preparing a nanocomposite loaded with a quorum sensing inhibitor, which is different from Example 1 in that:

[0086] The concentration of the polyallylamine hydrochloride aqueous solution used in step S2 is 1.5 mg / mL;

[0087] The concentration of the polyacrylic acid aqueous solution used in step S3 is 0.8 mg / mL;

[0088] Step S4 uses a 0.3 mol / L MgCl2 solution;

[0089] The constant temperature water bath in step S5 is 85 °C, and 5 mL of 15 mM chloroplatinic acid aqueous solution is slowly added dropwise at a rate of 0.7 mL / min using a micro-injection pump; the concentration of the ascorbic acid aqueous solution is 0.15 M, and the concentration of the pre-dispersed MnO2-Mg composite aqueous solution is 0.6 mg / mL;

[0090] The concentration of the MnO2-Mg / Pt nanocomposite aqueous dispersion used in step S6 is 3 mg / mL; the final concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 3 mmol / L; the final concentration of the 4-azidoaniline hydrochloride solution is 1.5 mmol / L; the reaction is gently stirred at 30 °C.

[0091] Experimental example: The MnO2 nanoparticles, MnO2-Mg composites, MnO2-Mg / Pt nanocomposites, and MnO2-Mg / Pt@BBF nanocomposites prepared in Example 1 were observed by transmission electron microscopy (TEM), and the results are as Figure 1 shown.

[0092] By mapping to compare the elemental compositions of different nanomaterials, the results are as Figure 2 shown.

[0093] The different materials prepared in each step of Example 1 were subjected to FT-IR testing, and the results are as Figure 3 shown.

[0094] The different materials prepared in each step of Example 1 were subjected to Zeta potential testing, and the results are as Figure 4 shown.

[0095] The different materials prepared in each step of Example 1 were subjected to XPS spectroscopy testing, and the results Figure 5 are shown.

[0096] The different materials prepared in each step of Example 1 were subjected to a Prussian blue agar plate detection experiment to detect the generation of H2O2 using a Prussian blue agar plate, and the results are as Figure 6 shown.

[0097] The relative expression levels of the S. gordonii spxB gene in the different materials obtained in each step of Example 1 were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the results are as Figure 7 shown.

[0098] The hydroxyl radical generation characteristics of the different materials obtained in each step of Example 1 were detected, and the results are as Figure 8 shown.

[0099] The different materials obtained in each step of Example 1 were used to intervene in the generation of reactive oxygen species in single- and dual-species biofilms, and the results are as Figure 9 shown.

[0100] The CCK-8 assay was performed on the MnO2-Mg / Pt@BBF nanocomposite prepared in Example 1, and the CCK-8 assay results at 24 h and 72 h are as Figure 10 shown.

[0101] The hemolysis assay was performed on the MnO2-Mg / Pt@BBF nanocomposite prepared in Example 1, and the results are as Figure 11 shown.

[0102] The antibacterial assays were performed on the various materials prepared in Example 1: the content of bacteria in the single-species biofilm of P. gingivalis and the multi-species biofilm of S. gordonii-P. gingivalis was evaluated by CFU, and the results are as Figure 13 shown; the bacterial state and membrane thickness of the single-species biofilm of P. gingivalis and the multi-species biofilm of S. gordonii-P. gingivalis were detected by live / dead staining, and the results are as Figure 14 shown; the SEM images of different bacteria are as Figure 15 shown; the metabolism of bacteria in the single-species biofilm of P. gingivalis and the multi-species biofilm of S. gordonii-P. gingivalis was evaluated by the MTT method, and the results are as Figure 16 shown; the eDNA content of the bacterial biofilm was detected by staining and kits, and the results are as Figure 17 shown; the leakage of nucleic acids and proteins in the single-species biofilm of P. gingivalis and the multi-species biofilm of S. gordonii-P. gingivalis was evaluated, and the results are as Figure 18 shown; the expression of P. gingivalis AI-2 is as Figure 19 shown; the RT-qPCR results of the adhesin molecule genes and virulence factor genes in the multi-species biofilm are as Figure 20 shown.

[0103] The MnO2-Mg / Pt@BBF nanocomposite prepared in Example 1 was used for animal experiments: The H&E staining results of the main organs of the animals are as Figure 12 shown; The antibacterial effects of different materials in rats were evaluated by CFU, and the results are as Figure 21 shown; Three-dimensional modeling of the alveolar bone in the maxillary molar area of rats based on Micro-CT, and the results are as Figure 22 shown; The pathological analysis results of H&E staining in the periodontal inflammation area are as Figure 23 shown; The evaluation results of the collagen state in the periodontal inflammation area based on Masson trichrome staining are as Figure 24 shown; The localization and quantitative analysis results of IL-6 inflammatory factor in gingival tissue are as Figure 25 shown; The localization and quantitative analysis results of Arg-1 inflammatory factor in gingival tissue are as Figure 26 shown.

[0104] It should be noted that the above experimental methods are all common knowledge in the art, and existing conventional experimental methods, experimental instruments and reagents can be used for experiments, which will not be elaborated here.

