CuTA-por epsilon-pl nano platform, preparation method and application thereof

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a CuTA-Por@epsilon-PL nano platform and a preparation method and application thereof. The nano platform is prepared by grafting protoporphyrin (Por) through amidation reaction of CuTA, and wrapping the CuTA by epsilon-polylysine (epsilon-PL) through electrostatic adsorption, so as to synthesize CuTA-Por@epsilon-PL NPs (CPP NPs), which can be used as a potential treatment method for treating peri-implantitis, destroying bacterial biofilm and regulating inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to the CuTA-Por@ε-PL nanoplatform, its preparation method, and its application. Background Technology

[0002] The successful clinical application of dental implants is one of the major advancements in 20th-century dentistry. Dental implants have dramatically changed the way humans restore missing teeth, making a significant contribution to maintaining the integrity of the maxillofacial organs, restoring oral function, and maintaining oral and overall health; hence, they are often referred to as "humanity's third set of teeth." Currently, dental implant restoration has become an important restorative method for patients with missing teeth, and the number of implants has increased dramatically.

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

[0004] Currently, non-surgical and surgical treatments are the main methods for treating implant-related plaque buildup (PI) in clinical practice. Non-surgical treatment typically involves mechanical debridement of the implant surface, supplemented by topical antibiotics or antibacterial agents. Repeated mechanical debridement can easily scratch the crown and implant surface, making it easier for bacteria to re-colonize and accumulate, resulting in stubborn plaque biofilm residue that is difficult to completely remove. While surgical treatment can thoroughly remove plaque biofilm, it carries risks such as significant trauma, high cost, and postoperative gingival recession and implant exposure. Exploring safer and more effective treatment methods for PI can help improve patients' quality of life, reduce the medical burden, and has important clinical value and social significance.

[0005] In recent years, antimicrobial photodynamic therapy (aPDT) has received increasing attention in antimicrobial applications due to its numerous advantages, such as minimally invasiveness, rapid onset and high efficacy, broad-spectrum antimicrobial activity, and lack of bacterial resistance. Some photosensitizers (PS), such as methylene blue, porphyrin (Por), and dihydroporphyrin e6, possess the ability to instantaneously generate large amounts of reactive oxygen species (ROS) under excitation light irradiation. They have been applied to the treatment of PI or periodontitis, achieving certain clinical results. However, due to the special anatomical location of the periodontal pocket around implants and the complex structure of plaque biofilms, aPDT still faces the following challenges in clinical application: First, excessive ROS generated by aPDT can exacerbate oxidative stress in peri-implant tissues, hindering clinical translation. Second, most PS lack specific targeting ability to plaque biofilms, resulting in non-specific phototoxicity to surrounding normal tissues. Third, extracellular polymers in the biofilm hinder the penetration and diffusion of PS, making it difficult for them to enter the biofilm interior, thus limiting their antimicrobial effect.

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

[0007] To address the aforementioned problems, this invention provides a CuTA-Por@ε-PL nanoplatform, its preparation method, and its applications. The nanoplatform of this invention involves grafting protoporphyrin (Por) onto CuTA via an amidation reaction, followed by electrostatic adsorption to encapsulate it with ε-polylysine (ε-PL), thereby synthesizing CuTA-Por@ε-PL NPs (CPP NPs). This can serve as a potential therapeutic method for treating PI (protoporphyrin-induced inflammation) by disrupting bacterial biofilms and regulating inflammation.

[0008] To achieve the above objectives, 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, CuTA nanosheets were prepared by reacting tannic acid with copper sulfate pentahydrate;

[0011] S2. Grafting protoporphyrin onto APTES-ammoniated CuTA nanosheets to form CuTA-Por;

[0012] S3 and ε-PL are used to coat CuTA-Por, thus obtaining the CuTA-Por@ε-PL nanoplatform.

[0013] Secondly, the present invention provides a CuTA-Por@ε-PL nanoplatform, which is prepared by the above-described preparation method.

[0014] Thirdly, this invention provides the application of the CuTA-Por@ε-PL nanoplatform in the treatment of peri-implantitis.

[0015] Fourthly, the present invention provides a drug comprising the above-described CuTA-Por@ε-PL nanoplatform.

[0016] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0017] This invention can serve as a potential therapeutic approach for treating PI (proliferative prophylaxis) by disrupting bacterial biofilms and modulating inflammation. On one hand, CuTA-Por@ε-PL NPs (CPP NPs) possess a highly positively charged surface, which significantly enhances their penetration and distribution within biofilms, as well as their preferential selectivity for bacteria, thereby clearing biofilms through aPDT-mediated ROS bursts. On the other hand, CPP NPs can remove ROS from PI tissues and those remaining after aPDT, and adsorb pathogen-associated molecular patterns, thereby inhibiting inflammatory responses, regulating macrophage polarization, and promoting tissue regeneration. This carefully crafted strategy holds promise for providing a new avenue for improving the therapeutic efficacy of PI in future clinical applications. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0019] Figure 1 This invention provides a performance analysis of CuTA-Por with different Por / CuTA mass ratios in this embodiment. A and C represent the characterization and normalization analysis of the in vitro photodynamic properties of CuTA-Por with different Por / CuTA ratios, respectively, and B represents the UV-vis spectra of CuTA-Por with different Por / CuTA ratios.

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

[0021] Figure 3 This is a schematic diagram of CPP NPs treating PI in an embodiment of the present invention;

[0022] Figure 4 The preparation and characterization of PI therapeutic material CPP NPs in this embodiment of the invention are shown in A, which is a schematic diagram of the synthesis of CPP NPs, B is a transmission electron microscope image, C is the ultraviolet-visible spectrum, D is the hydrated particle size, and E is the zeta potential.

