A polyphenol-protein self-assembled material, a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a kind of polyphenol-protein self-assembly material and its preparation method and application.The application effectively forms polyphenol-protein self-assembly material (NE APs) by using multiple physical interactions between EGCG and growth factor NRG-1.NRG-1 can be continuously released from NE APs, and EGCG protects NRG-1 from oxidative stress, maintains its biological activity related to stem cell recruitment, migration and angiogenesis.In addition, NE APs utilize the ability of EGCG to scavenge ROS, maintain mitochondrial homeostasis and promote osteogenic differentiation, while synergistically regulating immune response through TNF / NF-κB / JAK-STAT signaling pathways.In vivo experiments show that NE APs accelerate the repair of inflammatory bone defects by enhancing stem cell recruitment, angiogenesis and immune regulation, and have good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polyphenol-protein self-assembly material, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Inflammatory bone defects are a common clinical condition with a significant economic burden, typically involving damage to the structure and function of bone tissue due to infectious or non-infectious inflammatory responses. To overcome the limitations of natural bone implants, such as donor shortages and the risk of immune rejection, bone tissue engineering materials have emerged as a promising alternative. However, despite their potential, traditional metal, ceramic, and polymer-based materials face significant challenges at bone defect sites due to limited bioactivity. In particular, inflammatory diseases lead to the accumulation of reactive oxygen species (ROS) and immune imbalances, disrupting bone homeostasis and hindering tissue integration, thus complicating the treatment of inflammatory bone defects. Growth factor delivery strategies are widely used in bone regeneration due to their effectiveness in accelerating healing and restoring tissue function. Typically, growth factors are directly incorporated into biomaterials for sustained release, enabling them to perform their corresponding biological functions. However, a key challenge is the susceptibility to degradation and inactivation caused by oxidative stress, especially under inflammatory conditions during long-term bone regeneration, leading to the loss of growth factor bioactivity. Therefore, developing advanced delivery strategies to protect the bioactivity of growth factors in oxidative environments has become a critical and urgent focus in growth factor-based bone tissue engineering.

[0004] Epigallocatechin gallate (EGCG) is a polyphenolic compound extracted from tea with significant antioxidant, anti-inflammatory, and osteogenic properties, making it a promising drug for bone tissue engineering. For example, EGCG-modified poly(e-caprolactone) scaffolds have been shown to promote bone regeneration through ROS scavenging and immunomodulation. Furthermore, EGCG-loaded electrospun poly-L-lactic acid / gelatin-based nanofiber membranes accelerate osteogenic differentiation and bone repair through sustained EGCG release. However, the preparation processes of these materials are not only cumbersome but also require the use of various chemical reagents, which does not align with current green and environmentally friendly manufacturing principles. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides a polyphenol-protein self-assembled material, its preparation method, and its applications. Specifically, this invention utilizes various physical interactions between epigallocatechin-3-gallate (EGCG) and growth factors (such as neuroregulatory protein-1 / NRG-1) to effectively form self-assembled particles (NE APs), i.e., the aforementioned polyphenol-protein self-assembled material. This overcomes the limitations of traditional growth factor delivery strategies, providing a new, multifunctional, protective delivery strategy for growth factors, improving their utilization and therapeutic efficacy, and opening up new avenues for treating inflammatory bone defects. Based on the above research results, this invention is thus completed.

[0006] This invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a polyphenol-protein self-assembly material, wherein the raw materials of the polyphenol-protein self-assembly material include at least epigallocatechin gallate and neuroregulatory protein-1.

[0008] The mass ratio of the gallocatechin gallate to neuroregulatory protein-1 is 1-50:0.1, more specifically 10-30:0.1, and even more specifically 20:0.1.

[0009] A second aspect of the present invention provides a method for preparing the above-mentioned polyphenol-protein self-assembled material, wherein the preparation method comprises mixing and stirring an epigallocatechin gallate solution and a neuroregulatory protein-1 solution, followed by centrifugation. That is, the present invention, under mild conditions, simply mixes neuroregulatory protein-1 and polyphenol EGCG to form self-assembled particles, thereby achieving the protective delivery of growth factors.

[0010] The concentration of the epigallocatechin gallate solution is 1-50 mg / mL, more preferably 20 mg / mL; the concentration of the neuroregulatory protein-1 solution is 0.01-1 mg / mL, more preferably 0.1 mg / mL; when mixed, the volume ratio of the two is 0.1-10:1, preferably 1:1.

[0011] During the mixing process, the specific mixing conditions are: mixing at 600-1000 rpm for 0.1-5 min, preferably at 800 rpm for 1 min.

[0012] The specific centrifugation conditions are as follows: centrifuge at 10,000-15,000 rpm for 1-10 minutes, preferably at 12,000 rpm for 5 minutes.

[0013] A third aspect of the present invention provides the application of polyphenol-protein self-assembled materials in the preparation of products for repairing inflammatory bone defects.

[0014] The repair of inflammatory bone defects is specifically manifested as follows:

[0015] (a) Neuroregulatory protein-1 is sustainably released from polyphenol-protein self-assembly materials, and epigallocatechin gallate protects neuroregulatory protein-1 from oxidative stress, maintaining its biological activity associated with stem cell recruitment, migration and angiogenesis;

[0016] (b) Polyphenol-protein self-assembly materials utilize the ability of epigallocatechin gallate to scavenge ROS, maintain mitochondrial homeostasis and promote osteogenic differentiation, while synergistically regulating immune responses through the TNF / NF-κB / JAK-STAT signaling pathway.

[0017] (c) Polyphenol-protein self-assembly materials enhance the body's ability to recruit stem cells, angiogenesis, and immune regulation.

[0018] In another specific embodiment of the present invention, the product may be a drug.

[0019] In addition, when the product is a drug, the drug may also contain at least one non-drug active ingredient, which may include pharmaceutically acceptable excipients, carriers, etc., without specific limitations.

[0020] In this invention, the drug dosage form is not specifically limited. In one specific embodiment of this invention, the drug may be an injectable dosage form.

[0021] A fourth aspect of the present invention provides a method for repairing bone defects, the method comprising: applying the above-mentioned polyphenol-protein self-assembled material to the bone defect site of a subject.

[0022] The bone defect may be a periodontal bone defect, and the application method may be to apply the above-mentioned polyphenol-protein self-assembled material to the bone defect site by injection.

[0023] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0024] The aforementioned technical solution utilizes multiple physical interactions between EGCG and the growth factor NRG-1 to effectively form NE APs, developing a simple and universal protective delivery strategy. NRG-1 is sustainably released from NE APs, while EGCG protects NRG-1 from oxidative stress, maintaining its bioactivity related to stem cell recruitment, migration, and angiogenesis. Furthermore, NE APs leverage EGCG's ability to scavenge ROS, maintain mitochondrial homeostasis, and promote osteogenic differentiation, while simultaneously synergistically regulating immune responses through the TNF / NF-κB / JAK-STAT signaling pathway. In vivo experiments demonstrate that NE APs create a favorable microenvironment for bone regeneration by enhancing stem cell recruitment, angiogenesis, and immune regulation, thereby accelerating the repair of inflammatory bone defects. This self-assembly strategy combining polyphenols and growth factors offers a promising approach to improving the therapeutic efficiency of growth factors in regenerative medicine, thus possessing significant practical application value. Attached Figure Description

[0025] 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.

