CuTA-Poratepsilon-PL nano platform as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a CuTA-Por epsilon-PL nano platform as well as a preparation method and application of the CuTA-Por epsilon-PL nano platform. According to the nano platform, protoporphyrin (Por) is grafted through amidation reaction of CuTA, epsilon-polylysine (epsilon-PL) wraps the protoporphyrin (Por) through electrostatic adsorption, and therefore CuTA-Porat epsilon-PL NPs (CPP NPs) is synthesized, and the CuTA-Porat epsilon-PL NPs (CPP NPs) can be used as a potential treatment method for treating peri-implantitis, destroying bacterial biofilms and regulating inflammation.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and particularly relates to a CuTA-Por@ε-PL nano-platform and a preparation method and application thereof. Background Art

[0002] The successful clinical application of dental implants is one of the major advances in oral medicine in the 20th century. Dental implants have greatly changed the mode of repairing human tooth loss, and have made great contributions to maintaining the integrity of human oral and maxillofacial organs, restoring oral functions, and maintaining oral health and overall health. Therefore, they are called "the third set of teeth for humans." Currently, dental implant restoration has become an important repair method for patients with edentulous and missing teeth, and the number of implants has increased significantly.

[0003] The long-term stability of implants is affected by many factors, among which the occurrence of peri-implantitis (PI) is one of the main reasons for the loss of long-term stability of dental implants. PI is an inflammatory infectious disease caused by a combination of multiple factors. Plaque biofilm attacks the body as an initiating factor, triggering excessive inflammatory response in the tissues around the implant, leading to progressive destruction of the soft and hard tissues around the implant.

[0004] At present, non-surgical treatment and surgical treatment are the main methods for clinical treatment of PI. Non-surgical treatment usually includes mechanical debridement of the implant surface, assisted by local use of antibiotics or antimicrobial agents. Repeated mechanical debridement can easily scratch the crown and implant surface, and bacteria are prone to re-colonization and aggregation, resulting in stubborn residual plaque biofilm that is difficult to completely remove. Although surgical treatment can more thoroughly remove plaque biofilm, it has the risks of large trauma, high cost, postoperative gingival recession, and implant exposure. Seeking safer and more effective PI treatment methods will help improve the quality of life of patients and reduce the medical burden, and has important clinical value and social significance.

[0005] In recent years, antimicrobial photodynamic therapy (aPDT) has attracted increasing attention in antimicrobial applications due to its many advantages, such as minimally invasive, rapid and efficient onset, broad-spectrum antimicrobial activity, and no bacterial resistance. Some photosensitizers (PS), such as methylene blue, porphyrin (Por), and dihydrochlorin e6, have the ability to instantly produce a large number of reactive oxygen species (ROS) through photocatalysis under the irradiation of excitation light. They have been used in the treatment of PI or periodontitis and have achieved certain clinical effects. However, due to the special anatomical position of the peri-implant periodontal pocket and the complex structure of the plaque biofilm, aPDT still faces the following challenges in clinical application: First, the excessive ROS produced by aPDT will aggravate the oxidative stress of the peri-implant tissue and hinder clinical transformation. Second, most PS lack the ability to specifically target the plaque biofilm and will produce nonspecific phototoxicity to the surrounding normal tissues. Third, the extracellular polymers in the biofilm hinder the penetration and diffusion of PS, making it difficult for it to enter the biofilm, and the antibacterial effect is limited.

[0006] Therefore, seeking a nanoplatform with excellent antibacterial properties and the ability to effectively remove excess ROS to alleviate inflammation is of great significance for the clinical treatment of PI. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a CuTA-Por@ε-PL nanoplatform and its preparation method and application. The nanoplatform of the present invention is to graft protoporphyrin (Por) with CuTA through an amidation reaction, and use electrostatic adsorption to encapsulate it with ε-polylysine (ε-PL), thereby synthesizing CuTA-Por@ε-PL NPs (CPP NPs), which can be used as a potential treatment method for treating PI, destroying bacterial biofilms, and regulating inflammation.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for preparing a CuTA-Por@ε-PL nanoplatform, comprising the following steps:

[0010] S1, tannic acid reacts with copper sulfate pentahydrate to prepare CuTA nanosheets;

[0011] S2, grafting protoporphyrin onto APTES-aminated CuTA nanosheets to form CuTA-Por;

[0012] S3. ε-PL coated CuTA-Por to obtain CuTA-Por@ε-PL nanoplatform.

[0013] In a second aspect, the present invention provides a CuTA-Por@ε-PL nanoplatform prepared by the above preparation method.

[0014] In a third aspect, the present invention provides the use of the CuTA-Por@ε-PL nanoplatform in the treatment of peri-implantitis.

[0015] In a fourth aspect, the present invention provides a drug comprising the above-mentioned CuTA-Por@ε-PL nanoplatform.

[0016] Beneficial technical effects of one or more of the above technical solutions:

[0017] The present invention can be used as a potential therapeutic method for treating PI, destroying bacterial biofilms, and regulating inflammation. On the one hand, the surface of CuTA-Por@ε-PL NPs (CPP NPs) carries a high positive charge, which greatly enhances its penetration and distribution ability in the biofilm, as well as its preferential selectivity for bacteria, thereby clearing the biofilm with the help of aPDT-mediated ROS burst. On the other hand, CPP NPs can clear ROS remaining in PI tissues and after aPDT, and adsorb pathogen-related molecular patterns to inhibit inflammatory responses, regulate macrophage polarization, and promote tissue regeneration. This carefully planned strategy is expected to provide a new way to improve the therapeutic effect of PI in future clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 is a performance analysis of CuTA-Por with different Por / CuTA mass ratios in the embodiment of the present invention, wherein A and C are the characterization and normalized analysis of the in vitro photodynamic properties of CuTA-Por with different Por / CuTA ratios, respectively, and B is the UV-vis spectra of CuTA-Por with different Por / CuTA ratios;

[0020] Figure 2 is the CPP hydrated particle size with different CuTA-Por / ε-PL ratios in the embodiments of the present invention;

[0021] Figure 3 Schematic diagram of CPP NPs treating PI in an embodiment of the present invention;

[0022] Figure 4 The preparation and characterization of the PI therapeutic material CPP NPs in the embodiment of the present invention, wherein A is a schematic diagram of the synthesis of CPP NPs, B is a transmission electron microscope image, C is a UV-visible spectrum, D is a hydrated particle size, and E is a Zeta potential;

[0023] Figure 5The stability and biocompatibility verification results of CPP NPs in the examples of the present invention, wherein A is the particle size change of CPP NPs in different solutions within 7 days, B is the CCK-8 test of HGFs and L929 cells treated with different concentrations of CPP NPs, and C is the hemolysis test of different concentrations of CPP NPs;

[0024] Figure 6 To evaluate the photodynamic therapy performance of CPP NPs, CuTA-Por and Por in the examples of the present invention, wherein A is the absorption spectrum of DPBF in CPP solution under different laser irradiation times (0-7 min), and B is the normalized analysis of ROS generated by Por, CuTA-Por and CPP;