[0023] Figure 5The results of stability and biocompatibility verification of CPP NPs in the embodiments of the present invention are as follows: A is the particle size change of CPP NPs in different solutions over 7 days; B is the detection of CCK-8 in 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 this embodiment of the invention, 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 following are the verification results of the mechanism of ROS scavenging by CPP NPs in the embodiments of the present invention. In this figure, A is a schematic diagram of the antioxidant activity of CPP NPs, and B and C represent the oxidation of NBT by ·O in the presence of different concentrations of CPP NPs, respectively. 2- The absorption spectra and related normalized analyses of the reduction are shown. D and E represent the absorption spectra and related normalized analyses of the reaction between SA and ·OH at different concentrations of CPP NPs, respectively. F and G represent the dissolved oxygen concentrations catalyzed by different concentrations of CPP NPs and different solutions, respectively. H and I represent the dissolved oxygen concentrations of ·OH. 2- Absorption spectra and corresponding normalized analyses of NBT reduction in different solutions are presented. J and K represent the absorption spectra and corresponding normalized analyses of the reaction of SA with ·OH in different solutions, respectively. L and M represent the absorption spectra and corresponding normalized analyses of the reaction of SA with ·OH in different solutions, respectively. The imaging system provides in vitro fluorescence images of ROS generation and clearance under different treatments, as well as corresponding fluorescence intensity analysis. N and O represent the quantitative analysis of ROS levels in HGFs after different treatments and CLSM images (scale bar: 50 μm).

[0026] Figure 8In this embodiment of the invention, CuTA-Cy5.5 and CCP were synthesized to simulate the penetration and accumulation of CuTA-Por and CPP NPs in mature biofilms. A represents the biofilm penetration and selective adsorption scheme mediated by CPP NPs; B(i) represents the biofilm penetration scheme mediated by CuTA-Por and CPP NPs; (ii) and (iii) represent the 3D CLSM images and corresponding z-stack images of *P. gingivalis* biofilms treated with CuTA-Cy5.5 and CCP for different times, along with their relative fluorescence intensities; C represents the TEM images (scale bar: 1 μm) of *P. gingivalis* after treatment with CuTA-Por and CPP, and a partial magnification of these images (scale bar: 200 nm); D represents the Zeta potentials of *P. gingivalis* and HGFs cells after mixing different concentrations of CPP NPs for 30 minutes; and E represents the co-cultured *P. gingivalis* (Nile Red stained) and HGFs cells (Hoechst)... Flow cytometry analysis of 33342 staining (30 minutes after adding CPP NPs).

[0027] Figure 9 In this embodiment of the invention, the antibacterial effect of CPP NPs on free P. gingivalis was studied using plate count and TEM. A shows (i) colony images and (ii) TEM images (scale bar: 1 μm) of P. gingivalis treated with different concentrations of CPP NPs. B shows the quantitative analysis of P. gingivalis treated with different concentrations of CPP NPs. C shows the quantitative analysis of P. gingivalis after different treatments. D shows the absorbance curves of P. gingivalis suspensions at 260 nm after different treatments, indicating nucleic acid leakage detection. E shows (i) colony images, (ii) TEM images (scale bar: 1 μm), and (iii) photographs of P. gingivalis after different treatments.

[0028] Figure 10To investigate the antimicrobial effect of CPP NPs on established and forming *P. gingivalis* biofilms in this embodiment of the invention, A is a schematic diagram of the anti-biofilm function of CPP NPs, B is a representative 3D live / dead image of the established *P. gingivalis* biofilm, C is a representative image of the biofilm established after crystal violet staining, D is the ratio of dead to live *P. gingivalis* bacteria in the biofilm, E is the average thickness of the established *P. gingivalis* biofilm, F and G are representative colony photographs and related quantitative analyses of the established *P. gingivalis* biofilm, and H is a representative 3D image of *P. gingivalis* biofilm formation. Live / Dead images: I is a representative image of biofilm formed after crystal violet staining; J is the ratio of dead to live bacteria in P. gingivalis biofilm formation; K is the average thickness of P. gingivalis biofilm formation; L is a representative colony image of P. gingivalis biofilm formation; M is related quantitative analysis; N is the relative mRNA levels of adhesin genes and virulence factor genes in the established P. gingivalis biofilm detected by RT-qPCR.

[0029] Figure 11 In this embodiment of the invention, the transcriptomics analysis results before and after treatment with CPP NPs were compared using P. gingivalis as a model organism. Among them, A is a volcano map of upregulated and downregulated genes in the biomembrane after CPP NPs treatment, B is a heatmap of DEGs in the Control and CPP+L groups, C is the GO enrichment analysis of DEGs, D is the KEGG enrichment analysis of DEGs, and E is a heatmap of interactions of significantly differentially expressed genes in the ribosome pathway.

[0030] Figure 12This invention presents an example of stimulating RAW 264.7 cells with lipopolysaccharide derived from *P. gingivalis* to simulate acute inflammation, and describes the in vitro anti-inflammatory effect of CPP NPs treatment. In this example, A represents the anti-inflammatory activity regimen of CPP NPs; B represents 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 using RT-qPCR analysis; D represents the concentrations of IL-6 and TNF-α in the supernatant after 3 hours of different treatments; E represents the immunofluorescence image (scale bar: 50 μm) of macrophage NF-κB / p65 translocation and the quantification of (I) positive cells; and F represents the CPP NPs concentration. NPs-mediated macrophage polarization regulation protocol, J represents flow cytometry analysis of CD86, a specific marker for M1 macrophages, and CD206, a specific marker for M2 macrophages, and G and H represent quantitative analysis of CD86-positive and CD206-positive cells under different experimental treatments, respectively.

