Rhizoma drynariae extracellular vesicle for treating periodontitis by targeting macrophage ETS2 gene as well as preparation method and application of rhizoma drynariae extracellular vesicle

Abstract

The invention relates to the technical field of biological medicines, in particular to rhizoma drynariae extracellular vesicles for treating periodontitis by targeting macrophage ETS2 genes as well as a preparation method and application of the rhizoma drynariae extracellular vesicles. The extracellular vesicles are creatively extracted from the traditional Chinese medicine rhizoma drynariae by combining a differential ultracentrifugation method with a sucrose density gradient centrifugation method, the functions of the extracellular vesicles are studied, and it is found that the extracellular vesicles can effectively slow down alveolar bone resorption in a periodontitis state and reduce periodontal soft tissue inflammation and have a good treatment effect on periodontitis. It is found for the first time that the rhizoma drynariae extracellular vesicles can reduce the inflammatory ability of macrophages in an inflammatory state and can reduce expression of proinflammatory factors TNF-alpha and IL-6. The invention has important scientific research value and clinical significance for further development and application of various traditional Chinese medicine plant extracellular vesicles including rhizoma drynariae extracellular vesicles.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an extracellular vesicle of *Drynaria fortunei* cells that targets the ETS2 gene of macrophages for the treatment of periodontitis, its preparation method, and its application. Background Technology

[0002] Periodontitis is an infectious disease of the periodontal tissues caused by plaque biofilm. It is a chronic infectious disease affecting the four periodontal supporting tissues (gingiva, periodontal ligament, alveolar bone, and cementum), often leading to inflammatory destruction of these tissues. Periodontitis is mainly classified into chronic periodontitis, aggressive periodontitis, periodontitis reflecting systemic disease, and necrotizing periodontitis. It has become the sixth leading cause of non-communicable disease worldwide, a major cause of tooth loss in adults, affecting 743 million people globally. The complex interactions between oral microenvironment factors such as bacteria and inflammation, and their host immune response, disrupt the original balance of bone remodeling in the alveolar bone, ultimately leading to a certain degree of loss of periodontal supporting tissues.

[0003] Currently, the main treatments for periodontitis are antibiotics, such as nitroimidazoles, tetracyclines, and macrolides. Although these antibiotics have good antibacterial effects, long-term use increases the risk of oral flora imbalance and drug resistance, and is also accompanied by side effects such as gastrointestinal reactions. Traditional Chinese medicine (TCM) treatment for periodontitis is mostly administered orally and systemically, which has disadvantages such as unstable drug absorption and slow onset of action. In recent years, with the rapid development of separation technology and exosomes, many researchers have been able to isolate EVs with a particle size of approximately 30-500 nm from TCM plants. These herbal-derived EVs possess targeting and biological activity, and can facilitate interspecies communication, thereby regulating the body's biological functions.

[0004] Although there is a certain research basis for the efficacy of plant extracellular vesicle therapy and the clinical evidence of systemic use of traditional Chinese medicine as an adjunct to the treatment of periodontitis, there are no reports to date on the use of *Drynaria fortunei* extracellular vesicles for the treatment of periodontitis, especially the application of extracellular vesicle administration through local oral cavity. Summary of the Invention

[0005] The purpose of this invention is to provide a *Drynaria fortunei* extracellular vesicle for targeted macrophage ETS2 gene therapy in periodontitis, its preparation method, and its application, thereby addressing the problems existing in the prior art. The *Drynaria fortunei* extracellular vesicles provided by this invention can effectively slow down alveolar bone resorption in periodontal inflammation, reduce periodontal soft tissue inflammation, and have a good therapeutic effect on periodontitis.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing extracellular vesicles of Drynaria fortunei, comprising the steps of sequentially juicing Drynaria fortunei, differential ultracentrifugation, and sucrose density gradient centrifugation to obtain the extracellular vesicles of Drynaria fortunei.