[0025] Figure 7 The results of the mechanism verification of CPP NPs scavenging ROS in the embodiment of the present invention are shown in Figure 1, wherein A is a schematic diagram of the antioxidant activity of CPP NPs, and B and C are respectively the results of NBT being ·O 2- The absorption spectra of SA and the related normalized analysis of the reduction, D and E are the absorption spectra of SA and ·OH reaction under different concentrations of CPP NPs and the related normalized analysis, F and G are the dissolved oxygen concentrations catalyzed by different concentrations of CPP NPs and different solutions, H and I are ·O 2- Absorption spectra of NBT reduced in different solutions and the corresponding normalized analysis, J and K are the absorption spectra of SA reacting with ·OH in different solutions and the related normalized analysis, L and M are the absorption spectra of NBT reduced in different solutions and the corresponding normalized analysis, In vitro fluorescence images of ROS generation and clearance under different treatments and the corresponding fluorescence intensity analysis by the imaging system, N and O are the quantification and CLSM images of ROS levels in HGFs after different treatments, respectively (scale bar: 50 μm);

[0026] Figure 8The penetration and accumulation of CuTA-Por and CPP NPs in mature biofilms were simulated by synthesizing CuTA-Cy5.5 and CCP in an embodiment of the present invention, wherein A is a biofilm penetration and selective adsorption scheme mediated by CPP NPs, (i) in B is a biofilm penetration scheme mediated by CuTA-Por and CPP NPs, (ii) and (iii) are 3D CLSM images and corresponding z-stack images and relative fluorescence intensities of P.gingivalis biofilms treated with CuTA-Cy5.5 and CCP for different time periods, respectively, C is a TEM image of P.gingivalis treated with CuTA-Por and CPP (scale bar: 1 μm), and local magnifications of these images (scale bar: 200 nm), D is the Zeta potential of P.gingivalis and HGFs cells after mixing with different concentrations of CPP NPs for 30 minutes, and E is the co-cultured P.gingivalis (Nile red staining) and HGFs cells (Hoechst 33342 staining) flow cytometry analysis 30 min after the addition of CPP NPs.

[0027] Fig. 9 The antibacterial effect of CPP NPs on free P.gingivalis was studied by plate colony counting method and TEM in the examples of the present invention, wherein A is (i) colony image and (ii) TEM image (scale bar: 1 μm) after P.gingivalis was treated with CPP NPs at different concentrations, B is quantitative analysis of P.gingivalis after treatment with CPP NPs at different concentrations, C is quantitative analysis of P.gingivalis after different treatments, D is nucleic acid leakage detection of P.gingivalis, absorbance curve of P.gingivalis suspension at 260 nm after different treatments, and E is (i) colony image, (ii) TEM image (scale bar: 1 μm), and (iii) photo of P.gingivalis after different treatments.

[0028] Fig.10The antibacterial effect of CPP NPs on established and forming P.gingivalis biofilms in the examples of the present invention is studied, wherein A is a schematic diagram of the anti-biofilm function of CPP NPs, B is a representative 3D Live / Dead picture of an established P.gingivalis biofilm, C is a representative image of an established biofilm after crystal violet staining, D is the ratio of dead to live P.gingivalis in the biofilm, E is the average thickness of the established P.gingivalis biofilm, F and G are representative photos of colonies of the established P.gingivalis biofilm and related quantitative analysis, respectively, and H is a representative 3D image of the formation of a P.gingivalis biofilm. Live / Dead images, I is a representative image of biofilm formed after crystal violet staining, J is the ratio of dead to live bacteria of P. gingivalis forming biofilm, K is the average thickness of biofilm formed by P. gingivalis, L is a representative photo of colonies forming biofilm of P. gingivalis, M is related quantitative analysis, and N is the relative mRNA levels of adhesin molecule genes and virulence factor genes in P. gingivalis biofilm established by RT-qPCR detection;

[0029] Fig.11 In the embodiment of the present invention, P.gingivalis was used as a model organism to compare the transcriptomic analysis results before and after CPP NPs treatment, wherein A is a volcano plot of up-regulated and down-regulated genes in the biofilm after CPP NPs treatment, B is a heat map of DEGs in the Control and CPP+L groups, C is a GO enrichment analysis of DEGs, D is a KEGG enrichment analysis of DEGs, and E is an interaction heat map of significantly differentially expressed genes in the ribosomal pathway.

[0030] Fig.12Figure 3 is the in vitro anti-inflammatory effect of CPP NPs treatment in which RAW 264.7 cells were stimulated with lipopolysaccharide from P. gingivalis to simulate acute inflammation in the examples of the present invention, wherein A is the anti-inflammatory activity scheme of CPP NPs, B is the relative mRNA levels of pro-inflammatory factors (M1 markers: IL-6, IL-1β, TNF-α) and anti-inflammatory factors (M2 markers: IL-10, Arg-1, TGF-β) after different treatments by RT-qPCR analysis, D is the concentration of IL-6 and TNF-α in the supernatant after 3 h of different treatments, E is the immunofluorescence image of NF-κB / p65 translocation in macrophages (scale bar: 50 μm) and the quantification of (I) positive cells, and F is the CPP NPs. NPs-mediated regulation scheme of macrophage polarization. J is the flow cytometric analysis of M1 macrophage-specific marker CD86 and M2 macrophage-specific marker CD206. G and H are the quantitative analysis of CD86-positive cells and CD206-positive cells under different experimental treatments, respectively.

[0031] Fig.13 The results of the in vivo anti-inflammatory and antibacterial activities of CPP NPs were investigated by establishing a rat model of peri-implantitis in the embodiments of the present invention, wherein A is a diagram of the establishment of the PI model and the dosing regimen, B is an intraoral photograph of the model rats after treatment with different NPs (scale bar: 2 mm), C is an image of the colonies separated from the peri-implant tissue after different treatments, D is a related quantitative analysis, E and F are in vivo fluorescence imaging of the ROS levels at the implant site under different treatments and the corresponding quantification, respectively, (i) and (ii) in G are three-dimensional reconstructions by Micro-CT (scale bar: 1 mm) and buccal and palatal slice images of implants with different treatments, respectively, and HJ is a quantitative statistics of alveolar bone resorption, bone volume fraction, and trabecular number.

[0032] Fig.14 The results of the regulation of CPP NPs on inflammatory response verified by HE staining, Masson staining, and immunohistochemistry in the embodiments of the present invention, wherein A and B are HE staining images and immune cell counts, respectively, C and D are Masson staining images and collagen volume fraction percentages, respectively, E is an immunofluorescence image (red: IL-6 positive cells, blue: cell nuclei, scale bar: 100 μm), F is the relative immunofluorescence intensity of IL-6, G is an immunofluorescence image (green is Arg-1 positive cells, blue is cell nuclei, scale bar: 100 μm), and H is the relative immunofluorescence intensity of Arg-1. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0035] In a typical embodiment of the present invention, a method for preparing a CuTA-Por@ε-PL nanoplatform is provided, comprising the following steps:

[0036] S1, tannic acid reacts with copper sulfate pentahydrate to prepare CuTA nanosheets;

[0037] S2, grafting protoporphyrin onto APTES-aminated CuTA nanosheets to form CuTA-Por;

[0038] S3. ε-PL coated CuTA-Por to obtain CuTA-Por@ε-PL nanoplatform.

[0039] In another specific embodiment of the present invention, in S1, tannic acid and copper sulfate pentahydrate are dissolved in deionized water, and the CuTA nanosheets are obtained by adjusting the pH, heating and stirring, centrifuging, washing and drying.