[0031] Figure 13 In this embodiment of the invention, a rat model of peri-implantitis was established to investigate the in vivo anti-inflammatory and antibacterial activities of CPP NPs. A is a diagram of the PI model establishment and drug administration regimen; B is an intraoral photograph of the model rats after treatment with different NPs (scale bar: 2 mm); C is an image of bacterial colonies isolated from the peri-implant tissue after different treatments; D is the relevant quantitative analysis; E and F are in vivo fluorescence imaging and corresponding quantification of ROS levels at the implantation site under different treatments, respectively; G(i) and (ii) are three-dimensional reconstruction images of the buccal and palatal sections of the implants after different treatments using Micro-CT (scale bar: 1 mm); HJ is the quantitative statistics of alveolar bone resorption, bone volume fraction, and number of trabeculae.

[0032] Figure 14 In this embodiment of the invention, HE staining, Masson staining, and immunohistochemistry are used to verify the regulatory effect of CPP NPs on the inflammatory response. A and B are HE staining images and immune cell counts, respectively; C and D are Masson staining images and collagen volume 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: Arg-1 positive cells, blue: cell nuclei, scale bar: 100 μm); and H is the relative immunofluorescence intensity of Arg-1. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, 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 terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting 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, CuTA nanosheets were prepared by reacting tannic acid with copper sulfate pentahydrate;

[0037] S2. Grafting protoporphyrin onto APTES-ammoniated CuTA nanosheets to form CuTA-Por;

[0038] S3 and ε-PL are used to coat CuTA-Por, thus obtaining the 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 CuTA nanosheets are obtained by adjusting the pH, heating and stirring, centrifuging, washing and drying.

[0040] Specifically, tannic acid and copper sulfate pentahydrate were dissolved in deionized water. The pH of the mixture was adjusted to 7 ± 0.5 by adding sodium hydroxide solution. The solution was then heated to 60°C in an oil bath and stirred for 4–6 hours. The product was obtained by centrifugation at 5000 rpm / min for 4–6 minutes, followed by washing three times with deionized water and once with anhydrous ethanol. After vacuum drying in a 60°C oven, the product was stored at 4°C for later 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, the protoporphyrin needs to be activated, specifically by dissolving the protoporphyrin, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in deionized water, and stirring the mixture at 23-24°C for 25-35 minutes under a nitrogen atmosphere to activate the carboxyl groups of the protoporphyrin.

[0043] In another specific embodiment of the present invention, in S2, the preparation method of APTES-ammoniated CuTA nanosheets is as follows: CuTA is dispersed in ethanol, then APTES is added, the solution is heated and stirred in an oil bath, and the product "CuTA-NH2" is obtained by centrifugation.

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

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

[0046] Specifically, an aqueous solution of ε-PL is mixed with an aqueous solution of CuTA-Por. The mixture is then sonicated for 30 minutes and stirred at 25°C for 5–8 hours. Afterward, the product is purified using deionized water through multiple washing and centrifugal cycles, and then dried by lyophilization to obtain the final product.

[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 one specific embodiment of the present invention, the application of the above-described CuTA-Por@ε-PL nanoplatform in the preparation of products for treating peri-implantitis is provided.

[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 locally into the peri-implant pocket to achieve a therapeutic effect. Experiments have demonstrated that it possesses the ability to clear biofilms and regulate inflammation. The drug dosage form is an injection.

[0052] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0053] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0054] Example 1

[0055] I. 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 approximately 5 mL of 2 mol / L sodium hydroxide solution. The solution was then 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, followed by washing three times with deionized water and once with anhydrous ethanol. After vacuum drying in a 60 °C oven 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 via an 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. The mixture was stirred at 25 °C for 30 min under a nitrogen atmosphere to activate the carboxyl groups of the protoporphyrin. Subsequently, 20 mL of copper tannate (CuTA) solution (containing 100 mg CuTA) was added to the activated protoporphyrin solution, and the mixture was stirred at 25 °C for 6 h under nitrogen protection. The color of the reaction solution gradually changed from green to purple. After the reaction was complete, the solution was centrifuged (5000 rpm / min, 5 min) and washed three times with deionized water. After drying, the product CuTA-Por (CuTA-Por, Por / CuTA mass ratio of 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 effective antimicrobial photodynamic therapy (aPDT). 1,3-Diphenylisobenzofuran (DPBF) was used as a singlet oxygen sensor to assess extracellular reactive oxygen species (ROS) generation. DPBF was added to CuTA-Por solutions with different Por / CuTA mass ratios (1:10–1:80) to record absorbance at 420 nm. Absorbance was measured every 30 seconds during 660 nm laser irradiation.