[0008] The differential ultracentrifugation includes a first centrifugation process, a second centrifugation process, a third centrifugation process, and a fourth centrifugation process; the conditions for the first centrifugation process are: centrifugation at 100-1000×g for 10-30 min; the conditions for the second centrifugation process are: centrifugation at 1000-3000×g for 10-30 min; the conditions for the third centrifugation process are: centrifugation at 8000-10000×g for 30-60 min; and the conditions for the fourth centrifugation process are: centrifugation at 100000-150000×g for 60-120 min.

[0009] Preferably, the sucrose density gradient centrifugation process includes the step of density gradient centrifugation using a sucrose solution;

[0010] The sucrose solution is a 15 wt% sucrose solution, a 30 wt% sucrose solution, a 45 wt% sucrose solution, and a 60 wt% sucrose solution.

[0011] This invention provides extracellular vesicles of *Drynaria fortunei* cells prepared using the above-described preparation method.

[0012] This invention provides the application of the above-mentioned *Drynaria fortunei* extracellular vesicles in the preparation of anti-periodontitis products.

[0013] Preferably, the product achieves the purpose of treating periodontitis by reducing inflammation and improving alveolar bone resorption.

[0014] Preferably, the product includes a drug.

[0015] The present invention provides an anti-periodontitis product, the product comprising the above-mentioned *Drynaria fortunei* extracellular vesicles.

[0016] Preferably, the product includes a drug.

[0017] Preferably, the product includes pharmaceutically acceptable excipients.

[0018] Preferably, the dosage form of the product includes injectable liquid, gel, or ointment.

[0019] More preferably, the product achieves the purpose of treating periodontitis by reducing inflammation and improving alveolar bone resorption.

[0020] The present invention discloses the following technical effects:

[0021] This invention creatively extracts extracellular vesicles from the traditional Chinese medicine *Drynaria fortunei* using differential ultracentrifugation combined with sucrose density gradient centrifugation, and studies their functions. It was found that these vesicles can effectively slow down alveolar bone resorption and reduce periodontal soft tissue inflammation in periodontal inflammation, demonstrating a good therapeutic effect on periodontitis. Specifically: By constructing a mouse periodontitis model, this invention found that injecting *Drynaria fortunei* extracellular vesicles into the periodontal tissues of mice with periodontitis significantly slowed down alveolar bone resorption and reduced periodontal soft tissue inflammation, achieving the goal of alleviating or treating periodontal inflammatory diseases in mice. An inflammatory cell model was constructed by stimulating macrophages (RAW264.7) with bacterial lipopolysaccharide. After culturing *Drynaria fortunei* extracellular vesicles, these vesicles targeted and reduced ETS2 gene expression, while simultaneously reducing LPS-induced inflammation in RAW264.7 cells. Network pharmacology analysis of the target and active ingredients of *Drynaria fortunei* in preventing and controlling periodontitis, as well as the KEGG pathway, showed that *Drynaria fortunei* exerts its effects by regulating signaling pathways such as EGFR and VEGF.

[0022] In summary, this invention is the first to discover that extracellular vesicles of *Drynaria fortunei* can reduce the inflammatory capacity of macrophages in an inflammatory state and decrease the expression of pro-inflammatory factors TNF-α and IL-6. This invention has significant scientific research value and clinical significance for the further development and application of extracellular vesicles from various traditional Chinese medicinal plants, including those containing *Drynaria fortunei* cells. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A flowchart illustrating the overall process for preparing extracellular vesicles from *Drynaria fortunei* cells;

[0025] Figure 2 Precipitated products of extracellular vesicles from Drynaria fortunei (A) and TEM characterization (BC);

[0026] Figure 3 The particle size distribution (A) and zeta potential (B) of extracellular vesicles in *Drynaria fortunei* cells;

[0027] Figure 4 A schematic diagram of the experimental cycle for modeling periodontitis in mice; where A is a schematic diagram of the modeling cycle and drug administration method; and B is a schematic diagram of the mouse periodontitis model.

[0028] Figure 5The study aimed to demonstrate the efficacy of *Drynaria fortunei* extracellular vesicles in alleviating periodontitis in mice. Figure A shows the results of a Micro-CT experiment; Figure B shows the changes in buccal alveolar bone resorption in different groups; PD represents the periodontitis group; Nar represents the positive control naringin group; 2.25RDNVs represents the low-concentration *Drynaria fortunei* extracellular vesicle group; and 15RDNVs represents the high-concentration *Drynaria fortunei* extracellular vesicle group.