[0040] Specifically, tannic acid and copper sulfate pentahydrate are dissolved in deionized water, and the pH value of the mixture is adjusted to 7±0.5 by adding sodium hydroxide solution. Subsequently, the solution is placed in an oil bath, heated to 60°C and stirred for 4 to 6 hours, and centrifuged at a speed of 5000 rpm / min for 4 to 6 minutes to obtain a product, which is then washed three times with deionized water and once with anhydrous ethanol. After vacuum drying in an oven at 60°C, the product is stored at 4°C for future use.

[0041] In another specific embodiment of the present invention, in S1, the mass ratio of tannic acid to copper sulfate pentahydrate is 1:(30-35), preferably, the mass ratio of tannic acid to copper sulfate pentahydrate is 27:875.

[0042] In another specific embodiment of the present invention, in S2, protoporphyrin needs to be activated, specifically: protoporphyrin, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride are dissolved in deionized water, and the mixture is stirred at 23-24° C. for 25-35 minutes under a nitrogen atmosphere to activate the carboxyl group of protoporphyrin.

[0043] In another specific embodiment of the present invention, in S2, the preparation method of APTES-aminated CuTA nanosheets is: disperse CuTA in ethanol, then add APTES, place the solution in an oil bath, heat and stir, and obtain the product "CuTA-NH2" by centrifugation.

[0044] In another specific embodiment of the present invention, in S2, the mass ratio of protoporphyrin to CuTA nanosheets aminated with APTES is 1:(10-80), preferably, the mass ratio of protoporphyrin to CuTA nanosheets aminated with APTES is 1:20.

[0045] In another specific embodiment of the present invention, in S3, the ε-PL coating of CuTA-Por is performed during ultrasonic treatment.

[0046] Specifically, an aqueous solution of ε-PL and an aqueous solution of CuTA-Por are mixed. Subsequently, the mixture is ultrasonically treated for 30 minutes and stirred at 25° C. for 5 to 8 hours. Thereafter, the product is purified by multiple washing and centrifugation cycles using deionized water, and then dried by freeze drying.

[0047] In another specific embodiment of the present invention, in S3, the mass ratio of ε-PL to CuTA-Por is 1:(5-20), preferably, the mass ratio of ε-PL to CuTA-Por is 1:10.

[0048] In another specific embodiment of the present invention, a CuTA-Por@ε-PL nanoplatform is provided, which is prepared by the above preparation method.

[0049] In a specific embodiment of the present invention, there is provided the use of the CuTA-Por@ε-PL nanoplatform in the preparation of a product for treating peri-implantitis.

[0050] In another specific embodiment of the present invention, a drug is provided, which contains the above-mentioned CuTA-Por@ε-PL nanoplatform.

[0051] In another specific embodiment of the present invention, the CuTA-Por@ε-PL nanoplatform or drug can be in the form of an injection; specifically, the CuTA-Por@ε-PL nanoplatform or drug can be injected into the pocket around the implant to achieve a therapeutic effect. Experiments have shown that it has the ability to remove biofilms and regulate inflammation. The drug dosage form is an injection.

[0052] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0053] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0054] Example 1

[0055] 1. Preparation of CPP NPs

[0056] 1. Preparation of CuTA nanosheets: 54 mg of tannic acid and 1750 mg of copper sulfate pentahydrate were dissolved in 100 mL of deionized water. The pH of the mixture was adjusted to 7.4 by adding about 5 mL of 2 mol / L sodium hydroxide solution. Subsequently, the solution was heated to 60 ° C in an oil bath and stirred for 5 hours. The product was obtained by centrifugation at 5000 rpm / min for 5 minutes, then washed three times with deionized water and once with anhydrous ethanol. After vacuum drying in an oven at 60 ° C for 2 hours, the product was stored at 4 ° C for subsequent experiments.

[0057] 2. Screening of Por / CuTA ratio

[0058] CuTA-Por was prepared by amidation reaction. Protoporphyrin (Por, 5 mg, 0.007 mmol), N-hydroxysuccinimide (NHS, 4.09 mg, 0.036 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 13.6 mg, 0.071 mmol) were dissolved in 30 mL of deionized water. Under a nitrogen atmosphere, the mixture was stirred at 25 ° C for 30 minutes to activate the carboxyl group of protoporphyrin. Subsequently, 20 mL of copper tannate (CuTA) solution (containing 100 mg CuTA) was added to the activated protoporphyrin solution and stirred at 25 ° C for 6 hours under nitrogen protection. It can be observed that the color of the reaction solution gradually changes from green to purple. After the reaction is completed, the solution is centrifuged (5000 rpm / min, 5 minutes) and washed three times with deionized water. After drying, the product CuTA-Por (CuTA-Por, Por / CuTA mass ratio is 1:20) was obtained. Similarly, CuTA-Por with different Por / CuTA mass ratios (1:10, 1:40, 1:80) were prepared.

[0059] High photodynamic performance is a prerequisite for antimicrobial photodynamic therapy (aPDT), and 1,3-diphenylisobenzofuran (DPBF) was used as a singlet oxygen sensor to assess the generation of extracellular reactive oxygen species (ROS). DPBF was added to CuTA-Por solutions with different Por / CuTA mass ratios (1:10-1:80) to record its absorbance at 420 nm. During 660 nm laser irradiation, the absorbance was measured every 30 seconds.

[0060] like Figure 1 As shown in Figure A, we synthesized CuTA-Por NSs with different Por / CuTA mass ratios and used 1,3-diphenylisobenzofuran (DPBF) as a reactive oxygen species (ROS) probe to study their ROS production ability in vitro. The aPDT effect mainly depends on the transient burst of ROS. Figure 1 As shown in Figure B, there was no significant difference in the photodynamic performance of CuTA-Por with a Por / CuTA mass ratio of 1:10 and 1:20 in the first five minutes. However, CuTA-Por with a Por / CuTA mass ratio of 1:40 and 1:60 showed insufficient ROS production capacity in the first five minutes. Therefore, we decided to choose CuTA-Por with a Por / CuTA mass ratio of 1:20 for subsequent experiments.

[0061] 3. Preparation of CuTA-Por: Amino-functionalized CuTA nanosheets were prepared in the presence of 3-aminopropyltriethoxysilane (APTES). 100 mg of copper tannate (CuTA) was dispersed in 50 mL of ethanol, and then 200 μL of APTES was added to the nanoparticle suspension. The solution was heated to 60 ° C in an oil bath and stirred for 4 hours. The product "CuTA-NH2" was obtained by centrifugation at a speed of 5000 rpm / min for 5 minutes, followed by washing three times with anhydrous ethanol and vacuum drying in an oven at 60 ° C for 2 hours.

[0062] Protoporphyrin (Por, 5 mg, 0.007 mmol), N-hydroxysuccinimide (NHS, 4.09 mg, 0.036 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 13.6 mg, 0.071 mmol) were dissolved in 30 mL of deionized water. Under a nitrogen atmosphere, the mixture was stirred at 25 ° C for 30 minutes to activate the carboxyl group of protoporphyrin. Subsequently, 20 mL of copper tannate (CuTA) solution (containing 100 mg CuTA) was added to the activated protoporphyrin solution and stirred at 25 ° C for 6 hours under nitrogen protection. It can be observed that the color of the reaction solution gradually changes from green to purple. After the reaction is completed, the solution is centrifuged (5000 rpm / min, 5 minutes) and washed three times with deionized water. After drying, the product CuTA-Por (CuTA-Por, Por / CuTA mass ratio is 1:20) is obtained.