[0060] like Figure 1 As shown in Figure A, we synthesized CuTA-Por NSs with different Por / CuTA mass ratios and investigated their in vitro ROS generation capacity using 1,3-diphenylisobenzofuran (DPBF) as a reactive oxygen species (ROS) probe. The aPDT effect mainly depends on the instantaneous burst of ROS. Figure 1 As shown in Figure B, there was no significant difference in the photodynamic performance of CuTA-Por with Por / CuTA mass ratios of 1:10 and 1:20 within the first five minutes. However, CuTA-Por with Por / CuTA mass ratios of 1:40 and 1:60 showed insufficient ROS generation capacity within the first five minutes. Therefore, we decided to select 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 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. The mixture was stirred at 25 °C for 30 min under a nitrogen atmosphere to activate the carboxyl groups of the protoporphyrin. Subsequently, 20 mL of copper tannate (CuTA) solution (containing 100 mg CuTA) was added to the activated protoporphyrin solution, and the mixture was stirred at 25 °C for 6 h under nitrogen protection. The color of the reaction solution gradually changed from green to purple. After the reaction was complete, the solution was centrifuged (5000 rpm / min, 5 min) and washed three times with deionized water. After drying, the product CuTA-Por (CuTA-Por, Por / CuTA mass ratio 1:20) was 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 / mL). The mixture was then sonicated for 30 minutes and stirred at 25°C for 6 hours. The product was subsequently purified by repeated washing and centrifugation with deionized water, and then dried by lyophilization. CuTA-Por@ε-PL (CPP) with different ε-PL / CuTA-Por mass ratios (1:5 and 1:20) were prepared using a similar method.

[0065] The DLS results for three CPP NPs with mass ratios of 1:5, 1:10, and 1:20 are as follows: Figure 2As shown, the particle size of CPP (1:5) is mainly distributed in the range of 255 nm to 955 nm (polydispersity index PDI is 0.690). The particle size of CPP (1:10) is 204.6 nm, and the PDI is 0.291. The particle size of CPP (1:20) is 631.5 nm (PDI is 0.470), and a peak of larger-sized products appears at 5000 nm. 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 some CuTA-Por to be coated too thickly or to aggregate, thus forming products with larger particle sizes. When the ratio is 1:10, the product exhibits a suitable particle size and uniform distribution. 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 minutes and stirred at 25°C for 6 hours. The product was then purified by repeated washing and centrifugation with deionized water, and finally dried by lyophilization.

[0067] The hydrated particle size distribution, polydispersity index (PDI), zeta potential, and morphology were determined using dynamic light scattering and transmission electron microscopy. The UV-Vis spectra of intermediate and final products were measured using UV-Vis-NIR spectroscopy.

[0068] II. Stability and Biosafety of CPP NPs

[0069] The stability of CPP NPs was assessed 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 assessed using the CCK-8 assay and hemolysis test. Human gingival fibroblasts (HGFs) and L929 cells were first seeded in 96-well plates and cultured overnight. Then, different concentrations of CPP NPs were added, and the plates were incubated for 24 hours. After treatment, the old medium was replaced with fresh medium containing Cell Counting Kit-8 (CCK-8), and the cells were incubated for another 3 hours. Finally, cell viability was measured at 450 nm using a microplate reader.

[0071] For the hemolysis assay, red blood cells were separated by centrifugation at 1500 rpm for 15 minutes. The red blood cells were then washed three times with PBS and diluted tenfold to prepare a red blood cell suspension. Positive controls (+) were prepared with distilled water, while negative controls (-) were prepared with PBS. Equal volumes of the red blood cell suspension were mixed with sample solutions of different concentrations to be evaluated, incubated at 37°C for 2 hours, and then centrifuged at 1500 rpm for 15 minutes. The supernatant was then transferred to 96-well plates, and absorbance was measured at 576 nm using a microplate reader.

[0072] III. ROS time-space control based on CPP NPs

[0073] 1. In vitro ROS generation of CPP: Similar to the method described above, extracellular ROS generation in CPP cells was assessed using DPBF. DPBF was added to Por, CuTA-Por, and CPP solutions to record absorbance intensity at 420 nm. Absorbance was measured every minute during 660 nm laser irradiation.

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

[0075] 2.1 In vitro scavenging of superoxide anions (·O2) - CPP scavenging of O2 was evaluated by measuring the inhibition rate of NBT photoreduction. - The ability to [conduct experiments] was investigated. Here, 20 μM riboflavin, 12.5 mM methionine, 75 μM NBT, and different concentrations of CPPNPs were dissolved in PBS (pH 7.4). These mixtures were then exposed to UV radiation for 15 minutes. After irradiation, [the following parameters were observed]: O2 [reduction / reduction]. - NBT was reduced to a blue product with an absorption peak at 560 nm. This product was further quantitatively analyzed by UV-Vis absorption spectroscopy. It contained riboflavin, methionine, and NBT. Samples under UV-free and UV-illuminated conditions were designated as negative and positive controls, respectively. All experiments were conducted in darkness. CuTA and CPPNPs were detected using the same method after 5 minutes of 660 nm laser irradiation. - Clearance capability.

[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). First, 2 × 10⁻⁶ ions were added to the salicylic acid (SA) solution. -4 M H2O2 and 2×10 -4 M FeSO4 was mixed and ·OH was generated via the Fenton reaction. Subsequently, different concentrations of CPP NPs were added to the solution to eliminate the ·OH. Finally, 1×10⁻⁶ ppm was added...-3 MSA was used to detect the remaining ·OH. ·OH oxidizes SA to 2,3-dihydroxybenzoic acid, which is purple in color. The absorption peak at 510 nm was further determined by UV-Vis absorption spectroscopy. The same method was used to detect CuTA and CPP NPs after 5 minutes of 660 nm laser irradiation. · OH removal efficiency.

[0077] 2.3 Catalase-like Activity of CPP NPs: The CAT activity of CPP NPs was investigated by measuring the hydrogen peroxide (H2O2) scavenging rate. 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 analyzer. The CAT enzyme activity of CuTA and CPP NPs irradiated with a 660 nm laser for 5 minutes was also investigated.