[0026] Figure 1 Preparation and characterization of NE APs of the present invention. (A) Self-assembly of a mixture of EGCG and NRG-1 into particles after vigorous shaking. (B) SEM and TEM images of NE APs. (C) EDS elemental map of NE APs. (D) Hydrodynamic diameter of NE APs. (E) FT-IR spectra of EGCG / BSA, EGCG, and BSA NE APs. (F) XRD patterns of NE APs. (G) Interaction analysis at the EGCG and NRG1 binding interface in NE APs. These figures show 3D interaction maps and surface structure maps of the complex interface. (H) Cumulative release of NRG-1 from NE APs over time. (I) Scavenging activity of NE APs at different concentrations (μg / mL) against three typical free radicals (DPPH, ·OH, and ·O2-).

[0027] Figure 2This invention utilizes NE APs to protect NRG-1 cells from H2O2-induced damage. (A) Transwell chamber cell migration assay to evaluate the chemotactic behavior of PDLSCs in negative control (NC), NRG-1, EGCG, NE APs, H2O2 / NRG-1, and H2O2 / NE APs groups. (B) Cell migration counting and data analysis. (C) Representative images of PDLSCs in two-dimensional scratch analysis. (E) Chemotactic behavior of PDLSCs in negative control (NC), positive control (PC), NRG-1, H2O2 / NRG-1, and H2O2 / NE APs groups. (D) Quantitative analysis of scratch healing rate. (E) Tube formation of HUVECs 12 hours after negative control (NC), positive control (PC), NRG-1, EGCG, NE APs, H2O2 / NRG-1, and H2O2 / NE APs groups. (F) Semi-quantitative analysis describing the number of nodes. *p<0.05, **p<0.01 and ***p<0.001.

[0028] Figure 3 This invention demonstrates the intracellular ROS scavenging activity of NE APs. (A) Fluorescence intensity was detected by flow cytometry after ROS staining. (B) Quantitative statistical analysis of ROS positivity rate. (C) Relative mRNA expression levels of antioxidant enzymes (CAT and SOD1) after different treatments under H2O2 stimulation. (D) JC-1 staining of PDLSCs was performed to monitor mitochondrial membrane potential. (E) Representative fluorescence images of mitochondrial ROS stained with Mito-Sox under different treatments. (F) Representative TEM images show the mitochondrial morphology and distribution of PDLSCs after different treatments. ***p<0.001.

[0029] Figure 4 To demonstrate the anti-inflammatory effect of NE APs in this invention. (A) Immunofluorescence staining of iNOS in RAW264.7 cells. (B) Flow cytometry detection of the proportion of iNOS-positive RAW264.7 cells. (C) Relative mRNA expression level of iNOS. (D) Relative mRNA expression level of pro-inflammatory cytokines. (E) Immunofluorescence staining of CD206 in RAW264.7 cells. (F) Flow cytometry detection of the proportion of CD206-positive cells. (G) Quantitative analysis of the proportion of CD206-positive RAW264.7 cells. (H) Relative mRNA expression level of CD206. (I) Relative mRNA expression level of the anti-inflammatory cytokine IL-10. (J) RT-qPCR detection of the inflammatory response of PDLSCs stimulated with LPS after different treatments. (K) ELISA kit detection of the inflammatory response of PDLSCs stimulated with LPS after different treatments. *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 5 Transcriptome sequencing analysis for this invention. (A) Heatmap of distance between samples. (B) Volcano plot of transcriptomic analysis of differentially expressed genes between the control group and the NE APs group. (C) Heatmap of differentially expressed genes between the control group and the NE APs group. (D) GO analysis of DEGs after NEAPs treatment in PDLSCs. (E) Bubble plot of KEGG enrichment analysis. GSEA-based TNF(F), NF-κB(G), and IL-17(H) signaling pathways. (I) Western blot and quantitative analysis of p-p65 and p65. (J) Western blot and quantitative analysis of p-κBα and κBα. **p<0.01, ***p<0.001.

[0031] Figure 6 This invention demonstrates the osteogenic effect of NE APs on PDLSCs. (A) ALP staining of PDLSCs after 7 and 14 days of different treatments. (B) Expression of osteogenic differentiation-related genes ALP, BMP2, and OPN in PDLSCs after 7 and 14 days of different treatments. (C) Osteogenic mineralization-related ARS staining of PDLSCs after 21 and 28 days of different treatments. (D) Semi-quantitative analysis of relative ARS levels. ***P<0.001.

[0032] Figure 7 This invention demonstrates the therapeutic effect of NE APs in a rat model of periodontitis. (A) 3D reconstructed micro-CT images of the maxillary alveolar bone at 2 and 4 weeks post-surgery. (B) Quantitative micro-CT analysis of BV / TV, Tb.Sp, and BS / TV. (C) H&E staining images at 2 and 4 weeks post-surgery. (D) Quantitative analysis of CEJ-ABC distance assessed by H&E staining. (E) Masson staining images of maxillary alveolar bone defects at 2 and 4 weeks post-surgery. (F) Col I staining images of maxillary alveolar bone defects at 2 weeks post-surgery. **P<0.01, ***P<0.001.

[0033] Figure 8 This invention includes: (A) Immunohistochemical analysis of M1 macrophage marker CD80 and M2 macrophage marker CD163 in maxillary alveolar bone defects at 2 and 4 weeks post-surgery; (B) Quantitative analysis of CD80+ cells in different groups; (C) Quantitative analysis of CD206+ cells in different groups; (D) Immunofluorescence staining to observe the presence and distribution of PDLSCs in the bone defect area; (E) α-SMA immunofluorescence staining to assess angiogenesis in the bone defect area at 2 and 4 weeks post-surgery; and (F) Quantitative analysis of α-SMA expression in different groups. ***P<0.001.

[0034] Figure 9This is a schematic diagram illustrating the construction of NE APs and the treatment of periodontitis-related inflammatory bone loss according to the present invention. (A) Assembly process of NE APs. (B) Treatment of periodontitis-related inflammatory bone loss. (C) Mechanism of bone regeneration. Detailed Implementation

[0035] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] 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.

[0037] As previously mentioned, the efficacy of bone regeneration therapies based on growth factor delivery is often compromised by oxidative stress, especially under inflammatory conditions, which can lead to structural damage and functional inactivation of growth factors.

[0038] In view of this, the present invention proposes a simple and universal protective delivery strategy by utilizing the various physical interactions between epigallocatechin-3-gallate (EGCG) and growth factors (such as neuroregulatory protein-1 / NRG-1) to effectively form self-assembled particles (NE APs), i.e., polyphenol-protein self-assembled materials.