[0063] 4. Screening of ε-PL / CuTA-Por Ratio

[0064] An aqueous solution of ε-PL (25 mL, 20 mg / mL) was mixed with an aqueous solution of CuTA-Por (25 mL, 2 mg / ). The mixture was then sonicated for 30 min and stirred at 25 °C for 6 h. The product was then purified by multiple washing and centrifugation cycles with deionized water and then dried by lyophilization. A similar method was used to prepare CuTA-Por@ε-PL (CPP) with different ε-PL / CuTA-Por (1:5 and 1:20) mass ratios.

[0065] The DLS results of three CPP NPs with mass ratios of 1:5, 1:10, and 1:20 are shown in Figure 2. Figure 2As shown in the figure, the particle size of CPP (1:5) is mainly distributed in the range of 255nm to 955nm (polydispersity index PDI is 0.690). The particle size of CPP (1:10) is 204.6nm and PDI is 0.291. The particle size of CPP (1:20) is 631.5nm (PDI is 0.470), and a peak of larger size products appears at 5000nm. Based on the above results, it is believed that when the ratio is 1:5, CuTA-Por is difficult to be completely coated, resulting in a relatively wide particle size distribution range. When the ratio is 1:20, the excess ε-PL causes part of the CuTA-Por to be coated too thick or aggregated, thereby forming a product with a larger particle size. When the ratio is 1:10, the product presents a suitable particle size and is uniformly distributed. Therefore, 1:10 is selected as the optimal CuTA-Por / ε-PL ratio for synthesizing CPP.

[0066] Preparation of CPP NPs: An aqueous solution of ε-PL (25 mL, 20 mg / mL) was mixed with an aqueous solution of CuTA-Por (25 mL, 2 mg / mL). The mixture was then sonicated for 30 min and stirred at 25 °C for 6 h. The product was then purified by multiple washing and centrifugation cycles using deionized water and then dried by lyophilization.

[0067] The hydrated particle size distribution, polydispersity index (PDI), zeta potential and morphology were determined by dynamic light scattering and transmission electron microscopy. The UV-visible spectra of the intermediate and final products were measured by UV-visible-near infrared spectrometer.

[0068] 2. Stability and biosafety of CPP NPs

[0069] The stability of CPP NPs was evaluated by incubating them in water, phosphate buffered saline (PBS), and DMEM containing 10% serum at 37°C for 7 days. The particle size distribution was measured using dynamic light scattering (DLS).

[0070] The biosafety of CPP was evaluated using CCK-8 assay and hemolysis test. Human gingival fibroblasts (HGFs) and L929 cells were first seeded in 96-well plates and cultured overnight. Subsequently, different concentrations of CPP NPs were added and left for 24 hours. After the treatment, the old culture medium was replaced with fresh culture medium containing cell counting kit-8 (CCK-8), and the cells were incubated for another 3 hours. Finally, cell viability was measured at a wavelength of 450 nm using a microplate reader.

[0071] For the hemolysis test, erythrocytes were separated by centrifugation at 1500 rpm / min for 15 minutes. The erythrocytes were then washed three times with PBS and diluted tenfold to prepare an erythrocyte suspension. The positive control (+) was prepared with distilled water, while the negative control (-) was prepared with PBS. An equal volume of the erythrocyte suspension was mixed with the sample solution of different concentrations to be evaluated, incubated at 37°C for 2 hours, and then centrifuged at 1500 rpm / min for 15 minutes. Subsequently, the supernatant was transferred to a 96-well plate and the absorbance was measured at 576 nm using a microplate reader.

[0072] 3. Spatiotemporal regulation of ROS based on CPP NPs

[0073] 1. In vitro ROS generation of CPP: Similar to the method in the previous article, the generation of extracellular ROS of CPP cells was evaluated by using DPBF. DPBF was added to Por, CuTA-Por and CPP solutions to record their absorbance intensity at 420 nm. During the 660 nm laser irradiation, the absorbance was measured every minute.

[0074] 2. In vitro ROS scavenging effect of CPP:

[0075] 2.1 In vitro removal of superoxide anions (·O2 - ): Evaluation of CPP scavenging·O2 by measuring the inhibition rate of NBT photoreduction - Here, 20 μM riboflavin, 12.5 mM methionine, 75 μM NBT, and different concentrations of CPPNPs were dissolved in PBS (pH 7.4). Then, these mixtures were exposed to UV radiation for 15 minutes. After illumination, ·O2 - NBT will be reduced to a blue product with an absorption peak at 560 nm. The product was further quantitatively analyzed by UV-visible absorption spectroscopy. It contains riboflavin, methionine and NBT. The samples without and with UV illumination were designated as negative and positive controls, respectively. All experiments were performed in a dark environment. The same method was used to detect the ·O2 - Clearing ability.

[0076] 2.2 In vitro clearance · OH: The efficiency of CPP NPs in scavenging ·OH was evaluated by monitoring the amount of ·OH in salicylic acid (SA). -4 M H2O2 and 2×10 -4 M FeSO4 was mixed to generate ·OH through Fenton reaction. Subsequently, different concentrations of CPP NPs were added to the solution to eliminate ·OH. Finally, by adding 1×10-3 M SA was used to detect the remaining ·OH. ·OH can oxidize SA to 2,3-dihydroxybenzoic acid, which is purple. The absorption peak at 510 nm was further determined by UV-visible absorption spectroscopy. The same method was used to detect the absorption peak of CuTA and CPP NPs after 660 nm laser irradiation for 5 minutes. · OH scavenging efficiency.

[0077] 2.3 Catalase-like (CAT) activity of CPP NPs: The CAT activity of CPP NPs was studied by the scavenging rate of hydrogen peroxide (H2O2). Briefly, different concentrations of hydrogen peroxide (H2O2) were mixed with CPP NPs in 5 mL of phosphate buffered saline (PBS, pH 7.4). The generated oxygen (O2) was quantitatively measured using a dissolved oxygen meter. The CAT enzyme activity of CuTA and CPP NPs after 660 nm laser irradiation for 5 minutes was also studied.

[0078] 3. Dynamic monitoring of ROS generation and clearance in vitro: The experimental group was divided into five groups: control group (Control), CuTA group, CPP group, Por group irradiated with 660nm laser for 5 minutes (Por+L), and CPP group irradiated with 660nm laser for 5 minutes (CPP+L). The stock solutions of these five groups were prepared separately. The Por+L group and the CPP+L group were irradiated with 660nm laser for 5 minutes, while the other groups were not treated. Subsequently, 180μL of solution was immediately taken out from the stock solution and added to the well plate, followed by the addition of 2',7'-DCFH-DA. Immediately in Fluorescence imaging was recorded on an imaging system (excitation wavelength λex = 488 nm, emission wavelength λem = 525 nm) and marked as "0 min". After that, 180 μL of the stock solution was taken out every 3 minutes and added to the well plate, 2',7'-DCFH-DA was added, and the above detection process was repeated.