[0078] 3. Dynamic monitoring of ROS generation and clearance in vitro: The experimental group consisted of five groups: control group, CuTA group, CPP group, Por group (Por+L) irradiated with 660nm laser for 5 minutes, and CPP group (CPP+L) irradiated with 660nm laser for 5 minutes. Stock solutions for these five groups were prepared separately. The Por+L and CPP+L groups were irradiated with 660nm laser for 5 minutes, while the other groups received no treatment. Immediately afterwards, 180μL of the stock solution was added to the well plate, followed by the addition of 2',7'-DCFH-DA. Fluorescence imaging was recorded on the imaging system (excitation wavelength λex = 488 nm, emission wavelength λem = 525 nm) and marked as "0 minutes". Subsequently, every 3 minutes, 180 μL of solution was added from the stock solution to the well plate, followed by 2',7'-DCFH-DA, and the detection process was repeated.

[0079] DCFH-DA was used to measure intracellular and extracellular ROS levels. Intracellular ROS levels were also measured in five groups as described above: Control, CuTA, CPP, Por+L, and CPP+L. Human gingival fibroblasts (HGFs) were seeded at a density of 2 × 10⁵ cells / dish in confocal microscope culture dishes (35 mm) and cultured at 37°C for 24 hours. Then, PBS, CuTA, Por, and CPP were added, with some cells treated with laser and others 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 using a confocal laser scanning microscope (CLSM).

[0080] IV. Antibacterial Performance Test of CPP NPs

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

[0082] 1.1 CPP NPs-mediated biofilm penetration and dispersion: To form a mature *P. gingivalis* biofilm, the microbial concentration was set at 10⁸ CFU / mL and placed in a confocal microscope culture dish, followed by incubation in anaerobic medium at 37°C for 96 hours. To assess the ability of NPs to penetrate the biofilm, CuTA was labeled with Cy₅₅ to obtain CuTA-Cy₅₅, which was then synthesized as CuTA-Cy₅₅@ε-PL (CCP). CuTA-Cy₅₅ and CCP represent CuTA-Por and CPP NPs, respectively. Then, 1 mL of CuTA-Cy₅₅ and CCP solutions were added to the biofilm, respectively, and incubated for 0.5 h, 1 h, and 2 h, respectively. After incubation, the biofilm was washed three times with PBS to remove unbound NPs. Subsequently, the biofilm was stained with DMAO for 20 minutes in the dark. The penetration of NPs into the biofilm was observed using a confocal laser scanning microscope (CLSM).

[0083] 1.2 Selective Adsorption of CPP NPs to Cells and Bacteria: Human gingival fibroblasts (HGFs) were stained with Hoechst 33342 for 20 minutes and then washed with phosphate-buffered saline (PBS). *P. gingivalis* was stained with Nile Red for 20 minutes and also washed with PBS. HGFs and *P. gingivalis* were mixed in PBS at concentrations of 10⁵ cells / mL and 10⁷ CFU / mL, respectively. CCPs were then added to the co-incubation solution, and the mixture was incubated for another 30 minutes. Finally, the samples were quantitatively analyzed by flow cytometry.

[0084] 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 co-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. Cells / bacteria were collected, resuspended, and their zeta potentials were measured to investigate the effect of CPPNPs on the surface charge of cells / bacteria.

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

[0086] 2. Evaluation of the antibacterial and antibiofilm properties of CPP NPs

[0087] 2.1 Evaluation of the antibacterial activity of CPP NPs against free bacteria: Different concentrations of CPP NPs were mixed with 1 mL of *Gingivalis* bacterial suspension (10⁶ CFU / mL). After 5 minutes of 660 nm laser irradiation, the mixture was incubated for 24 hours. After serial dilution, the solutions were evenly spread on blood agar plates and then anaerobically cultured at 37 °C for colony counting. For TEM imaging, the bacterial suspension was fixed with 2.5% (w / v) glutaraldehyde. Subsequently, images were taken to observe bacterial morphology and its interaction with CPP NPs. Similarly, the concentration of CPP was fixed at 300 μg / mL, and five groups were established (control group, CuTA group, Por+L group, CPP group, and CPP+L group), with and without laser irradiation. 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 / 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 at 260 nm.

[0089] 2.3 Evaluation of anti-biofilm properties: Five groups were established: control group, CuTA group, Por+L group, CPP group, and CPP+L group. Plates containing biofilms that were forming or had already formed were treated with different NPs (NPs) and then subjected to a 660nm laser (1W / cm²). 2Irradiation was performed for 5 minutes. 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 sonication. Subsequently, the biofilm was continuously diluted tenfold. Then, 100 μL of the diluted bacterial solution was spread on blood agar and placed in an incubator for anaerobic culture. For live / dead fluorescent staining, the biofilm was first rinsed to remove unattached bacteria. Then, a mixture of 2.5 μM SYTO 9 and 2.5 μM propidium iodide (PI) was prepared according to the manufacturer's instructions and used to stain each sample for 20 minutes. Three-dimensional images of the biofilm were obtained using a confocal laser scanning microscope (CLSM). For crystal violet staining, the treated biofilm was first fixed for 15 minutes. After air drying, 1 mg / mL crystal violet solution was added and stained for 15 minutes. Excess dye was then thoroughly rinsed off. Finally, the stained biofilm was dissolved in 95% ethanol, and the absorbance at 590 nm was measured using a microplate reader.

[0090] 2.4 Evaluation of extracellular protein gene expression in *P. gingivalis*, specifically, analysis of genes such as FimAII, FimAIV, RgpA, RgpB, and Kgp using RT-qPCR. A 2- ΔΔCt The relative gene expression levels were determined using a method. For data standardization, the 16S rRNA gene was used as an internal control, 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 NP treatment, the formed *P. gingivalis* biofilm was treated with CPP NPs and irradiated with a 660 nm laser. After treatment, the biofilm was collected by centrifugation (10,000 rpm, 3 min), then frozen in liquid nitrogen for 15 min, and then placed in dry ice for further RNA extraction.