[0029] Figure 6 The results are from single-cell combined transcriptome analysis; A is a Umap image depicting single-cell samples clustered into 11 clusters; B is a Umap image depicting macrophages before annotation; C is the expression level of specific genes (IL-1β, IL-6, MMP9, and ETS2 genes) in macrophages.

[0030] Figure 7 This study investigated the expression levels of TNF-α, IL-6, and ETS2 genes in RAW264.7 cells after treatment of periodontitis with macrophage-targeted therapy using *Drynaria fortunei* extracellular vesicles. The results showed that Con was the blank control group, LPS was the model group, LPS+Nar was the naringin group, LPS+0.75RDNVs was the low-concentration *Drynaria fortunei* extracellular vesicle group, LPS+2.25RDNVs was the medium-concentration *Drynaria fortunei* extracellular vesicle group, and LPS+15RDNVs was the high-concentration *Drynaria fortunei* extracellular vesicle group.

[0031] Figure 8 Venn diagram of the target intersection of *Drynaria fortunei* targeting macrophages in the treatment of periodontitis;

[0032] Figure 9 PPI diagram of target genes and ETS2 protein interactions for Drynaria fortunei-targeted macrophage therapy for periodontitis;

[0033] Figure 10 KEGG enrichment analysis of *Drynaria fortunei*-targeted macrophage therapy for periodontitis. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] The present application will be further described in detail below with reference to embodiments, performance testing, and network pharmacology methods.

[0040] Unless otherwise specified, all materials used in this invention are commonly purchased by those skilled in the art, and all methods used in this invention are well known to those skilled in the art.

[0041] Example 1: Extraction and purification of extracellular vesicles from *Drynaria fortunei* cells by ultracentrifugation and sugar density gradient centrifugation

[0042] The preparation process of extracellular vesicles from *Drynaria fortunei* is as follows: Figure 1 As shown, the details are as follows:

[0043] 1 kg of fresh *Drynaria fortunei* was washed clean with deionized water, cut into pieces, and juiced using a juicer. The juice was filtered, and the supernatant was collected. The supernatant was then centrifuged at 500 g for 15 min at 4°C (first centrifugation) to remove impurities and floating cells. The supernatant was then collected again at 2000 × g for 30 min at 4°C (second centrifugation) to remove dead cells and detached vesicles. The supernatant was then centrifuged at 10000 × g for 45 min at 4°C (third centrifugation) to remove dead cells, detached vesicles, and apoptotic bodies. The supernatant was collected in centrifuge tubes for a differential ultracentrifuge and centrifuged at 130000 × g for 90 min at 4°C (fourth centrifugation). The supernatant was discarded, and the precipitate was resuspended in pre-cooled 1×PBS buffer to obtain *Drynaria fortunei* extracellular vesicles.

[0044] Four different concentrations (15wt%, 30wt%, 45wt%, and 60wt%) of sucrose density gradient solutions were prepared. The resuspension was added to the top of the density gradient solutions (10 mL of each of the four different concentrations of sucrose density gradient solutions were added sequentially) using a syringe, and centrifuged at 130,000 × g for 60 min. The extracellular vesicle precipitate enriched at the 30wt% and 45wt% interface was recovered, and an equal volume of pre-cooled 1×PBS buffer was added. The mixture was centrifuged again at 130,000 × g for 60 min to remove the sucrose solution. The bottom precipitate was resuspended in a small amount of PBS to obtain the purified *Drynaria fortunei* extracellular vesicle solution.

[0045] Example 2 characterized the morphology, particle size, and protein concentration of the extracellular vesicles of *Drynaria fortunei* cells extracted in Example 1.