[0079] DCFH-DA was used to measure the levels of intracellular and extracellular ROS. The detection of intracellular ROS levels was also divided into the above five groups: Control, CuTA, CPP, Por+L, and CPP+L. Human gingival fibroblasts (HGFs) were seeded into confocal microscope culture dishes (35 mm) at a density of 2×105 cells / dish and cultured at 37°C for 24 hours. Then, PBS, CuTA, Por, and CPP were added, some of which were laser-treated and some were not. After irradiation, the cells were incubated at 37°C for 6 hours. Subsequently, DCFH-DA was added and incubated for 20 minutes. Next, the cells were fixed with 4% paraformaldehyde for 10 minutes. Finally, the cells were stained with DAPI for 5 minutes and observed by confocal laser scanning microscopy (CLSM).

[0080] 4. Antibacterial property test of CPP NPs

[0081] 1. Evaluation of CPP NPs-mediated biofilm penetration and bacterial selectivity

[0082] 1.1 Penetration and dispersion of biofilm mediated by CPP NPs: To form mature P.gingivalis biofilm, the microbial concentration was set to 108 CFU / mL in a confocal microscopy dish and then cultured in anaerobic medium at 37°C for 96 h. To evaluate the ability of NPs to penetrate the biofilm, CuTA was labeled with Cy5.5 to obtain CuTA-Cy5.5, and then CuTA-Cy5.5@ε-PL(CCP) was synthesized. CuTA-Cy5.5 and CCP were used to represent CuTA-Por and CPP NPs, respectively. Then, 1 mL of CuTA-Cy5.5 and CCP solutions were added to the biofilm and incubated for 0.5 h, 1 h, and 2 h, respectively. After incubation, the biofilm was washed three times with PBS to remove NPs that were not bound to the biofilm. Subsequently, the biofilm was stained with DMAO for 20 min in a dark environment. Confocal laser scanning microscopy (CLSM) was used to observe the penetration of NPs (NPs) into the biofilm.

[0083] 1.2 Selective adsorption of CPP NPs on cells and bacteria: Human gingival fibroblasts (HGFs) were stained with Hoechst33342 for 20 minutes and then washed with phosphate buffered saline (PBS), and P.gingivalis was stained with Nile-Red for 20 minutes and washed with phosphate buffered saline (PBS). Human gingival fibroblasts (HGFs) and P.gingivalis were mixed in phosphate buffered saline (PBS) at concentrations of 105 / mL and 107CFU / mL, respectively. Subsequently, CCP was added to the co-incubation solution and incubated for another 30 minutes. Finally, the samples were quantitatively analyzed by flow cytometry.

[0084] 1.3 Human gingival fibroblasts (HGFs) at a concentration of 105 cells / mL and P.gingivalis at a concentration of 107 colony-forming units / mL (CFU / mL) were incubated with different concentrations of CPPNPs for 30 minutes. Afterwards, they were centrifuged at 5000 rpm / min for 3 minutes and washed three times to remove unbound CPPNPs. The cells / bacteria were collected and resuspended, and the effect of CPPNPs on the surface charge of cells / bacteria was investigated by measuring their Zeta potential.

[0085] 1.4 In addition, after P. gingivalis was incubated with CPPNPs for 30 min, the bacteria were fixed with glutaraldehyde solution for 30 min and then observed by transmission electron microscopy (TEM).

[0086] 2. Evaluation of the antibacterial and anti-biofilm properties of CPP NPs

[0087] 2.1 Evaluation of the antibacterial ability of CPP NPs against free bacteria: CPP NPs at different concentrations were mixed with 1 mL P.gingivalis bacterial solution (106 CFU / mL). After 5 minutes of 660 nm laser irradiation, the mixture was incubated for 24 h. After gradient dilution, the solution was evenly spread on a blood agar plate and then anaerobically cultured at a constant temperature of 37 ° C for colony counting. For TEM imaging, the bacterial suspension was fixed with 2.5% (mass fraction) glutaraldehyde. Subsequently, images were taken to observe the bacterial morphology and its interaction with CPP NPs. Similarly, the concentration of CPP was fixed at 300 μg / mL, and five groups (control group, CuTA group, Por+L group, CPP group, CPP+L group) were set up with and without laser irradiation, and the antibacterial efficacy of CPP NPs was further studied by CFU counting and TEM.

[0088] 2.2 Nucleic acid leakage detection: The concentration of P.gingivalis bacterial suspension was adjusted to 108 CFU per mL and treated with different NPs (NPs) and laser irradiation. Subsequently, the bacterial suspension was collected every 10 minutes and the supernatant was diluted. Finally, the release of nucleic acid was determined by measuring the optical density value at 260 nm.

[0089] 2.3 Evaluation of anti-biofilm properties: Five groups were set up: control group, CuTA group, Por+L group, CPP group, and CPP+L group. Plates containing developing or formed biofilms were treated with different NPs (NPs) and then irradiated with a 660 nm laser (1 W / cm 2, 5min). The control group, CuTA group, and CPP group did not receive laser treatment. For CFU counting, after different treatments, the attached biofilm was removed by ultrasonic treatment. Subsequently, it was diluted tenfold continuously. Then, 100 μL of the diluted bacterial solution was spread on blood agar and placed in a constant temperature incubator for anaerobic culture. For live / dead fluorescent staining, the biofilm was first rinsed to remove non-adherent bacteria. Subsequently, according to the manufacturer's instructions, a mixture of 2.5 μM SYTO 9 and 2.5 μM propidium iodide (PI) was prepared and used to stain each sample for 20 minutes. A three-dimensional image of the biofilm was obtained using a confocal laser scanning microscope (CLSM). For crystal violet staining, the treated biofilm was first fixed for 15 minutes. After natural air drying, 1 mg / mL crystal violet solution was added to stain it for 15 minutes. Subsequently, the excess dye was thoroughly rinsed off. Finally, the stained biofilm was dissolved with 95% ethanol, and the absorbance at 590 nm was measured with a microplate reader.

[0090] 2.4 Evaluation of the expression of extracellular protein genes in P.gingivalis, specifically, RT-qPCR technology was used to analyze genes such as FimAII, FimA IV, RgpA, RgpB, Kgp, etc. ΔΔCt The relative gene expression was determined by the PCR method. For data normalization, the 16S rRNA gene was used as an internal reference, and the Ct value of the control group was used as the calibration value. All experiments were repeated three times.

[0091] 2.5 For transcriptome analysis of P.gingivalis after CPP NPs treatment, the formed P.gingivalis biofilms were treated with CPP NPs and irradiated with 660 nm laser. After treatment, the biofilms were collected by centrifugation (10000 rpm, 3 min), then frozen in liquid nitrogen for 15 min and placed in dry ice for further RNA extraction.

[0092] 5. Anti-inflammatory activity of CPP NPs against LPS- and aPDT-induced pro-inflammatory responses

[0093] 1. Phenotypic conversion of macrophage polarization:

[0094] 1.1 The M1 / M2 phenotype of macrophages was detected by detecting relevant cytokines. Porphyromonas gingivalis-LPS (P.gingivalis-LPS) was selected as a stimulant to create a basic inflammatory state and simulate PI conditions. RAW 264.7 cells were seeded in 6-well plates at a density of 1×105 cells per well and cultured for 1 day. Subsequently, cells were stimulated with P.gingivalis-LPS (1μg / mL) for 3h before adding different NPs (NPs) to simulate acute inflammation in vitro. The CuTA group, CPP group, and non-irradiated LPS group were used to compare their anti-inflammatory properties against PI. The Por group and the CPP group irradiated with 660nm laser were used to compare the protective ability against aPDT-exacerbated inflammation during treatment.