[0039] NE APs provide sustained release of NRG-1 while protecting it from oxidative damage, maintaining its biological functions of cell recruitment, migration, and angiogenesis. Furthermore, NE APs utilize the ability of EGCG to scavenge reactive oxygen species and maintain mitochondrial homeostasis, while synergistically regulating the TNF / NF-κB / JAK-STAT signaling pathway, mediating M2 macrophage polarization, and suppressing inflammation to support immune responses and osteogenic differentiation. In vivo experiments demonstrate that NE APs create a favorable microenvironment for bone regeneration through stem cell recruitment, angiogenesis, and immune modulation, effectively promoting the repair of inflammatory bone defects. This multifunctional protective delivery strategy based on the self-assembly of polyphenols and growth factors offers potential for promoting the application of growth factors in regenerative medicine.

[0040] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] Example

[0042] 1. Materials and Methods

[0043] 1.1 Materials

[0044] Neuroregulatory protein-1 (NRG-1) was purchased from R&D Systems (USA). Epigallocatechin-3-gallate (EGCG) was purchased from Yuanyue Biotechnology (Shanghai, China) Co., Ltd. CuCl2, 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from Macklin (Shanghai, China). Calcein AM / PI live / dead staining kit (CA1630), superoxide anion activity assay kit (BC1290), 2,2-diphenyl-1-picrylhydrazyl (DPPH, purity >97%, 394.32MW), JC-1 mitochondrial membrane potential assay kit. Mito Tracker Green (C1048), ROS assay kit (S0033), GSH and GSSG assay kits. Cell counting kit 8 (CCK8) and ATP chemiluminescence assay kit were purchased from Elabscience (Wuhan, China). Rabbit monoclonal antibody against NF-κB p65 (C22B4), rabbit monoclonal antibody against phosphorylated NF-κB p65 (Ser536)(93H1), GAPDH monoclonal antibody, recombinant antibody against Bata tubulin, IKB-Alpha monoclonal antibody, recombinant antibody against phosphorylated IKB-Alpha (Ser32 / 36), HRP-conjugated goat anti-mouse IgG (H+L), and HRP-conjugated goat anti-rabbit IgG (H+N) were purchased from Abcam (USA).

[0045] 1.2 Synthesis of NE APs (NRG-1 and EGCG self-assembled particles)

[0046] A 20 mg / mL EGCG solution was mixed with a 100 μg / mL NRG-1 solution at a 1:1 volume ratio. The mixture was vigorously stirred at 800 rpm for 1 minute on a shaker, followed by centrifugation at 12000 rpm for 5 minutes to form white NE APs. The resulting product was stored in a refrigerator at 4°C.

[0047] 1.3 Molecular Dynamics Simulation

[0048] The interaction between NRG1 protein and EGCG was simulated using the GROMACS 2019.6 software package. The binding free energy between NRG1 and EGCG was calculated using molecular dynamics simulations.

[0049] 1.4 NE APs Characterization

[0050] Fourier transform infrared spectroscopy: Fourier transform infrared (FTIR) spectroscopy (PerkinElmer Spectrum 100, USA) is used to characterize the chemical structures of NRG-1, BSA and synthesized NE APs.

[0051] XPS: X-ray photoelectron spectroscopy is used to analyze lyophilized NE APs, perform peak fitting, and valence state calibration.

[0052] SEM and TEM: NE APs were prepared by resuspending the precipitate in PBS after centrifugation. A few drops of the sample suspension were deposited on a silicon wafer or copper grid. The morphology of the NE APs was observed using a scanning electron microscope (SEM, S4800, Hitachi Ltd., Japan) and a transmission electron microscope (TEM, JEM-2100, JEOL, Japan).

[0053] EDS: The elemental composition of NEAPs was examined using an energy-dispersive X-ray spectrometer (EDS, Phenom ProX, G5, Eindhoven, Netherlands).

[0054] DLS and Zeta Potential: The average hydrodynamic particle size of NE APs was measured using dynamic light scattering (DLS, Zetasizer Nano ZS90, UK). NE APs were diluted to 10 μg / mL with ultrapure water, and the hydrodynamic size and Zeta potential were recorded on a Zetasizer Nano ZSP (Malvern Instruments, UK).

[0055] In vitro release behavior of 1.5NRG-1 from NE APs

[0056] The release behavior of NRG-1 from NE APs was assessed using dialysis. Sample solutions (1 mg / mL) were incubated at 37°C with gentle agitation. After centrifugation at specific time intervals, 500 μL of PBS was collected, and an equal volume of fresh PBS was added to the sample for remixing. The collected PBS was stored at -80°C prior to analysis. The concentration of NRG-1 in the samples was measured using an ELISA kit.

[0057] Antioxidant capacity of 1.6NE APs

[0058] The antioxidant capacity of NE APs was evaluated using three different methods. In the DPPH assay, solutions of NE APs at different concentrations (0, 5, 10, 20, 40, and 80 μg / mL) were mixed with a DPPH radical working solution. After incubation, absorbance was measured at 510 nm to assess radical scavenging activity. The conversion of H₂O₂ to hydroxyl radicals (·OH) was measured using a CuCl₂-mediated Fenton-like reaction. Absorbance was recorded at 562 nm to assess antioxidant capacity. The ·O₂⁻ scavenging capacity of NE APs was determined using a superoxide anion activity assay kit.

[0059] 1.7 Characterization of the biocompatibility of NE APs

[0060] Cell viability assay: PDLSC extraction was approved by the Ethics Committee of the Stomatological Hospital of Shandong University, Jinan, China (Program No.: NO.20201007). The effect of NE APs on PDLSC viability was assessed using the CCK8 kit. PDLSCs were seeded in 96-well plates, and NE APs were added at different concentrations (0, 10, 20, and 40 μg / mL). After incubation for 1, 2, and 3 days, CCK8 working solution was added, followed by incubation for 2 hours. Absorbance (OD) was measured at 450 nm using a microplate reader (SPECTROstar Nano, Germany) to determine cell viability.

[0061] Live / dead cell staining: PDLSCs were treated in DMEM medium in 24-well plates with different conditions (NRG-1 (50 ng / mL), EGCG (10 μg / mL), and NE APs (10 μg / mL)) for 24 hours. After treatment, double staining of live and dead cells was performed. After incubation in the dark for 20 minutes, the cells were washed three times with PBS and observed under a fluorescence microscope (OLYMPUS IX73, Japan).

[0062] Cytoskeleton staining: PDLSCs were incubated for 24 hours in DMEM medium containing NRG-1 (50 ng / mL), EGCG (10 μg / mL), and NE APs (10 μg / mL) in 24-well plates. The cytoskeleton was stained with phalloidin, while the nuclei were co-stained with DAPI. Cells were then observed under a fluorescence microscope (OLYMPUS IX73, Japan).

[0063] 1.8 PDLSCs homing test

[0064] Transwell migration assay: The homing ability of PDLSCs to NE APs was assessed using an extrawell migration assay. A total of 3 × 10⁵ cells were seeded in the upper cavity of an insert with 8 μm wells, while NRG-1 (50 ng / mL), EGCG (10 μg / mL), NE APs (10 μg / mL), H₂O₂+NRG-1, and H₂O₂+NE APs were added to the lower cavity. NRG-1, H₂O₂+NRG-1, and H₂O₂+NE APs were pre-incubated at 4 °C for 24 h, with a final H₂O₂ concentration of 500 μM. After 20 hours of co-culture, cells that had migrated to the basal side of the insert membrane were stained with crystal violet. The stained cells were observed under an optical microscope (Olympus, Japan), and the number of migrated cells was counted. The negative control (NC) received 2% FBSDMEM, and the positive control (PC) received 10% FBSDMEM.