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

[0093] 1. Phenotypic switching of macrophage polarization:

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

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

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

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

[0098] Using LPS as a typical PAMP, we investigated the ability of CPP NPs to block PAMP-induced inflammatory responses. RAW 264.7 cells were seeded at a density of 1 × 10⁵ cells per well in 6-well plates and cultured for 1 day. Subsequently, *P. gingivalis*-LPS (1 μg / mL) was mixed with different NPs, and this 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 an ELISA kit.

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

[0100] To further verify the anti-inflammatory and aPDT protective mechanisms of CPP NPs, the NF-κB signaling pathway was evaluated by translocation of the NF-κB / p65 subunit. 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 were laser-irradiated, while others were not, 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. Next, the cells were incubated with rabbit anti-NF-κB p65 primary antibody (1:500) at 37 °C for 1.5 h, and then incubated with goat anti-rabbit Alexa 488-conjugated IgG secondary antibody (1:1000) at 37 °C for 1 h. Cell nuclei were stained using DAPI after incubation in the dark for 5 minutes. Finally, imaging was performed 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 were ethically reviewed and 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 acclimatization, the left maxillary first molar of the rats was completely extracted, and an implant was immediately placed. Four weeks later, when the implants had achieved osseointegration, the rats were randomly divided into six groups: control, PI, CuTA, Por+L, CPP, and CPP+L. Except for the control group, all other groups were treated for two weeks by ligating the implant neck with silk sutures and inoculating with *P. gingivalis* to establish the PI model. Subsequently, the control and PI groups received no treatment, while the other groups received the appropriate substance once daily into the periimplant pocket. In addition, the Por+L and CPP+L groups received laser irradiation for 5 minutes. After two weeks of treatment, the rats were euthanized, and the left maxilla was collected for Micro-CT scanning and immunohistochemical analysis.

[0103] 2. Measure the bone retraction height at the proximal and distal ends of the implant separately, and use the average of the two measurements to represent the implant bone retraction height.

[0104] 3. In vivo animal fluorescence imaging: ROS levels in peri-implant tissues after different treatments were investigated. After 2 weeks of treatment, 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 over 30 minutes. Afterwards, using… An imaging system (excitation filter: 525nm; emission filter: 495nm, PerkinElmer) recorded fluorescence imaging.

[0105] All data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's unpaired t-test and two-way ANOVA. 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 within biofilms.

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

[0108] UV spectroscopy results as follows Figure 4 As shown in Figure C, CuTA-Por exhibits both 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 (approximately 110 nm) in the dry state. However, poor solubility of CuTA-Por in aqueous solution and its tendency to precipitate are observed. Dynamic light scattering (DLS) detection results are shown in Figure B. Figure 4As shown in Figure D, the hydrated particle size of CuTA-Por is 1009 nm. This characteristic is unfavorable for the penetration and dispersion of NPs within biofilms. To address this issue, 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, ε-PL coating not only improves the water solubility of CuTA-Por but also endows CPP NPs with a strong positive charge. After amino modification, the zeta potential of CuTA increases from -14.87 mV to -0.478 mV. Furthermore, after Por grafting, its zeta potential further changes to -6.04 mV, confirming the success of the modification.

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

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

[0112] This invention investigated the changes in particle size stability and biocompatibility of CPP NPs in water, phosphate-buffered saline (PBS), and Dalberg modified Eagle medium (DMEM). Figure 5 As shown in Figure A, CPP NPs exhibit excellent particle size stability. The slight increase in particle size in DMEM solution can be attributed to protein adsorption. Figure 5 As shown in B, after synthesizing CPP NPs, the good biocompatibility of CPP NPs was verified using a cell counting kit-8 (CCK-8) and a hemolysis assay. Figure 5 As shown in C, even at concentrations as high as 800 μg / mL, more than 80% of human gingival fibroblasts (HGFs) and L929 cells remained viable, with a hemolysis rate of less than 5%.

[0113] Result 3: CPP NPs have a ROS regulatory effect.

[0114] The instantaneous burst of ROS induced by excitation light is the main mechanism by which aPDT exerts its antibacterial effect. Under excitation light irradiation, photosensitizers (PSs) can instantaneously generate a large amount of ROS through photocatalysis. Therefore, it is important to prioritize investigating the effects of copper tannate (CuTA) and ε-polylysine (ε-PL) coating on the ROS generation performance of porphyrins (Por) under laser irradiation.

[0115] The photodynamic therapy (PDT) performance of composite polyelectrolyte particles (CPP), copper tannate-porphyrin tannate (CuTA-Por), and Por was evaluated using 1,3-diphenylisobenzofuran (DPBF). Figure 6 As shown in A, compared to Por, the PDT performance of CPP is not significantly different, which is of great significance for CPP NPs to achieve aPDT through a short-term instantaneous ROS burst. Figure 6 As shown in Figure B, CuTA-Por's poor water solubility reduces its PDT effect in water. Therefore, ε-PL coating not only enhances penetration and distribution within bacterial biofilms due to its positive charge and small size, but also improves CuTA-Por's PDT effect, further enhancing ROS bursts.