[0095] After a 24-hour incubation period, total RNA from macrophages was extracted using an RNA extraction kit. The M1 / M2 phenotype inflammatory response of RAW 264.7 was evaluated by qPCR based on the mRNA expression levels of chemokine interleukin-1β (IL-1β), chemokine interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), chemokine interleukin-10 (IL-10), transforming growth factor-β (TGF-β) and arginase-1 (Arg-1). Data were normalized to the housekeeping gene β-actin (β-actin).

[0096] 1.2 Detection of the expression of macrophage surface markers by flow cytometry. RAW 264.7 cells were inoculated, cultured and treated as described above, and then the cells were collected and stained with PE-labeled anti-mouse CD86 and APC-labeled anti-mouse CD206 for 15 minutes at room temperature in a dark environment. The cells were washed three times with staining buffer (PBS / 2% FBS), then resuspended and detected by the instrument.

[0097] 2. Blocking the inflammatory response induced by pathogen-associated molecular patterns (PAMPs):

[0098] Using LPS as a typical PAMPs, we investigated the ability of CPP NPs to block PAMPs-induced inflammatory responses. RAW 264.7 cells were seeded in 6-well plates at a density of 1×105 cells per well and cultured for 1 day. Subsequently, P.gingivalis-LPS (1 μg / mL) was first mixed with different NPs, and then the mixture was added to the cells for 3 hours of stimulation. The concentrations of IL-6 and TNF-α in the supernatant of RAW 264.7 cells after treatment were detected using ELISA kits.

[0099] 3. Inhibition of NF-κB activation in macrophages induced by LPS and aPDT:

[0100] To further verify the anti-inflammatory and aPDT protective mechanisms of CPP NPs, the NF-κB signaling pathway was evaluated by translocation of NF-κB / p65 subunits. RAW264.7 cells (1×105 cells / well) were seeded into 6-well plates and cultured for 24 h. The seeded cells were stimulated with P.gingivalis-LPS (1 μg / mL) for 3 h, and then different NPs were added to each well, some with laser irradiation and some without, followed by incubation for 24 h. After fixation with 4% paraformaldehyde for 10 min, the cells were treated with 0.25% Triton X-100 for 10 min, followed by blocking with blocking goat serum at 37°C for 1 h. Subsequently, the cells were incubated with rabbit anti-NF-κB p65 primary antibody (1:500) at 37°C for 1.5 h, followed by incubation with goat anti-rabbit Alexa 488-conjugated IgG secondary antibody (1:1000) at 37°C for 1 h. The cell nuclei were stained with DAPI after incubation for 5 min in the dark. Finally, images were taken using CLSM.

[0101] VI. In vivo evaluation of the antibacterial and anti-inflammatory properties of CPP NPs

[0102] 1. Animal model establishment and drug administration: All animal experiments have been approved by the Ethics Committee of the Affiliated Hospital of Qingdao University. Six-week-old male Sprague-Dawley rats were purchased from Beijing HFK Bioscience Co. Ltd. After one week of adaptive feeding, the left maxillary first molar of the rat was completely extracted, and the implant was implanted immediately after tooth extraction. After 4 weeks, when the implant formed bone integration, the rats were randomly divided into 6 groups, namely the control group (Control), PI group, CuTA group, Por+L group, CPP group and CPP+L group. Except for the control group, the remaining groups were treated by ligating the neck of the implant with silk thread and inoculating P.gingivalis for 2 weeks to construct the PI model. Subsequently, the Control group and the PI group were not treated, while the other groups were given the corresponding substances once a day into the pocket around the implant. In addition, the Por+L group and the CPP+L group were required to be irradiated with laser for 5 minutes. After 2 weeks of treatment, the rats were euthanized, and the left maxilla was harvested for Micro-CT scanning and immunohistochemical analysis.

[0103] 2. Measure the bone recession height at the proximal and distal ends of the implant respectively, and use the average value of the two to represent the implant bone recession height.

[0104] 3. In vivo fluorescence imaging: The ROS levels of the peri-implant tissues after different treatments were studied. After the 2-week treatment, the rats in each group were anesthetized with sodium pentobarbital. Subsequently, the ROS probe DCFH-DA was intravenously injected at a dose of 1.8 mg / kg for 30 min. The fluorescence images were recorded by an imaging system (excitation filter: 525 nm; emission filter: 495 nm, PerkinElmer).

[0105] All data are presented as mean ± SD. Statistical analysis was performed using Student's unpaired t-test and two-way ANOVA. The significance levels were set at *P < 0.05, *P < 0.01, *P < 0.001, and *P < 0.0001.

[0106] Result 1: CPP NPs can deeply penetrate and disperse in biofilms

[0107] The synthetic route of CPP is as follows Figure 4 As shown in Figure A, CuTA NSs are first synthesized, and then modified with aminopropyltriethoxysilane (APTES) to obtain amino-modified CuTA-NH2, which is then covalently linked to the carboxyl group of porphyrin (Por) to obtain CuTA-Por NSs with photodynamic therapy (PDT) function. TEM images are shown in Figure 1. Figure 4 As shown in B(i-iii) in the figure, the morphology of CuTA, CuTA-NH2 and CuTA-Por did not change significantly, showing a good lamellar structure, which indicates that the treatment with APTES and the modification with Por have no obvious effect on the lamellar structure of CuTA.

[0108] UV spectrum results are as follows Figure 4 As shown in Figure C, CuTA-Por simultaneously exhibits the peak shape of CuTA and the characteristic absorption band of Por at 410 nm, further confirming the successful synthesis of CuTA-Por nanosheets.

[0109] like Figure 2 As shown in Figure B, CuTA-Por exhibits a relatively small size (about 110 nm) in the dry state. However, it was observed that CuTA-Por has poor solubility in aqueous solution and is easily precipitated. The results of dynamic light scattering (DLS) are shown in Figure 2. Figure 4As shown in D in Figure 1, the hydrated particle size of CuTA-Por is 1009 nm. This property is not conducive to the penetration and dispersion of NPs in the biofilm. To solve this problem, CuTA-Por was coated with ε-PL to prepare CPP NPs. After coating with ε-PL, no precipitation was observed in the CPP solution after centrifugation at 5000 rpm for 5 min. Figure 4 As shown in Figure E, the coating of ε-PL not only improves the water solubility of CuTA-Por, but also gives CPP NPs a strong positive charge. After amino modification, the zeta potential of CuTA increases from -14.87mV to -0.478mV. In addition, after Por grafting, its zeta potential further changes to -6.04mV, confirming the success of the modification.

[0110] like Figure 4 As shown in Figures D and E, CPP has a well-hydrated particle size (204.6 nm) and a positive surface charge (+36.4 mv), which makes CPP NPs more conducive to deep penetration and dispersion in the biofilm.

[0111] Result 2: CPP NPs have excellent stability and biocompatibility.

[0112] The present invention detects the particle size stability of CPP NPs in water, phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) and its biocompatibility. Figure 5 As shown in Figure A, CPP NPs exhibited excellent particle size stability. The slight increase in particle size in DMEM solution can be attributed to protein adsorption. Figure 5 As shown in Figure B, after the synthesis of CPP NPs, cell counting kit-8 (CCK-8) and hemolysis test were used to verify the good biocompatibility of CPP NPs. Figure 5 As shown in Figure C, even at a concentration as high as 800 μg / mL, more than 80% of human gingival fibroblasts (HGFs) and L929 cells remained viable and the hemolysis rate was less than 5%.