[0065] Scratch assay: A scratch assay was performed to further evaluate the migration ability of PDLSCs. Once a monolayer of cells was formed, scratches were created using the tip of a 100 μL pipette. Cells were washed twice with PBS and fresh medium containing 2% FBS was added. Cells were incubated for 12 hours with NRG-1 (50 ng / mL), EGCG (10 μg / mL), NE APs (10 μg / mL), H2O2 + NRG-1, and H2O2 + NE APs. Images of the scratched areas were captured under a microscope, and the scratch closure rate was quantitatively analyzed using ImageJ software.

[0066] 1.9 Angiogenesis Analysis

[0067] Human umbilical vein endothelial cells (HUVECs) were seeded into 6-well plates (3 × 10⁵ cells / well) and treated with NRG1, EGCG, NE APs, H₂O₂+NRG-1, and H₂O₂+NE APs for 48 hours. Growth factor-depleted Matrigel (Corning, Inc.) was added to ice-cold 96-well plates and incubated at 37°C for 30 minutes to induce gelation. HUVECs were then reseeded into Matrigel plates at a rate of 1 × 10⁵ cells / well. After 12 hours of incubation, tube formation was observed under an optical microscope. Total tube length, nodes, and grid were quantitatively analyzed using ImageJ software. RT-PCR analysis was performed to quantify the expression levels of angiogenesis-related genes (CD31, KDR, and VEGF).

[0068] 1.10 NE APs' ability to scavenge and protect against intracellular ROS

[0069] Intracellular ROS changes were detected using a ROS detection kit. PDLSCs were administered at 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 g / mL in 6-well plates and cultured with NRG-1 (50 ng / mL), EGCG (10 μg / mL), and NE APs (10 μg / mL). After 24 hours, the medium was replaced with DMEM containing 400 μM H2O2 and incubated for 2 hours. Subsequently, cells were treated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 60 minutes and then washed three times with PBS. Fluorescence imaging was performed using fluorescence microscopy, and fluorescence intensity was quantified using ImageJ 1.44 software (National Institutes of Health). In addition, fluorescence intensity was further analyzed by flow cytometry. Mitochondrial ROS were detected by Mito-Sox staining, and mitochondrial morphology was simultaneously examined by transmission electron microscopy (TEM).

[0070] The expression of antioxidant-related genes (CAT, NQO, and SOD-1) in different treatment groups was analyzed by quantitative real-time PCR (RT-PCR) using an ABI 7500 RT-PCR system (Applied Biosystems, Massachusetts, USA) and SYBR GreenPCR Master Mix (AG, China). Relative gene expression was calculated using the 2-ΔΔCt method and normalized to the housekeeping gene β-Actin.

[0071] 1.11 NE APs' anti-inflammatory ability

[0072] PDLSCs with 2×10 per orifice 5 Cells were seeded at a density of 100 ng / mL in 6-well plates and treated with LPS (100 ng / mL), NRG1, EGCG, and NE APs for 24 hours. After this treatment, specific pro-inflammatory genes (IL-8, IL-6, and CCL2) were characterized using an ELISA kit.

[0073] 1.12 Macrophage polarization

[0074] Inhibition of M1 polarization: Mouse-derived macrophages (RAW264.7) were injected at a dose of 3 × 10⁻⁶. 5 Cells were seeded at density in 6-well plates and treated for 24 hours with normal DMEM medium, LPS, LPS+NRG-1, LPS+EGCG, and LPS+NE-APs. For flow cytometry analysis, 1×10⁶ cells were... 6Cells were resuspended in 100 μL PBS, and then pre-conjugated antibody FITC-CD86 and rat isotype IgG control antibody were added. After incubation in the dark for 30 min, cells were washed twice and resuspended in flow buffer for analysis using a BD LSR Fortessa flow cytometer. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde and infiltrated with 0.1% Triton X-100 (Solarbio, China). After blocking with 10% goat serum, cells were incubated overnight at 4°C with anti-iNOS2 primary antibody (1:200 dilution). The next day, cells were incubated for 1 h in the dark at 25°C with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200 dilution). The nuclei were counterstained with DAPI (Abcam) before observation under a fluorescence microscope. RT-PCR was used to assess the expression of specific pro-inflammatory genes (IL-6 and TNFα) and M1 macrophage marker genes (iNOS).

[0075] Promoting M2 polarization: RAW264.7 macrophages were treated with normal DMEM medium, LPS, NRG-1, EGCG, and NE APs for 24 hours. M2 polarization was analyzed by flow cytometry using the pre-conjugated antibody APC-CD206 and a rat isotype IgG control antibody. Immunofluorescence staining involved incubation with anti-CD206 primary antibody (1:200 dilution) and Alexa Fluor488-conjugated goat anti-rabbit IgG (1:200 dilution). The expression of the anti-inflammatory gene (IL-10) and the M2 macrophage marker gene (CD206) was quantified using RT-PCR.

[0076] 1.13 Osteogenic Differentiation

[0077] PDLSCs were cultured in osteogenic differentiation medium supplemented with NRG1, EGCG, and NE APs. ALP staining was performed at 7 and 14 days of culture, and Alizarin Red S staining at 21 and 28 days. The expression of osteoblast-related genes (ALP, OPN, and BMP-2) was analyzed by RT-PCR at 7 and 14 days of culture. Semi-quantitative analysis of ALP and Alizarin Red S staining was performed using ImageJ.

[0078] 1.14 RNA-Seq and Bioinformatics

[0079] Transcriptome sequencing and analysis were performed by OE Biotechnology Co., Ltd. RNA-seq analysis was performed using 1g of total RNA for each sample. Raw reads were processed using the bioinformatics pipeline tool BMKCloud (www.biocloud.net) online platform. Differential expression between samples was analyzed using edgeR software with significance thresholds of FDR < 0.05 and Fold Change ≥ 2. Gene Ontology (GO), Kyoto Gene and Genome Encyclopedia (KEGG) pathway enrichment, and Gene Set Enrichment Analysis (GSEA) were performed using the GOstats software package.

[0080] 1.15 Promotes in vivo repair of bone defects caused by periodontitis

[0081] All animal experiments were approved by the Ethics Committee of the Stomatological Hospital of Shandong University, Jinan, China (Program No.: NO.20231202). Seventy 8-week-old male Wistar rats were randomly divided into five groups: untreated group (blank), perinatal group, NRG-1 group, EGCG group, and NE APs group. Rats were anesthetized using inhalation anesthesia, and periodontitis was induced by ligating the maxillary left second molar with 3-0 silk suture for 2 weeks. After establishing the periodontitis model, the ligation was removed, and PBS, NRG-1, EGCG, or NE APs solution was injected into the gingival sulcus every other day for 2 or 4 weeks.

[0082] 1.16 Blood Biochemistry Tests

[0083] Complete blood count and biochemical tests were performed to assess the in vivo toxicity of NE APs. Whole blood was collected from the tail vein of SD rats 2 weeks post-surgery. For complete blood count measurements, 500 μL of whole blood was centrifuged (4°C, 45,000 rpm, 10 min) to obtain serum, and various blood parameters were compared among all experimental groups.