[0105] According to the above experimental results, it can be known that: In the embodiment of the present invention, a MnO2-Mg / Pt nanozyme loaded with a quorum sensing inhibitor is designed to enhance the chemodynamic efficiency, synergize the biofilm inhibition effect, and achieve efficient treatment of periodontitis. To improve the CDT efficiency of manganese dioxide, first, Mg 2+ is introduced to enhance the activity of SpxB enzyme of Streptococcus gordonii. This strategy greatly improves the pyruvate metabolism rate of Streptococcus gordonii, thereby increasing the local H2O2 concentration and providing sufficient substrates for the subsequent Fenton reaction. Subsequently, by introducing platinum nanoparticles (Pt NPs), the Mn 2+ content is increased - platinum nanoparticles can induce the generation of oxygen vacancies on the surface of manganese dioxide, promoting the reduction of Mn 4+ to Mn 2+ , and increasing the proportion of Mn 2+ . The constructed MnO2-Mg / Pt nano-system greatly improves the ROS generation efficiency of CDT. However, due to the structural barrier effect of the bacterial biofilm, the hydroxyl radicals (·OH) generated by MnO2-Mg / Pt nanoparticles are difficult to directly target the core of pathogenic bacteria. Therefore, in the embodiment of the present invention, BBF is introduced - this molecule can inhibit the synthesis of AI-2 signal molecules of P. gingivalis by covalently modifying the key site (Cys84 residue) of LuxS enzyme, thereby disrupting the co-aggregation of pathogenic bacteria and greatly reducing the ability of plaque biofilm formation. This innovative design significantly enhances the CDT efficacy through a triple synergistic mechanism of metabolic regulation-catalytic regulation-quorum sensing inhibition.

[0106] Based on the above-mentioned ideal embodiments of the present invention as inspiration, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of this invention. The technical scope of this invention is not limited to the content in the specification.

Claims

1. A preparation method of a nanocomposite loaded with a quorum sensing inhibitor, characterized in that, It includes the following steps: Disperse MnO2 nanoparticles in an aqueous solution of polyallylamine hydrochloride for surface modification, and then perform centrifugation to collect the product to obtain the MnO2 / PAH composite; Disperse the MnO2 / PAH composite in an aqueous solution of polyacrylic acid for secondary modification, and then perform centrifugation to collect the product to obtain the modified nanoparticles; Mix the modified nanoparticles with a MgCl2 solution, and perform ultrasonic treatment and aging treatment, and then perform centrifugation to collect the product to obtain the MnO2-Mg composite; Use a chemical reduction method to in-situ grow and load platinum nanoparticles on the surface of the MnO2-Mg composite to obtain the MnO2-Mg / Pt nanocomposite; Select a surface immobilization strategy based on azide-nitrene chemistry to load (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone on the MnO2-Mg / Pt nanocomposite to obtain the MnO2-Mg / Pt@BBF nanocomposite.

2. The preparation method of the nanocomposite material of the load population sensing inhibitor according to claim 1, characterized in that, The preparation method of the MnO2 nanoparticles includes the following steps: Mix the KMnO4 solution with oleic acid and magnetically stir the reaction in a constant temperature water bath; after the reaction system naturally cools to room temperature, perform centrifugation to collect the brownish-black precipitate; then remove the residual reactants to obtain the MnO2 nanoparticles.

3. The preparation method of the nanocomposite material of the load population sensing inhibitor according to claim 1, characterized in that, The concentration of the aqueous solution of polyallylamine hydrochloride is 0.5-1.5 mg / mL; the concentration of the aqueous solution of polyacrylic acid is 0.2-0.8 mg / mL.

4. The preparation method of the nanocomposite material of the 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 preparation method of the nanocomposite of the load population sensing inhibitor according to claim 1, characterized in that, The step of using a chemical reduction method to in-situ grow and load platinum nanoparticles on the surface of the MnO2-Mg composite to obtain the MnO2-Mg / Pt nanocomposite specifically includes: Dissolve polyvinylpyrrolidone and sodium citrate together in ultrapure water to obtain a mixed solution; Transfer the above mixed solution to a constant temperature water bath at 75-85 °C, maintain mechanical stirring, and slowly dropwise add an aqueous solution of chloroplatinic acid at a rate of 0.3-0.7 mL / min for reaction; Dropwise add an aqueous solution of ascorbic acid to the reaction system, and then immediately add the pre-dispersed aqueous solution of the MnO2-Mg composite for reaction to in-situ grow and load platinum nanoparticles on the surface of the MnO2-Mg composite, and then obtain the MnO2-Mg / Pt nanocomposite through separation and purification.

6. The preparation method of the nanocomposite of the quorum sensing inhibitor according to claim 5, characterized in that, The concentration of the aqueous solution of chloroplatinic acid is 5-15 mmol / L; the concentration of the aqueous solution of ascorbic acid is 0.05-0.15 mol / L; the concentration of the aqueous solution of the MnO2-Mg composite is 0.2-0.6 mg / mL.

7. The preparation method of the nanocomposite material of the load population sensing inhibitor according to claim 1, characterized in that, The step of selecting a surface immobilization strategy based on azide-nitrene chemistry to load (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone on the MnO2-Mg / Pt nanocomposite to obtain the MnO2-Mg / Pt@BBF nanocomposite specifically includes: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution to the aqueous dispersion of MnO2-Mg / Pt nanocomposite, and perform oscillating activation in a constant temperature shaker at 3-5 °C; Centrifuge the activated dispersion, discard the supernatant, and then perform repeated washing; Under light-shielded conditions, redisperse the washed composite nanomaterials in Tris-HCl buffer, and then add 4-azidoaniline hydrochloride solution, and carry out a stirring reaction at 20-30 °C; Add acetone to the reaction system, mix well and let it stand to allow the solvent to evaporate naturally. Subsequently, perform irradiation treatment under an ultraviolet lamp to induce the conversion of azide groups into highly reactive nitrene intermediates and capture (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone molecules, and then obtain the MnO2-Mg / Pt@BBF nanocomposite through separation and purification.

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

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

10. Use of a nanocomposite material as described in claim 9 in the preparation of a periodontitis treatment drug and / or an antibacterial drug, characterized in that, The bacteria are bacteria in P.gingivalis single-species biofilms and S.gordonii-P.gingivalis multi-species biofilms.

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