[0046] The morphology of the extracellular vesicles of *Drynaria fortunei* cells extracted in Example 1 was observed under a transmission electron microscope, and the results are as follows: Figure 2 As shown. The results showed that the extracellular vesicles of *Drynaria fortunei* cells were round or elliptical in shape, with typical goblet vesicle structure and complete cell membrane structure, and the particle size ranged from 0 to 200 nm (see...). Figure 2 The particle size range of extracellular vesicles (scale bar 50, 100 nm) is consistent with the reported range in the literature, proving the successful extraction of extracellular vesicles from Drynaria fortunei.

[0047] Dynamic light scattering was used to analyze the particle size distribution and zeta potential of extracellular vesicles in *Drynaria fortunei* cells. The results are as follows: Figure 3 As shown in the figure. The results showed that the particle size distribution was between 0-200 nm, which was consistent with the results observed by transmission electron microscopy, further proving the successful extraction of extracellular vesicles from *Drynaria fortunei* cells; its zeta potential was -11.3 mV.

[0048] Example 3: Investigating the effect of extracellular vesicles of *Drynaria fortunei* on in vivo treatment of periodontitis in mice.

[0049] The experimental cycle of this embodiment is as follows: Figure 4 As shown, the details are as follows:

[0050] A mouse periodontitis model was established using the suture embedding method: Twenty male 20-25g mice aged 6-7 weeks were anesthetized by intraperitoneal injection of 6μL / g sodium pentobarbital. Under sterile conditions, 2.5mm of 5-0 suture was cut, the two ends were tied, and the suture was placed between the first and second molars in the right maxilla of the mouse, ensuring that the suture was below the contact point.

[0051] After successful modeling, the mouse periodontitis model was divided into a periodontitis group (PD), a positive control naringin group (Nar), a low-concentration *Drynaria fortunei* extracellular vesicle group (2.25 RDNVs), and a high-concentration *Drynaria fortunei* extracellular vesicle group (15 RDNVs). A blank control group (Con) was also set up. There were 5 mice in each group. The following treatments were performed on each group:

[0052] Blank control group: The same volume of physiological saline was injected and irrigated;

[0053] Periodontitis group: The mouse periodontitis model was injected with and irrigated with the same amount of physiological saline;

[0054] Positive control naringin group: Naringin was injected locally into the periodontal pockets and buccal and lingual periodontal tissues of the mouse periodontitis model. The administration method was to alternate between injection and irrigation for 10 days (i.e., injection on day 1, irrigation on day 2, injection on day 3, irrigation on day 4, and so on). The dosage was 25 μL each time, and the concentration of naringin was 1 μg / mL.

[0055] Low-concentration *Drynaria fortunei* extracellular vesicle group: *Drynaria fortunei* extracellular vesicles extracted in Example 1 were injected locally into the periodontal pockets and buccal and lingual periodontal tissues of a mouse periodontitis model. The administration method was alternating between injection and irrigation for 10 days (i.e., injection on day 1, irrigation on day 2, injection on day 3, irrigation on day 4, and so on). The dosage was 25 μL each time, and the concentration of *Drynaria fortunei* extracellular vesicles was 2.25 μg / mL.

[0056] High-concentration *Drynaria fortunei* extracellular vesicle group: The *Drynaria fortunei* extracellular vesicles extracted in Example 1 were injected locally into the periodontal pockets and buccal and lingual periodontal tissues of a mouse periodontitis model. The administration method was to alternate between injection and irrigation for 10 days (i.e., injection on day 1, irrigation on day 2, injection on day 3, irrigation on day 4, and so on). The dosage was 25 μL each time, and the concentration of *Drynaria fortunei* extracellular vesicles was 15 μg / mL.

[0057] Micro-CT results are as follows Figure 5 As shown in the figure. The results showed that both high and low concentrations of *Drynaria fortunei* extracellular vesicles injected into the periodontal tissues of mice with periodontitis significantly slowed down alveolar bone resorption. Specifically, the buccal alveolar bone resorption height was 575.40±2.92 μm in the PD group, 293.66±22.94 μm in the 2.25RDNVs group, 342.10±9.96 μm in the 15RDNVs group, and 403.30±1.83 μm in the Nar group. Therefore, the *Drynaria fortunei* extracellular vesicles extracted in this invention can alleviate or treat periodontal inflammatory diseases in mice.