[0084] 1.17 Micro-CT Analysis

[0085] Three weeks after treatment, the animals were euthanized, and the maxillae were harvested, fixed with 4% (w / v) buffered paraformaldehyde, and subjected to micro-CT analysis (Quantum GX, PerkinElmer, USA) to assess bone repair. Scanning parameters were set to 36 μm resolution, 90 kV voltage, and 88 μA current. Imaging data were collected, and 3D reconstructions were generated using the manufacturer's proprietary software. Following reconstruction, parameters such as bone mineral density (BMD), bone volume to tissue volume ratio (BV / TV), bone surface area density (BS / TV), and trabecular spacing / split spacing (Tb.Sp) were calculated.

[0086] 1.18 Histological, immunohistochemical and immunofluorescence analyses

[0087] Histological examinations were performed on the liver, spleen, kidneys, heart, and lungs. These organs were removed, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) using standard techniques. To assess the effectiveness of periodontitis treatment, maxillary samples were decalcified in 10% (w / v) ethylenediaminetetraacetic acid (EDTA), dehydrated via graded ethanol series, embedded in paraffin, and longitudinally sectioned into 5 μm thick sections. Sections were stained with H&E and Masson's stain to observe neotissue formation. Quantitative analysis was performed on the CEJ-ABC distance (the junction of cementum and alveolar ridge) and collagen area. Furthermore, these sections underwent immunohistochemical staining for various markers, including type I collagen (COL I) for assessing collagen production, CD80 and CD206 for assessing immunomodulation, CD44+CD34- for analyzing recruitment of endogenous periodontal ligament stem cells (PDLSCs), and α-SMA for assessing angiogenesis in bone defect areas. After staining, the slides were observed and examined using an optical Roche microscope (Olympus X71-F22PH, Japan).

[0088] 1.19 Statistical Analysis

[0089] Data are expressed as mean ± standard deviation (SD). Student's t-test was used for two-group comparisons and one-way ANOVA, followed by post-hoc Bonferroni test for multiple-group comparisons to determine statistical significance. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

[0090] 2. Results and Discussion

[0091] 2.1 Synthesis and Characterization of NE APs

[0092] Functionalized NE APs were synthesized via a one-step self-assembly reaction. In short, a mixture of EGCG and NRG-1 spontaneously assembled into particles through physical interactions after vigorous shaking. White NE APs were collected by centrifugation. Figure 1 A). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the morphology of NE APs. For example... Figure 1 As shown in Figure B, NE APs exhibit an aggregation of nanoparticles. Energy-dispersive X-ray spectroscopy (EDS) was used to examine the elemental composition of the NE APs. Figure 1C). EDS elemental mapping revealed the co-localization of C, O, and N in NE APs, indicating a uniform compositional distribution. Quantitative analysis showed that the atomic percentages of C, O, N, and S in NE APs were approximately 50.6%, 31.2%, 16.7%, and 1.4%, respectively. Dynamic light scattering (DLS) analysis indicated that the average hydrodynamic diameter of NE APs ranged from 2 to 4 μm. Figure 1 D). Fourier transform infrared spectroscopy (FT-IR) spectroscopy ( Figure 1 E) highlights the changes in the self-assembly process of EGCG molecules into APs, using BSA instead of NRG-1 due to limited availability and high cost. During self-assembly, the out-of-plane C-OH bending vibrations (700 and 600 cm⁻¹) of the phenolic ring in the EGCG monomer are observed. -1 The characteristic peak of carbonyl vibration (1691 cm⁻¹) and carbonyl vibration. -1 ) disappeared. Furthermore, at 1600cm -1 The peak broadening observed nearby is attributed to the asymmetric stretching of the carboxyl group (C=O) in the protein. (1450-1510 cm⁻¹) -1 The spectral regions between these regions are related to aromatic C=C stretching and remain essentially unchanged, indicating that the EGCG molecular framework is preserved. X-ray photoelectron spectroscopy (XPS) is used to analyze the valence states of elements in NE APs. Figure 1 F). C1s spectra showed the presence of C=C (284.8 eV), C=C (285.0 eV), and C=O (288.5 eV) bonds, confirming different carbon structures. Peaks at 531.7 eV and 533.0 eV in the O1s spectrum correspond to the C=O in the amide bond and the hydroxyl group in EGCG, respectively. The N1s spectrum showed a peak at 398.5 eV, which can be attributed to nitrogen in NRG-1. XPS analysis indicated that the assembly process did not change the valence states of the atoms, ensuring the bioactivity of the components. To further understand the interaction mechanism, the interaction between NRG-1 and EGCG was simulated using the Gromacs 2019.6 software package. Figure 1 (G). Simulation results show that EGCG molecules can stably bind to the surface of NRG-1. Molecular dynamics simulations show a binding free energy of -6.01 kcal / mol, primarily driven by hydrogen bonds, Coulomb forces, and hydrophobic interactions. These interactions help maintain biological activity and promote the efficient release of growth factors. The system remained stable throughout the simulation, confirming the strong binding between EGCG and NRG-1 and supporting the potential of polyphenolic protein APs as protective carriers of growth factors.

[0093] The cumulative release curve of NRG-1 from NE APs is as follows: Figure 1As shown in H. The initial explosive release occurred within the first three hours, likely due to rapid leakage of NRG-1 near the surface. Subsequently, NRG-1 molecules gradually diffused from the AP, with release stabilizing at approximately 336 hours. This prolonged and gradual release characteristic supports sustained long-term effects in both in vivo and in vitro applications. The intrinsic bioactivity of EGCG in NE APs was evaluated by measuring the radical scavenging capabilities of 1,1-diphenyl-2-picrylhydrazido (DPPH), 2,2′-azobis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium salt (ABTS), and superoxide anion (·O2-). Figure 1 As shown in Figure I, the free radical scavenging capacity of NE APs increases significantly with concentration. This trend was consistently observed in DPPH, ABTS, and ·O2- assays, where higher concentrations of NE APs resulted in more effective free radical scavenging and inhibition of ROS production. The strong antioxidant capacity exhibited by NE APs suggests that they may be therapeutic agents for ROS-related diseases.

[0094] 2.2NE APs protect NRG-1 from oxidative stress

[0095] First, the effect of different concentrations of NE APs on the survival of periodontal ligament stem cells (PDLSCs) was evaluated. On day 1, no significant difference in cell proliferation was observed within the concentration range of 0–40 μg / mL. On days 2 and 3, cell proliferation in the 20 and 40 μg / mL groups was slightly lower than that in the 10 μg / mL group, but not significantly different from the control group. Based on these results, 10 μg / mL of NE APs was selected for further experiments. Live / dead assays and cytoskeleton staining confirmed that PDLSCs exhibited good growth and typical spindle-shaped morphology in all groups, indicating that NE APs have excellent biocompatibility.