[0116] like Figure 7 As shown in Figure A, the main mechanism by which CPP NPs scavenge ROS originates from the SOD / CAT enzyme activity of CuTA. To verify that CPP NPs possess SOD / CAT enzyme activity and that this activity is retained after laser irradiation, thus achieving the mode of "generating ROS for antibacterial purposes under laser irradiation and scavenging ROS for anti-inflammatory purposes without laser irradiation," the catalytic superoxide radical disproportionation catalyzed by CPP NPs was determined by measuring the inhibition rate of NBT photoreduction. The results are shown below. Figure 7 As shown in B and C, the absorbance at 560 nm decreased with different concentrations of CPP, indicating SOD-like activity. The scavenging efficiency of CPP NPs on hydroxyl radicals was determined by measuring salicylic acid, and the results are shown in Figure 1. Figure 7 As shown in D and E, it demonstrates its excellent... · OH scavenging ability; the hydrogen peroxide decomposition performance was evaluated by adding CPPNPs to PBS (0.1M, pH 7.4) containing H2O2, and the results are as follows: Figure 7 As shown in F, the decomposition rate increases with increasing CPP NPs concentration, indicating that CPP NPs have inherent CAT-like activity and good scavenging ability for various ROS. Their SOD / CAT activity facilitates the conversion of ROS in the PI bag into O2 and H2O, improving hypoxia and enhancing the aPDT effect; Figure 7 As shown in G, the ability to remove H2O2 remained at 67.8%, indicating that CPP NPs can still perform ROS removal after exerting aPDT function, avoiding side effects and continuously reducing ROS levels. The ROS removal capabilities of CPP NPs before and after irradiation were compared using the same method, and the results are shown in G. Figure 7 The values ​​H, I, J, and K in the figure indicate the removal of O2 after irradiation. - and · The ability of OH to react did not change significantly.

[0117] To visually demonstrate ROS generation and removal, five stock solutions were prepared. The Por+L and CPP+L groups were irradiated with a 660nm laser for 5 minutes, while the other groups received no treatment. Solutions from the stock solutions were added to well plates, and 2',7'-dichlorofluorescein diacetate (DCFH-DA) was added. The imaging system recorded fluorescence imaging. The results are as follows: Figure 7 As shown in the figure, the fluorescence intensity of the Por+L group and the CPP+L group increased significantly and by a similar magnitude after irradiation, confirming the ROS generation capability of CPP NPs under laser irradiation; as shown in the figure. Figure 7 As shown in Figure M, after laser irradiation, the fluorescence intensity of the CPP+L group continued to decrease, and 78.1% of the ROS was cleared after 9 minutes, verifying the ROS clearance ability of CPP NPs without laser irradiation. The same grouping method was used to detect intracellular ROS production and clearance, and the results are shown in Figure M. Figure 7 As shown in the N and O values, the Por+L group had a high proportion of positive cells, indicating that aPDT treatment caused intracellular oxidative stress, while the CPP+L group had a low proportion, indicating that CPP NPs could effectively alleviate the high intracellular ROS state caused by aPDT.

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

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

[0120] CuTA-Cy5.5 and CCP were synthesized to simulate CuTA-Por and CPP NPs, and their penetration and accumulation in mature biofilms were investigated. Results are as follows: Figure 8 As shown in B, CPP NPs have a strong ability to penetrate biofilms and can effectively solve the problem of insufficient penetration of PSs. This may be due to their small hydrated particle size and positive charge.

[0121] The positive charge of CPP NPs enhances their penetration and dispersion within biofilms, promoting preferential interaction with bacteria. Experimental results are as follows: Figure 8 As shown in C and D, this indicates that CPP NPs have a strong interaction with bacteria, such as Figure 8 As shown in E and F, the binding rate of CPP NPs to P. gingivalis is about 12.1 times that to HGFs, indicating a clear preference for binding to bacteria.

[0122] The antibacterial effect of CPP NPs on free *P. gingivalis* was studied using plate count and TEM. Results are as follows: Figure 9 As shown in A, B, C, and E, CPP NPs can kill free bacteria, exhibiting better antibacterial effects than the CuTA group. They can effectively kill bacteria through the PDT effect and can also capture and kill free bacteria, blocking their recolonization process. Nucleic acid leakage experiment results are shown below. Figure 9 As shown in D, it is confirmed that in addition to causing membrane damage through interaction with bacteria, CPP NPs can also cause bacterial rupture and damage through ROS bursts generated by laser irradiation.

[0123] The antibacterial effect of CPP NPs on established and forming *P. gingivalis* biofilms was studied using CLSM, crystal violet staining, and CFU counting. CPP NPs exhibited excellent biofilm clearance capabilities. Figure 10 As shown in B to M, this indicates that the anti-biofilm effect of CPP NPs was 2.95 times higher than that of the Por+L group, effectively killing bacteria within the biofilm and reducing bacterial count. *P. gingivalis* can produce various virulence factors that trigger host inflammatory responses; therefore, the inhibitory effect on the expression of virulence factors of *P. gingivalis* within the CPP NPs biofilm was shown in the results. Figure 10 The presence of N in the figure indicates that CPP NPs can inhibit the expression of virulence factors in *P. gingivalis* within biofilms, disrupting its adhesion, aggregation, and parasitic processes. They also inhibit biofilm formation, significantly reducing biofilm thickness after treatment. Overall, CPP NPs can damage bacterial cell membranes and nucleic acids, capturing and killing bacteria, thus achieving biofilm clearance and inhibition of biofilm formation.