[0058] Example 4: Investigating the effect of extracellular vesicles of *Drynaria fortunei* cells on in vitro treatment of an inflammatory cell model.

[0059] The inventors constructed an inflammatory cell model by stimulating macrophages (RAW264.7) with bacterial lipopolysaccharide. They then detected the relative mRNA expression levels of pro-inflammatory cytokines (ETS2), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) in the macrophages of this model. The specific experimental steps are as follows:

[0060] Construction of the inflammatory cell model: RAW264.7 cells were grown at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and allowed to adhere overnight. They were then divided into four groups: a model group (LPS), a naringin group (LPS + Nar), a low-concentration *Drynaria fortunei* extracellular vesicle group (LPS + 0.75 RDNVs), a medium-concentration *Drynaria fortunei* extracellular vesicle group (LPS + 2.25 RDNVs), and a high-concentration *Drynaria fortunei* extracellular vesicle group (LPS + 15 RDNVs). A blank control group (Con) was also included. Each group underwent the following treatments:

[0061] Blank control group: macrophages (RAW264.7) cultured in normal cell culture medium (90% DMEM medium + 10% fetal bovine serum);

[0062] Model group: Inflammatory cell model cultured in cell culture medium containing 1 μg / mL bacterial lipopolysaccharide (1 μg / mL-LPS);

[0063] Naringin group: Inflammation + drug cell model cultured in cell culture medium containing 1 μg / mL-LPS + 1 μg / mL naringin (Nar);

[0064] Low-concentration *Drynaria fortunei* extracellular vesicle group: Inflammatory + drug cell model cultured in cell culture medium containing 1 μg / mL LPS + 0.75 μg / mL RDNVs;

[0065] Medium-concentration *Drynaria fortunei* extracellular vesicle group: Inflammatory + drug cell model cultured in cell culture medium containing 1 μg / mL LPS + 2.25 μg / mL RDNVs;

[0066] High-concentration *Drynaria fortunei* extracellular vesicle group: Inflammatory + drug cell model cultured in cell culture medium containing 1 μg / mL-LPS + 15 μg / mL RDNVs.

[0067] Forty-two samples from the GSE164241 single-cell dataset were analyzed, including buccal mucosa and gingival mucosa biopsy samples from 21 healthy volunteers (Normal) and gingival mucosa samples from 21 patients with untreated severe periodontitis (Periodontitis). The samples underwent batch de-batch processing, standardization, PCA dimensionality reduction, and cell clustering, and macrophage clusters of interest were extracted. Differentially expressed genes in macrophages were obtained using the Findmarker function and the default Wilcoxon rank-sum analysis, and differentially expressed genes upregulated in periodontitis (MacDEG) were selected for further analysis. The results are as follows: Figure 6 As shown in the figure. The results showed that the expression of the ETS2 gene in the periodontitis group was significantly higher than that in the healthy group.

[0068] Depend on Figure 7 It was found that in this inflammatory cell model, the relative mRNA expression levels of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), in macrophages ranged from 4.3 to 6.8, and the relative mRNA expression levels of interleukin-6 (IL-6) ranged from 99.5 to 147.7. By co-culturing the above inflammatory cell model with extracellular vesicles of *Drynaria fortunei* cells at concentrations of 0.75, 2.25, and 15 μg / mL, the relative mRNA expression levels of TNF-α and IL-6 in macrophages were significantly downregulated. Among them, the relative mRNA expression level of TNF-α decreased to 1.8-3.3 in the low-concentration *Drynaria fortunei* extracellular vesicle group, decreased to 1.7-2.7 in the medium-concentration group, and decreased to 1.1-1.2 in the high-concentration group. The relative mRNA expression level of IL-6 decreased to 22.5-28.1 in the low-concentration group, decreased to 63.4-66.5 in the medium-concentration group, and decreased to 127.1-154.3 in the high-concentration group. In the inflammatory cell model, the relative mRNA expression level of the proto-oncogene ETS2 ranged from 1.9 to 2.6. In the low-concentration *Drynaria fortunei* extracellular vesicle group, the relative mRNA expression level of ETS2 decreased to 0.6-0.9; in the medium-concentration group, it decreased to 0.3-0.6; and in the high-concentration group, it decreased to 0.6-1.0. This indicates that *Drynaria fortunei* extracellular vesicles can target and reduce ETS2 gene expression, while simultaneously reducing LPS-induced inflammation in RAW264.7 cells.