[0096] Stem cell recruitment and migration are crucial for initiating bone regeneration. Transwell assays were performed to assess the chemotactic effect of NEAPs on PDLSCs. NE APs recruited more PDLSCs than the control and EGCG groups, but slightly fewer than the NRG-1 group due to the slower release of NRG-1 from NE APs. The protective effect of NE APs against H2O2-induced growth factor inactivation was also investigated. Figure 2(A and B). Compared with the untreated NRG-1 group, exposure to H2O2 significantly reduced the recruitment capacity of NRG-1. In contrast, even after H2O2 treatment, NE APs maintained a strong recruitment capacity for PDLSCs, indicating that NE APs can mitigate oxidative damage and maintain the bioactivity of NRG-1. Similarly, in vitro scratch assays showed that PDLSCs in the NE APs group exhibited enhanced migration ability, with a scratch healing rate of 38.7% after 24 hours, lower than the 65.6% in the pure NRG-1 group due to the slower release of NRG-1 from NE APs. Figure 2 (C and D). In contrast, the healing rates in the EGCG group and the control group were only 13.7% and 6.6%, respectively. Furthermore, the healing capacity of NRG-1 was significantly reduced when exposed to H2O2, but NE APs maintained a higher recruitment capacity under oxidative stress, which was superior to the use of NRG-1 alone.

[0097] Angiogenesis plays a crucial role in supporting bone regeneration. To simulate angiogenesis in a three-dimensional environment, a tube formation experiment was conducted using HUVEC to evaluate the development of interconnected tubular networks. Figure 2 E). The NE APs and NRG-1 groups exhibited the highest tubular network density and branching, significantly outperforming the control and EGCG groups. Specifically, the NE APs group had 6.4 times and 3.5 times the total tube length of the control group, respectively. Figure 2 F). Notably, NRG-1 almost completely lost its pro-angiogenic capacity when treated with H2O2, while the H2O2 / NE-APs group retained a strong angiogenic capacity. Furthermore, RT-qPCR analysis was performed to quantify the expression levels of angiogenesis-related genes. The results showed that CD31, KDR, and VEGF were significantly upregulated in the H2O2 / NE APs, NE APs, and NRG-1 groups compared to the control, EGCG, and H2O2 / NRG-1 groups. These results indicate that NE APs protect NRG-1 from H2O2-induced damage and inactivation, retaining its biological activity in recruiting and inducing PDLSC migration, and promoting angiogenesis in HUVECs.

[0098] 2.3 Intracellular antioxidant and mitochondrial protective effects of NE APs

[0099] Effective clearance of excess intracellular ROS is crucial for maintaining cellular function. H2O2 was used to induce excessive intracellular ROS production in PDLSCs, and intracellular ROS levels and the ROS clearance capacity of NE APs were measured using the dichlorodihydrofluorescein diacetate (DCFH-DA) probe. Compared to the H2O2 group, there was no difference in fluorescence intensity in the NRG-1 group, while EGCG showed a decrease in fluorescence intensity due to its strong antioxidant capacity. Notably, the fluorescence intensity in the NE APs group was lower, only slightly higher than that in the EGCG group. Flow cytometry analysis confirmed a significant reduction in mean fluorescence intensity after NE AP treatment. Figure 3 The levels of A and B decreased to approximately 11.8%, indicating that NE APs eliminated approximately 73.3% of ROS. These results demonstrate that NE APs effectively preserve the antioxidant function of EGCG, providing robust protection for PDLSCs against H2O2-induced damage. Furthermore, the expression levels of antioxidant enzyme-specific genes (CAT and SOD1, which are key enzymes for maintaining ROS homeostasis in organisms) were analyzed by RT-qPCR. Figure 3 C). H2O2 stimulation led to ROS accumulation and downregulation of CAT and SOD1, disrupting redox balance. However, compared with the H2O2 and NRG-1 treatment groups, both NE APs and EGCG significantly upregulated the expression of CAT and SOD1, suggesting that enhanced antioxidant capacity helps restore cellular redox homeostasis.

[0100] Mitochondria, as the center of energy metabolism and a key target for ROS-induced damage, were also analyzed. To assess the protective effect of NE APs on mitochondrial function in PDLSCs, JC-1 mitochondrial membrane potential (MMP) was measured, and the fluorescence ratio (A / M ratio) of JC-1 aggregates to JC-1 monomers was measured. Figure 3 As shown in Figure D, healthy PDLSCs exhibited strong red fluorescence and minimal green fluorescence. However, exposure to H2O2 led to a significant decrease in red fluorescence and an increase in green fluorescence. NE APs and EGCG significantly restored the A / M ratio. The A / M ratio of healthy PDLSCs was initially 8.70, which decreased sharply to 1.74 after H2O2 stimulation. NE APs and EGCG upregulated the A / M ratio to 6.49 and 5.60, respectively, both significantly higher than the 1.69 observed in the NRG-1 group, and showed no difference compared to the H2O2 group. These results indicate that NE APs can maintain the stability of MMPs, thereby reducing the occurrence of mitochondrial dysfunction. Furthermore, mitochondrial ROS (mtROS) were detected using Mito-Sox red staining. Figure 3(E). Although NRG-1 did not reduce mtROS levels, NE APs and EGCG reduced mtROS by 6.83-fold and 4.99-fold, respectively, compared to the H2O2 group, indicating that NE APs effectively reduced mtROS production.

[0101] TEM is used to further examine the mitochondrial morphology and number in PDLSCs. For example... Figure 3 As shown in Figure F, compared to the control group, the H2O2 group exhibited numerous damaged mitochondria with reduced and broken cristae. In contrast, the NRG-1 and EGCG groups showed newly formed small mitochondria. NE APs significantly reduced H2O2-induced damage, restoring mitochondrial morphology to levels comparable to the control group and enabling cells to maintain their basic functions. Furthermore, the number of mitochondria with normal morphology and cristae was significantly increased in the NE AP groups, further enhancing mitochondrial homeostasis. These findings suggest that NE APs protect growth factors and possess strong antioxidant capabilities, effectively scavenging ROS and protecting mitochondrial function, highlighting their potential to control inflammation and promote tissue regeneration.

[0102] 2.4 NE APs regulate the inflammatory response and polarization of RAW264.7 macrophage cells.

[0103] Treatment with lipopolysaccharide (LPS, 100 ng / mL) was used to simulate an inflammatory environment, and the proportion of M1 macrophages was assessed using iNOS as a marker by immunofluorescence staining and flow cytometry. Figure 4 As shown in Figure A, the LPS group exhibited the highest iNOS expression level, while the iNOS expression levels in the NRG-1 and EGCG groups were significantly reduced. Notably, the iNOS expression level in the NE APs group was even lower than that in the NRG-1 and EGCG groups, which may be due to the synergistic effect of EGCG and NRG-1. Flow cytometry results were consistent, with the proportion of iNOS-positive macrophages decreasing from 80.7% to 53.5% in the NE APs group. Figure 4 B). RT-qPCR analysis further confirmed that NE APs downregulated the RNA expression levels of iNOS and pro-inflammatory cytokines IL-6, TNF-α, and CCL-2. Figure 4 C, D).