[0113] Result 3: CPP NPs have ROS regulating effects.

[0114] The transient burst of ROS induced by excitation light is the main mechanism for aPDT to exert its antibacterial effect. Under the irradiation of excitation light, photosensitizers (PSs) can instantly produce a large amount of ROS through photocatalysis. Therefore, it is important to first explore the effect of copper tannate (CuTA) and ε-polylysine (ε-PL) coating on the ROS generation performance of porphyrin (Por) under laser irradiation.

[0115] The photodynamic therapy (PDT) performance of composite polyelectrolyte particles (CPP), copper tannate-porphyrin (CuTA-Por), and Por was evaluated using 1,3-diphenylisobenzofuran (DPBF). Figure 6 As shown in Figure A, the PDT performance of CPP is not significantly different from that of Por, which is of great significance for CPP NPs to achieve aPDT through transient ROS burst in a short period of time. Figure 6 As shown in Figure B, due to the poor water solubility of CuTA-Por, its PDT effect in water is reduced. Therefore, ε-PL coating not only helps to enhance the penetration and distribution in the bacterial biofilm by virtue of its positive charge and small size, but also improves the PDT effect of CuTA-Por and further enhances the ROS burst.

[0116] like Figure 7 As shown in Figure A, the main mechanism of CPP NPs to remove ROS is derived from the SOD / CAT enzyme activity of CuTA. In order to verify that CPP NPs have SOD / CAT enzyme activity and that this activity is still retained after laser irradiation, the model of "generating ROS for antibacterial purposes under laser irradiation and removing ROS for anti-inflammatory purposes without laser irradiation" was achieved. The inhibition rate of NBT photoreduction was measured to determine the dismutation of superoxide radicals catalyzed by CPPNPs. The results are shown in Figure 1. Figure 7 As shown in Figures B and C, when different concentrations of CPP exist, the absorbance at 560 nm decreases, indicating SOD-like activity. The scavenging efficiency of CPP NPs on hydroxyl radicals was determined by measuring salicylic acid. Figure 7 As shown in D and E, it has excellent · OH scavenging ability; CPPNPs were added to PBS (0.1M, pH 7.4) containing H2O2 to evaluate the hydrogen peroxide decomposition performance. The results are shown in Figure 7 As shown in F, the decomposition rate increases with the increase of CPP NPs concentration, indicating that CPP NPs have inherent CAT-like activity and good scavenging ability for various ROS. Its SOD / CAT activity is beneficial to convert ROS in the PI bag into O2 and H2O, improve the hypoxia condition and enhance the aPDT effect. Figure 7 As shown in G, the ability to scavenge H2O2 is still retained by 67.8%, indicating that CPP NPs can still perform ROS scavenging function after exerting aPDT function, avoiding side effects and continuously reducing ROS levels. The same method was used to compare the ROS scavenging ability of CPP NPs before and after irradiation. The results are shown in Figure 7 As shown in H, I, J, and K, it is shown that after irradiation, O2 - and · There was no significant change in OH capacity.

[0117] In order to visually present the generation and clearance of ROS, five groups of stock solutions were prepared. The Por+L group and the CPP+L group were irradiated with 660nm laser for 5 minutes, and the other groups were not treated. The solution was taken from the stock solution and added to the well plate and 2',7'-dichlorofluorescein diacetate (DCFH-DA) was added. The imaging system records the fluorescence images. Figure 7 As shown in L, the fluorescence intensity of the Por+L group and the CPP+L group increased significantly and the amplitude was comparable after irradiation, confirming the ROS production ability of CPP NPs under laser irradiation; Figure 7 As shown in M, after the laser irradiation, the fluorescence intensity of the CPP+L group continued to decrease, and 78.1% of ROS had been cleared at 9 minutes, verifying the ROS scavenging ability of CPP NPs without laser irradiation. The same grouping method was used to detect the generation and clearance of ROS in cells. The results are shown in Figure 7 As shown in N and O, the proportion of positive cells in the Por+L group was high, indicating that aPDT treatment caused intracellular oxidative stress, while the proportion in the CPP+L group was low, indicating that CPP NPs could effectively alleviate the high intracellular ROS state caused by aPDT.

[0118] Result 4: CPP NPs have excellent bacterial selectivity, which is beneficial for their antibacterial and anti-biofilm properties

[0119] like Figure 8 As shown in A in the figure, the bacterial surface is usually negatively charged, while the CPP NPs surface is positively charged. It is speculated that it can strongly interact with typical pathogens of peri-implantitis, enhance its ability to penetrate the biofilm, and preferentially bind to bacteria, thereby enhancing its selectivity for bacteria.

[0120] CuTA-Cy5.5 and CCP were synthesized to mimic CuTA-Por and CPP NPs, and their penetration and accumulation in mature biofilms were studied. Figure 8 As shown in Figure B, CPP NPs have strong penetration ability into biomembranes and can effectively solve the problem of insufficient penetration of PSs, which may be due to their small hydrated particle size and positive charge.

[0121] The positive charge of CPP NPs enhances their penetration and dispersion capabilities within biofilms, promoting preferential interactions with bacteria. Figure 8 As shown in C and D, it shows that CPP NPs have a strong interaction with bacteria, such as Figure 8 As shown in Figures E and F, the binding ratio of CPP NPs to P.gingivalis is approximately 12.1 times that of CPP NPs to HGFs, indicating that they are significantly more inclined to bind to bacteria.

[0122] The antibacterial effect of CPP NPs on free P.gingivalis was studied by plate colony counting method and TEM. Fig. 9 As shown in A, B, C, and E in Figure 1, CPP NPs can kill free bacteria, and its antibacterial effect is better than that of the CuTA group. It can effectively kill bacteria through the PDT effect, and can also capture and kill free bacteria to block their recolonization process. The results of the nucleic acid leakage experiment are shown in Fig. 9 As shown in Figure 5D, it is confirmed that in addition to the interaction between CPP NPs and bacteria causing membrane damage, the ROS burst generated by laser irradiation can also cause bacteria to rupture and be damaged.

[0123] The antibacterial effect of CPP NPs on established and emerging P.gingivalis biofilms was studied by CLSM, crystal violet staining, CFU counting and other experimental methods, and CPP NPs showed excellent biofilm removal ability. Fig.10 As shown in Figures B to M, the anti-biofilm effect of CPP NPs was 2.95 times higher than that of the Por+L group, which could effectively kill bacteria in the biofilm and reduce the number of bacteria. P.gingivalis can produce a variety of virulence factors to induce host inflammatory response, so the CPP NPs inhibited the expression of virulence factors of P.gingivalis in the biofilm. Fig.10 As shown in N, CPP NPs can inhibit the expression of P.gingivalis virulence factors in biofilms and disrupt their adhesion, aggregation and parasitism. It also has an inhibitory effect on biofilm formation, and the thickness of biofilms is significantly reduced after treatment. In general, CPP NPs can damage bacterial cell membranes and nucleic acids, capture and kill bacteria, achieve biofilm removal and inhibit biofilm formation.