[0124] To investigate the antibacterial mechanism of CPP NPs, comparative transcriptomics analysis was conducted using *P. gingivalis* as a model organism. For example... Figure 11 As shown in A and B, unsupervised principal component analysis revealed significant transcriptomic differences between the control group and the CPP+L group, detecting a total of 431 significantly differentially expressed genes. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment pathway analysis results are as follows... Figure 11 As shown in C and D, ribosome damage was most pronounced after treatment with CPP+L NPs, attributed to a strong ROS burst. Ribosomes are the site of intracellular protein synthesis; *P. gingivalis* depends on protein synthesis for survival and reproduction. Impaired ribosome function inhibits synthesis, effectively controlling bacterial growth and reproduction, reducing pathogenicity, and minimizing the likelihood of antibiotic resistance. Figure 11As shown in Figure E, the significantly differentially expressed genes related to ribosomes after CPP+LNP treatment are listed in detail in the interactive heatmap. Furthermore, KEGG pathway analysis revealed that CPP+LNP treatment simultaneously affected the biosynthesis of arabinogalactan, ubiquinone, and other terpenoid quinones. Arabinogalactan is an important component of biofilms; its inhibition hinders biofilm formation and reduces bacterial colonization. Ubiquinone and methylnaphthoquinone are key electron carriers in cellular respiration; their inhibition impairs bacterial energy production, reduces growth rate, decreases the production and secretion of virulence factors, and weakens the ability to damage tissues surrounding the implant.

[0125] Result 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's immune system, triggering an inflammatory response in which macrophages play a crucial role. M1 macrophages participate in defending against pathogens but exacerbate inflammation and alveolar bone resorption. Traditional antibacterial photodynamic therapy, while effective in killing bacteria, generates large amounts of reactive oxygen species (ROS), which intensifies tissue oxidative stress. M2 phenotype macrophages possess regressive, anti-inflammatory, and regenerative activities, and regulating macrophage polarization is of great significance for the treatment of peri-implantitis.

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

[0128] Factors associated with M2 phenotype macrophages were detected by RT-qPCR, and the results are as follows: Figure 12 As shown in F, G, H, and J, the group containing copper tannate upregulated the expression of M2 markers and promoted the M1-to-M2 phenotype transition, which was also verified by flow cytometry results. Overall, CPP NPs remodeled the immune microenvironment of peri-implantitis and exhibited significant anti-inflammatory activity.

[0129] Result 6:

[0130] This study investigated the in vivo anti-inflammatory and antibacterial activities of CPP NPs by establishing a rat model of peri-implantitis.

[0131] like Figure 13 As shown in Figure A, this study induced peri-implantitis in rats by extracting the left first molar, preparing the alveolar socket, implanting the implant, ligating the implant neck with silk sutures, and inoculating with *P. gingivalis* 4 weeks later. Two weeks later, the rats' gingiva showed swelling, dark red color, food debris accumulation, and bleeding, demonstrating the successful establishment of the peri-implantitis model. Treatment and analysis were then conducted after the model was established.

[0132] like Figure 13 As shown in B, after 2 weeks of CPP NPs treatment, the symptoms of the CPP+L group were effectively relieved; the colony-forming unit count results are as follows. Figure 13 As shown in C and D, the CPP+L group had a level more than 75 times lower than the control group, effectively clearing biofilm and reducing the number of pathogens; results from microcomputed tomography and other methods are as follows. Figure 13 As shown in G to J, the CPP+L group showed a better inhibitory effect on alveolar bone resorption; in vivo imaging results in living animals are as follows: Figure 13 As shown in E and F, CPP NPs can alleviate tissue oxidative stress; HE staining affects histological results as follows: Figure 14 Figures A and B show that the CPP and CPP+L groups had the fewest inflammatory cells and exhibited a strong inhibitory effect on local inflammation; Masson staining results are shown in Figure 1. Figure 14 As shown in C and D, the CPP+L group is effective in maintaining collagen integrity; the immunohistochemical results are as follows. Figure 14 As shown in E to H, the expression of arginase-1 is increased and the expression of interleukin-6 is decreased, indicating that CPP NPs have the ability to regulate the inflammatory response and have the potential to remodel the inflammatory microenvironment in vivo.

[0133] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a CuTA-Por@ε-PL nanoplatform, characterized in that, Includes the following steps: S1, CuTA nanosheets were prepared by reacting tannic acid with copper sulfate pentahydrate; S2. Protoporphyrin is grafted onto APTES-ammoniated CuTA nanosheets to form CuTA-Por; the mass ratio of protoporphyrin to APTES-ammoniated CuTA nanosheets is 1:(10~60). S3, ε-polylysine is used to coat CuTA-Por to obtain CuTA-Por@ε-PL nanoplatform; the mass ratio of CuTA-Por to ε-polylysine is 1:(5~20). The CuTA-Por@ε-PL nanoplatform is used to prepare drugs for the treatment of peri-implantitis.

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 CuTA nanosheets are obtained by adjusting the 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).

4. The preparation method according to claim 3, characterized in that, In S1, the mass ratio of tannic acid to copper sulfate pentahydrate is 27:

875.

5. The preparation method according to claim 1, characterized in that, In S2, the protoporphyrin also needs to be activated. Specifically, the 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 groups of the protoporphyrin.

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

7. The preparation method according to claim 1, characterized in that, In S2, the mass ratio of protoporphyrin to APTES-ammoniated CuTA nanosheets is 1:

20.

8. The preparation method according to claim 1, characterized in that, In S3, the mass ratio of CuTA-Por to ε-polylysine is 1:

10.

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

10. The use of the CuTA-Por@ε-PL nanoplatform of claim 9 in the preparation of a medicament for treating peri-implantitis.

11. A drug, characterized in that, It includes the CuTA-Por@ε-PL nanoplatform as described in claim 9.

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