[0104] Next, the effects of NE APs on M2 macrophage polarization were evaluated. Figure 4 As shown in E and S16, there was no significant difference between the NRG-1 group and the control group. Interestingly, EGCG and NE-APs significantly upregulated CD206 expression, indicating that both EGCG and NE-APs promoted macrophage M2 polarization. Flow cytometry results ( Figure 4F and G) confirmed that the NE APs group had the highest proportion of CD206-positive cells. RT-qPCR analysis showed that NE APs significantly upregulated CD206 and the anti-inflammatory cytokine IL-10 (F206 and F206). Figure 4 These findings indicate that NE APs effectively enhance M2 macrophage polarization, contributing to the creation of an anti-inflammatory environment. Furthermore, the effects of NE APs on the secretion of pro-inflammatory cytokines by PDLSCs were assessed under LPS-induced inflammatory conditions. RT-qPCR results showed that NE APs significantly downregulated the expression of IL-8 and IL-6 (H and I). Figure 4 J). ELISA results further confirmed that, compared with other groups, the NEAPs group had significantly lower concentrations of IL-8 and IL-6 (J). Figure 4 These results indicate that NE APs significantly enhance the anti-inflammatory capacity of RAW264.7 and PDLSCs cells, thereby modulating the inflammatory response and promoting bone regeneration.

[0105] 2.5NE APs-mediated anti-inflammatory mechanism

[0106] To investigate the anti-inflammatory mechanism of NE APs, whole transcriptome sequencing was performed on RAW264.7 cells treated with and without NE APs. The results of the three independent experiments were highly consistent. Figure 5 A). A total of 4195 differentially expressed genes (DEGs) were identified, of which 2009 were upregulated and 2186 were downregulated (false discovery rate / FDR < 0.05, fold change / FC ≥ 2), indicating significant transcriptomic differences between the NE APs group and the LPS group. Figure 5 B). The heatmap highlights the top 20 DEGs, emphasizing significant associations of the changed genes. Figure 5 C). Compared with the LPS group, cells treated with NE APs showed upregulated expression levels of anti-inflammatory genes Stc1 and Hsd11b2, while the pro-inflammatory gene F5 showed a trend of downregulation. These observations suggest that NE APs enhance cellular resistance to inflammatory challenges.

[0107] Furthermore, gene ontology (GO) analysis revealed that DEG-related pathways were significantly enriched in inflammation-related biological processes, such as inflammatory responses, positive regulation of IL-17 production, negative regulation of IL-2 production, and cellular responses to IL-1, all of which were downregulated. Figure 5 D). These findings suggest that NE APs can modulate inflammation by influencing various inflammation-related pathways. To better understand the biological function of NE AP-induced DEGs, enrichment analysis was performed using the Kyoto Genome and Encyclopedia of Genomes (KEGG) pathway. Figure 5E). This analysis highlights the significant involvement of the TNF / NF-κB / JAK-STAT axis.

[0108] Gene set enrichment analysis (GSEA) further confirmed that DEGs are enriched in processes such as TNF, NF-κB, and JAK-STAT signaling pathways. Figure 5 FH). Meanwhile, as the core of the TNF / NF-κB / JAK-STAT axis, the expression of key proteins related to the NF-κB signaling pathway was assessed using Western blotting. After treatment with NE APs, the expression levels of p-p65 and p-κBα were significantly downregulated (FH). Figure 5 The results (I and J) indicate that NE APs effectively inhibit the NF-κB signaling pathway. Furthermore, NE APs also suppress inflammation by modulating the IL-17 and MAPK signaling pathways. These findings suggest that NE APs effectively reduce LPS-induced inflammatory responses. Therefore, the potent anti-inflammatory properties of NE APs offer significant therapeutic potential for the repair of inflammatory bone tissue.

[0109] 2.6NE APs promote in vitro osteogenic formation of PDLSCs

[0110] Besides controlling inflammation, osteogenic differentiation is crucial for inflammatory bone regeneration. Alkaline phosphatase (ALP) expression is a key marker of osteoblast activity and the early stages of bone formation. To assess osteogenic activity, ALP staining and semi-quantitative analysis were performed. Figure 6 A). PDLSCs treated with NE APs and EGCG showed similarly strong staining at 7 and 14 days, both significantly stronger than NRG-1 and the control group. These results indicate that EGCG promotes early osteogenic differentiation of PDLSCs, and NE APs further enhance this effect.

[0111] To explore the role of NE APs in regulating osteogenic differentiation of PDLSCs at the mRNA level, RT-qPCR analysis was performed on several osteogenic-related genes, including ALP, BMP2, and OPN, at 7 and 14 days after osteogenic induction. Figure 6 (B) Gene expression levels in the NRG-1 group were comparable to those in the control group, indicating that NRG-1 alone cannot induce osteogenic differentiation. In contrast, the EGCG group showed significant upregulation of osteogenic genes, consistent with the known osteogenic properties of EGCG. Notably, PDLSCs cultured with NE APs showed even greater upregulation of osteogenic genes compared to the EGCG group.

[0112] Calcium deposition is a marker of osteoblast differentiation. Alizarin Red S (ARS) staining was used to assess mineralization under different treatments. Figure 6(C and D). Increased calcium nodule formation was observed in all groups at 21 and 28 days after induction. However, NE APs resulted in the most abundant staining and the highest number of calcium nodules. These findings, consistent with ALP staining and RT-qPCR results, confirm the excellent osteoinductive activity of NE APs in vitro. These results indicate that the self-assembly of polyphenols with growth factors not only preserves the osteogenic induction potential of EGCG but also enhances its osteogenic activity. This improvement is attributed to NE APs, which promote sustained release of EGCG while improving its stability.

[0113] The above results collectively demonstrate that NE APs achieved the highest regulatory efficiency in promoting NRG-1-driven cell recruitment, migration, and angiogenesis, and effectively enhanced the anti-inflammatory, antioxidant, ROS scavenging, and osteogenic differentiation effects of EGCG on PDLSCs.

[0114] 2.7 In vivo therapeutic effect

[0115] Based on the positive results observed in vitro, we explored the bone regeneration capacity of NE APs in a periodontitis-induced in vivo bone loss model established in rats by local injection of LPS and suture ligation to induce inflammatory bone defects. Figure 7 As shown in Figure A, compared with the control group, the periodontitis (Perio) group exhibited significant alveolar bone resorption, with the distance from the cementum-cementum junction to the alveolar ridge (CEJ-ABC) increasing to 345 μm, confirming the successful establishment of the periodontitis model. Although both NRG-1 and EGCG partially inhibited alveolar bone resorption, the NE APs group showed the most effective inhibitory effect. After 4 weeks of treatment, the defects in the NE APs group were almost completely restored, and the CEJ-ABC distance (90.5 μm) was similar to that in the normal group (75.7 μm). Furthermore, the quality of newly formed bone was assessed by measuring bone volume fraction (BV / TV), trabecular bone separation (Tb.Sp), and bone surface area to volume ratio (BS / TV). Figure 7 (B) Among the four groups, the NE APs group exhibited the highest BV / TV and BS / TV values, as well as the lowest Tb-Sp value. Notably, all three parameters in the NE APs group nearly returned to normal levels after four weeks. These findings highlight the significant advantages of NE APs in promoting alveolar bone regeneration.