[0069] Example 5: Drynaria fortunei may exert its effects by regulating signaling pathways such as EGFR, VEGF, and MAPK.

[0070] Differentially expressed genes in macrophages were obtained using the Findmarker function and the default Wilcoxon rank-sum analysis, and differentially expressed genes upregulated in periodontitis (MacDEG) were selected for further analysis. Further network pharmacology analysis was conducted using the TCMSP, PubChem, and Swiss Target-Prediction databases to obtain the active components and target molecules (RD-Target) of *Drynaria fortunei*. The intersection of MacDEG and RD-Target was used to construct a protein-protein interaction network (PPI) graph by importing the above targets and ETS2 into the STRING database, followed by KEGG enrichment analysis. The results are as follows: Figures 8-10 As shown. The intersection results indicate that there are 25 potential targets for RD-targeted macrophage therapy in the treatment of periodontitis. Figure 8 Protein-protein interaction network analysis revealed significant interactions between ETS2 and target genes such as EP300, MMP12, and MAPKAPK2. Further KEGG enrichment analysis identified the important MAPK pathway by which *Drynaria fortunei* regulates macrophages in periodontitis. Figure 10 ).

[0071] In summary, the inventors analyzed the targets and active ingredients of Drynaria fortunei in preventing and controlling periodontitis through network pharmacology, and obtained 18 key active ingredients, mainly naringenin, horny stigmasterol, kaempferol, and (+)-catechin, as well as 170 core targets, mainly CYP19A1, ESR1, ESR2, ABCG2, and CYP1B1. Furthermore, KEGG pathway analysis showed that Drynaria fortunei may exert its effects by regulating signaling pathways such as EGFR, VEGF, and MAPK.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing extracellular vesicles of *Drynaria fortunei* cells, characterized in that, The process includes sequentially juicing, differential ultracentrifugation, and sucrose density gradient centrifugation of Drynaria fortunei to obtain the extracellular vesicles of Drynaria fortunei. The differential ultracentrifugation includes a first centrifugation process, a second centrifugation process, a third centrifugation process, and a fourth centrifugation process; the conditions for the first centrifugation process are: centrifugation at 100-1000×g for 10-30 min; the conditions for the second centrifugation process are: centrifugation at 1000-3000×g for 10-30 min; the conditions for the third centrifugation process are: centrifugation at 8000-10000×g for 30-60 min; and the conditions for the fourth centrifugation process are: centrifugation at 100000-150000×g for 60-120 min.

2. The preparation method according to claim 1, characterized in that, The sucrose density gradient centrifugation process includes the step of performing density gradient centrifugation using a sucrose solution. The sucrose solution is a 15 wt% sucrose solution, a 30 wt% sucrose solution, a 45 wt% sucrose solution, and a 60 wt% sucrose solution.

3. Extracellular vesicles of *Drynaria fortunei* cells prepared using the preparation method described in claim 1 or 2.

4. The use of the extracellular vesicles of *Drynaria fortunei* as described in claim 3 in the preparation of products for treating periodontitis.

5. The application according to claim 4, characterized in that, The product aims to treat periodontitis by reducing inflammation and improving alveolar bone resorption.

6. The application according to claim 4, characterized in that, The products include pharmaceuticals.

7. A product for treating periodontitis, characterized in that, The product includes the extracellular vesicles of *Drynaria fortunei* as described in claim 3.

8. The product according to claim 7, characterized in that, The products include pharmaceuticals.

9. The product according to claim 8, characterized in that, The product includes pharmaceutically acceptable excipients.

10. The product according to claim 9, characterized in that, The dosage forms of the product include injectable liquids, gels, or ointments.

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