[0124] To explore the antibacterial mechanism of CPP NPs, comparative transcriptomic analysis was performed using P. gingivalis as a model organism. Fig.11 As shown in A and B in Figure 1, unsupervised principal component analysis showed that there were significant transcriptome differences between the control group and the CPP+L group, and a total of 431 significantly differentially expressed genes were detected. The results of gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment pathway analysis are shown in Figure 1. Fig.11 As shown in C and D in Figure 1, ribosome damage is most prominent after CPP+L NPs treatment, which is attributed to the strong ROS burst. Ribosomes are the site of protein synthesis in cells. The survival and reproduction of P.gingivalis depends on protein synthesis. If the ribosome function is damaged, its synthesis is inhibited, which can effectively control the growth and reproduction of bacteria, reduce pathogenicity, and reduce the possibility of bacteria developing drug resistance. Fig.11As shown in Figure E, the significantly differentially expressed genes related to ribosomes after CPP+L NPs treatment are listed in detail in the interactive heat map. In addition, KEGG pathway analysis showed that CPP+LNPs treatment simultaneously affected the biosynthesis of arabinogalactan, ubiquinone and other terpenoid quinones. Arabinogalactan is an important component of biofilm. Its synthesis inhibition hinders biofilm formation and reduces bacterial colonization ability. Ubiquinone and menaquinone are key electron carriers of cellular respiration. Their synthesis inhibition hinders bacterial energy production, reduces growth rate, reduces the production and secretion of virulence factors, and weakens the ability to destroy tissues around implants.

[0125] Results 5: Anti-inflammatory activity of CPP NPs against lipopolysaccharide (LPS) and antimicrobial photodynamic therapy (aPDT)-induced inflammation

[0126] In the pathogenesis of peri-implantitis, bacterial colonization activates the host immune system and triggers an inflammatory response, in which macrophages play an important role. M1 macrophages participate in the defense against pathogens but aggravate inflammation and alveolar bone resorption. Although traditional antibacterial photodynamic therapy can kill bacteria, the large amount of ROS produced will aggravate tissue oxidative stress. M2 phenotype macrophages have pro-resolving, anti-inflammatory and pro-regenerative activities. Regulating macrophage polarization is of great significance for the treatment of peri-implantitis.

[0127] CPP NPs inhibit M1 macrophage polarization by scavenging ROS and blocking pathogen-associated molecular patterns (PAMPs). RAW 264.7 cells were stimulated with lipopolysaccharide from P.gingivalis to simulate acute inflammation, and then treated with different NPs. Fig.12 As shown in B and C, CPP NPs can inhibit the expression of pro-inflammatory factors, such as Fig.12 As shown in D in Figure , CPP NPs can overcome the ROS side effects produced by antimicrobial photodynamic therapy and can also alleviate inflammatory responses by adsorbing PAMPs, such as Fig.12 As shown in Figures E and I, CPP NPs can inhibit the translocation of nuclear factor-κB (NF-κB) / p65 subunit and exert anti-inflammatory effects.

[0128] RT-qPCR was used to detect factors associated with M2 phenotype macrophages. Fig.12 As shown in F, G, H, and J in Figure 3, the group containing copper tannate can upregulate the expression of M2 markers and promote the transition from M1 to M2 phenotype, which is also verified by flow cytometry results. Overall, CPP NPs reshaped the immune microenvironment of peri-implantitis and showed significant anti-inflammatory activity against inflammation.

[0129] Result 6:

[0130] In this study, a rat model of peri-implantitis was established to investigate the anti-inflammatory and antibacterial activities of CPP NPs in vivo.

[0131] like Fig.13 As shown in A, in this study, the left first molar of the rat was extracted, the alveolar socket was prepared, and the implant was implanted. After 4 weeks, the neck of the implant was ligated with silk thread and inoculated with P.gingivalis to induce peri-implantitis. After 2 weeks, the rat gums became swollen, dark red, and food residues accumulated and bled, proving that the peri-implantitis model was successfully established. After the model was established, treatment and analysis were carried out.

[0132] like Fig.13 As shown in Figure B, after 2 weeks of CPP NPs treatment, the symptoms of the CPP+L group were effectively alleviated; the colony forming unit count results were shown in Fig.13 As shown in C and D in Figure 1, the CPP+L group was more than 75 times lower than the control group, which can effectively remove biofilm and reduce the number of pathogens; micro-computed tomography and other results are shown in Figure 1. Fig.13 As shown in G to J in Figure 1, the CPP+L group has a better effect in inhibiting alveolar bone resorption; the in vivo imaging results of living animals are shown in Fig.13 As shown in E and F in Figure 3, CPP NPs can alleviate tissue oxidative stress; HE staining for histological results is shown in Figure 3. Fig.14 As shown in A and B, the number of inflammatory cells in the CPP group and the CPP+L group was the least, and the inhibitory effect on local inflammation was strong; the Masson staining results are shown in the figure Fig.14 As shown in C and D, the CPP+L group was effective in maintaining collagen integrity; the results of immunohistochemistry were shown in Fig.14 As shown in Figures E to H, the expression of arginase-1 increased and the expression of interleukin-6 decreased, indicating that CPP NPs have the ability to regulate inflammatory responses and have the potential to reshape the inflammatory microenvironment in vivo.

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a CuTA-Por@ε-PL nanoplatform, characterized in that: The steps include: S1, tannic acid reacts with copper sulfate pentahydrate to prepare CuTA nanosheets; S2, grafting protoporphyrin onto APTES-aminated CuTA nanosheets to form CuTA-Por; S3. ε-PL coated CuTA-Por to obtain CuTA-Por@ε-PL nanoplatform.

2. The preparation method according to claim 1, characterized in that In S1, tannic acid and copper sulfate pentahydrate are dissolved in deionized water, and the CuTA nanosheets are obtained by adjusting pH, heating and stirring, centrifuging, washing and drying.

3. The preparation method according to claim 1, characterized in that: In S1, the mass ratio of tannic acid to copper sulfate pentahydrate is 1:(30-35), preferably, the mass ratio of tannic acid to copper sulfate pentahydrate is 27:

875.

4. The preparation method according to claim 1, characterized in that: In S2, protoporphyrin needs to be activated, specifically: dissolving protoporphyrin, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in deionized water, stirring the mixture at 23-24° C. for 25-35 minutes under a nitrogen atmosphere to activate the carboxyl group of protoporphyrin.

5. The preparation method according to claim 1, characterized in that: In S2, the preparation method of APTES-aminated CuTA nanosheets is as follows: CuTA is dispersed in ethanol, and then APTES is added, the solution is heated and stirred in an oil bath, and the product "CuTA-NH2" is obtained by centrifugation.

6. The preparation method according to claim 1, characterized in that: In S2, the mass ratio of protoporphyrin to the CuTA nanosheets aminated with APTES is 1:(10-80), preferably, the mass ratio of protoporphyrin to the CuTA nanosheets aminated with APTES is 1:

20.

7. The preparation method according to claim 1, characterized in that: In S3, the mass ratio of ε-PL to CuTA-Por is 1:(5-20), preferably, the mass ratio of ε-PL to CuTA-Por is 1:

10.

8. A CuTA-Por@ε-PL nanoplatform, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the CuTA-Por@ε-PL nanoplatform according to claim 8 in the preparation of a product for treating peri-implantitis.

10. A drug, characterized in that Comprising the CuTA-Por@ε-PL nanoplatform as described in claim 8.

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