[0116] H&E staining was performed to assess periodontal tissue healing at the second molar furcation, confirming histological improvement. Figure 7C). In the NE APs group, a large amount of new bone tissue formed along the defect boundary two weeks postoperatively, and almost all residual defects were filled within four weeks. In contrast, new bone formation was limited in other groups. Particularly in the Periodo group, there was extensive fibrous connective tissue and inflammatory cell infiltration at the defect site. CEJ-ABC distance measurement based on H&E staining results ( Figure 7 D) reflects the trend observed by microCT.

[0117] Masson staining and quantitative analysis of collagen volume fraction showed that the NE APs group achieved significant therapeutic effects in periodontal tissue repair, characterized by a thicker epithelial layer and denser collagen fiber arrangement. Figure 7 E). Subsequently, immunohistochemical staining with COL I (IHC) was performed to assess alveolar bone repair. Figure 7 F). Uniform brown staining indicates a high COL I content in the newly regenerated tissue of the NE APs group, with significantly stronger staining intensity compared to other groups. These findings suggest that NE APs provide the most effective treatment outcome for periodontal tissue repair.

[0118] To elucidate the regeneration process, we first evaluated whether NE APs could modulate the M1 / M2 phenotype of macrophages to establish a local pro-regenerative immune microenvironment. Figure 8 AC). IHC was used to analyze the expression of M1 macrophages (labeled with CD80) and M2 macrophages (labeled with CD206) within periodontal defects. At 2 weeks post-surgery, the Periodo group showed the highest number of M1 macrophages, which remained elevated at 4 weeks, indicating a prolonged inflammatory response. The NRG-1 and EGCG groups showed partially decreased M1 macrophage levels at both 2 and 4 weeks. In contrast, the NE APs group showed a significant decrease in M1 macrophage expression. Regarding M2 macrophages, although the Periodo, NRG-1, and EGCG groups showed increases at both 2 and 4 weeks, the NE APs group gradually normalized its M2 macrophage count at 4 weeks, approaching levels similar to the control group. These findings suggest that NE APs effectively modulate the immune microenvironment, reduce inflammation, and promote tissue healing. The recruitment of CD44+CD34-PDLSCs in the defective region was further investigated using dual immunofluorescence staining. Figure 8(D) Compared with the control and Period groups, EGCG led to a slight increase in stem cell recruitment, while NRG-1 significantly enhanced the green fluorescence intensity, reflecting its strong stem cell recruitment capacity. More notably, the green fluorescence intensity of the NE APs group was significantly higher than that of the NRG-1 group. This enhancement can be attributed to the unique function of NE APs, which can continuously release and protect NRG-1, thereby enhancing its stem cell recruitment capacity. Quantitative analysis showed that CD44 expression was significantly increased in the NE APs group compared with other treatment groups, indicating a large enrichment of PDLSCs in the bone defect area. NE APs effectively delivered NRG-1, promoting the directed migration of endogenous PDLSCs to the bone defect site, thereby creating a microenvironment favorable to EGCG osteogenic activity and ultimately enhancing bone regeneration.

[0119] The role of NE APs in promoting angiogenesis in vivo was also investigated using immunofluorescence staining targeting the angiogenesis-related biomarker α-SMA. Figure 8 (E and F). The NE APs group exhibited the highest α-SMA expression level, indicating enhanced angiogenesis compared to other groups. Quantitative analysis confirmed increased angiogenesis in the NE APs group. These results suggest that NE APs modulate the immune microenvironment, recruit endogenous stem cells, promote angiogenesis, and accelerate alveolar bone repair and regeneration. Furthermore, H&E staining of major organs and routine blood tests indicated that NE APs possess good biocompatibility and safety, further supporting their potential for treating inflammatory bone defects.

[0120] It should be noted that the above examples are only used to illustrate the technical solutions of this embodiment and are not intended to limit it. Although this embodiment has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of this embodiment as needed, without departing from the spirit and scope of the technical solutions of this embodiment.

Claims

1. A polyphenol-protein self-assembled material, characterized in that, The polyphenol-protein self-assembly material contains at least epigallocatechin gallate and neuroregulatory protein-1 as its raw materials; The mass ratio of the gallocatechin gallate to neuroregulatory protein-1 is 1-50:0.

1.

2. The polyphenol-protein self-assembled material as described in claim 1, characterized in that, The mass ratio of gallocatechin gallate to neuroregulatory protein-1 is 10-30:0.

1.

3. The polyphenol-protein self-assembly material according to claim 2, wherein the mass ratio of gallic catechin gallate to neuroregulatory protein-1 is 20:0.

1.

4. The method for preparing the polyphenol-protein self-assembled material according to claim 1 or 2, characterized in that, The preparation method includes mixing and stirring an epigallocatechin gallate solution and a neuroregulatory protein-1 solution, followed by centrifugation.

5. The preparation method according to claim 4, characterized in that, The concentration of the epigallocatechin gallate solution is 1-50 mg / mL, and the concentration of the neuroregulatory protein-1 solution is 0.01-1 mg / mL.

6. The preparation method according to claim 5, wherein the concentration of the epigallocatechin gallate solution is 20 mg / mL.

7. The preparation method according to claim 5, wherein the concentration of the neuroregulatory protein-1 solution is 0.1 mg / mL.

8. The preparation method according to claim 4, characterized in that, When mixed, the volume ratio of the two is 0.1-10:

1.

9. In the preparation method according to claim 8, the volume ratio of the two components during mixing is 1:

1.

10. The preparation method according to claim 4, wherein the specific stirring conditions during the mixing process are: stirring at 600-1000 rpm for 0.1-5 min.

11. The preparation method according to claim 10, wherein the specific stirring conditions during the mixing process are 800 rpm for 1 min.

12. The preparation method according to claim 4, characterized in that, The specific centrifugation conditions are: centrifuge at 10000-15000 rpm for 1-10 minutes.

13. The preparation method according to claim 12, wherein the specific centrifugation conditions are 12000 rpm for 5 min.

14. The use of the polyphenol-protein self-assembled material according to any one of claims 1-3 or the polyphenol-protein self-assembled material prepared by the preparation method according to any one of claims 4-13 in the preparation of products for repairing inflammatory bone defects.

15. The application as described in claim 14, characterized in that, The repair of inflammatory bone defects is specifically manifested as follows: (a) Neuroregulatory protein-1 is sustainably released from polyphenol-protein self-assembly materials, and epigallocatechin gallate protects neuroregulatory protein-1 from oxidative stress, maintaining its biological activity associated with stem cell recruitment, migration and angiogenesis; (b) Polyphenol-protein self-assembly materials utilize the ability of epigallocatechin gallate to scavenge ROS, maintain mitochondrial homeostasis and promote osteogenic differentiation, while synergistically regulating immune responses through the TNF / NF-κB / JAK-STAT signaling pathway; (c) Polyphenol-protein self-assembly materials enhance the body's ability to recruit stem cells, angiogenesis and immune regulation.

16. The application as described in claim 14, characterized in that, The product is a medicine.

17. The application as described in claim 16, characterized in that, The drug also contains at least one non-pharmacological active ingredient; the drug dosage form is an injectable dosage form.

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