A pteris exosome-like vesicle and its application in the treatment of prostate cancer and / or bladder cancer

Abstract

The application discloses a pteris multifida exosome-like vesicle and application thereof in treatment of prostate cancer and / or bladder cancer. The pteris multifida exosome-like vesicle is prepared by differential ultracentrifugation combined with filter membrane filtration. Further, the pteris multifida exosome-like vesicle is subjected to anti-cancer effect test in multiple prostate cancer and bladder cancer cell lines. The results show that the pteris multifida exosome-like vesicle can effectively inhibit the proliferation, migration and invasion of prostate cancer cells, and block the cell cycle of prostate cancer cells, thereby inhibiting the growth of cancer cells and playing a role in treating prostate cancer. In addition, the pteris multifida exosome-like vesicle can also inhibit the proliferation of bladder cancer cells. It is shown that the pteris multifida exosome-like vesicle can have the treatment effect on prostate cancer and / or bladder cancer, and provides a new strategy and idea for the treatment of prostate cancer and bladder cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a fern-like exosome vesicle and its application in the treatment of prostate cancer and / or bladder cancer. Background Technology

[0002] Prostate cancer (PCa) is one of the most common malignant tumors in elderly men, ranking second in cancer incidence among men worldwide. Currently, the main treatments for prostate cancer include active surveillance, surgery, radiotherapy, chemotherapy, and endocrine therapy. While these treatments have some efficacy, they also have significant side effects. Many patients are prone to developing castration-resistant prostate cancer (CRPC) and metastasizing throughout the body in the later stages of treatment, severely impacting their quality of life and survival prognosis.

[0003] Bladder cancer, as the most common malignant tumor of the urinary system, is characterized by a high recurrence rate. The recurrence rate within 5 years after transurethral resection of bladder tumor (TURP) is 50%–70%, significantly shortening survival time. Currently, intravesical instillation of biologics such as Bacillus Calmette-Guerin (BCG) has become an important clinical intervention. However, some patients still develop drug resistance (including refractory, recurrent, and unresponsive cases), requiring further radical cystectomy, which carries high surgical risks and a high complication rate. Therefore, existing treatment strategies for urinary system tumors such as prostate cancer and bladder cancer face common challenges including limited efficacy, frequent drug resistance, and significant toxic side effects.

[0004] Plant-derived extracellular vesicles (PDEVs) are nanoscale membrane vesicles actively secreted by plant cells, ranging in diameter from 30 to 500 nanometers. Commonly referred to as "plant exosomes," they have attracted considerable attention due to their unique biological characteristics. Composed of a lipid bilayer, they contain various bioactive components such as nucleic acids, proteins, and lipids. Numerous studies have demonstrated that PDEVs possess cross-species regulatory functions and exhibit characteristics such as good biocompatibility, high stability, transdermal absorption, strong targeting, and high safety, making them promising for applications in drug delivery systems and disease treatment. Exosomes have been successfully isolated and identified from various plants, such as ginger, grape, green tea, pomegranate, and garlic, and have been extensively studied. Different plant PDEVs exhibit functional differentiation due to the unique bioactive molecules they carry. The core of this differentiation lies in the specificity of their contents (e.g., differences in miRNAs, metabolites, and membrane proteins). Therefore, PDEVs from different sources exhibit unique therapeutic potential due to the specific bioactive molecules they carry. This functional diversity provides a wealth of natural nanomaterials for precision medicine, allowing for the selection of appropriate PDEV sources based on different disease needs.

[0005] Pteris multifida (Latin name: Pteris multifida) Poir Pteris multifida, also known as well fence grass or chicken foot grass, is a plant belonging to the genus Pteris in the family Pteridaceae. It has a bland or slightly bitter taste, is cold in nature, and enters the large intestine, liver, and heart meridians. Pteris multifida contains various active ingredients such as flavonoids, terpenoids, and sterols. The total flavonoid extract of Pteris multifida has been reported to have anti-benign prostatic hyperplasia effects (Dai GC, HuB, Zhang WF, Peng F, Wang R, Liu ZY, Xue BX, Liu JY, Shan YX. Chemical characterization, anti-benign prostatic hyperplasia effect and subchronictoxicity study of total flavonoid extract of Pteris multifida. Food ChemToxicol. 2017 Oct;108(Pt B):524-531. doi: 10.1016 / j.fct.2016.11.010. Epub2016 Nov 12. PMID: 27845168.). However, there are currently no reports on exosome-like vesicles derived from Pteris vittata, and research on the application of Pteris vittata-derived exosome-like vesicles in the treatment of prostate and bladder cancer remains lacking. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for preparing exosome-like vesicles from *Pteris vittata*.

[0007] A second objective of this invention is to provide *Pteris vittata* exosome-like vesicles prepared by the aforementioned preparation method.

[0008] A third objective of this invention is to provide the application of the aforementioned *Pteris vittata* exosome-like vesicles.

[0009] The above-mentioned objective of the present invention is achieved through the following technical solution: A method for preparing exosome-like vesicles from *Pteris vittata* includes the following steps: mixing and crushing *Pteris vittata* and buffer solution to form a homogenate and filtering; centrifuging at 3000–5000 rpm for 20–40 min; centrifuging the supernatant at 10000–14000 rpm for 20–40 min; filtering the supernatant through a 0.2–0.8 μm filter membrane; and then ultracentrifuging at 140000–160000 g for 1–2 h; resuspending in buffer solution to obtain the vesicle.

[0010] This invention prepares exosome-like vesicles from *Pteris vittata* using differential ultracentrifugation combined with membrane filtration. Specifically, the process includes: plant homogenization and filtration; primary impurity removal: centrifuging the extract at 3000–5000 rpm to remove large particles such as fiber debris and cell remnants, obtaining a supernatant; medium-speed centrifugation: further centrifuging the supernatant at 10000–14000 rpm to further remove large vesicles and residual cellular components; re-filtration and ultracentrifugation enrichment: after further removing particulate impurities using a 0.20–0.8 μm filter membrane, ultracentrifugation at 140000–160000 g precipitates nanoscale vesicle particles. This invention represents the first successful isolation and purification of extracellular vesicles from *Pteris vittata*. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that these extracellular vesicles have a typical bilayer membrane structure that is intact and stable, with a particle size distribution of approximately 156.6 ± 7.87 nm and a zeta potential of -17.45 ± 0.78 mV.

[0011] Furthermore, the described Pteris vittata is the whole fresh Pteris vittata plant.

[0012] Further, the preparation method includes the following steps: mixing and crushing the fern and buffer solution to form a homogenate and filtering, centrifuging at 4000 rpm for 30 min, centrifuging the supernatant at 12000 rpm for 30 min, filtering the supernatant sequentially through 0.8 μm, 0.45 μm and 0.22 μm filter membranes, then ultracentrifuging at 150000 g for 90 min, and resuspending in buffer solution to obtain the final product.

[0013] Furthermore, the buffer solution is PBS buffer.

[0014] Furthermore, the ratio of the fern root to the buffer solution is 1:1 to 3 g / mL.

[0015] The present invention also provides *Pteris vittata* exosome-like vesicles prepared by any of the above-described preparation methods.

[0016] This invention further revealed through experiments using the CCK-8 assay, cell colony formation, apoptosis, migration, and invasion that the aforementioned exosome-like vesicles derived from *Fernonia cinerea* significantly inhibited the proliferation of prostate and bladder cancer cells, suppressed cell cycle arrest and colony formation in prostate cancer cells, thereby inhibiting cancer cell growth and playing a therapeutic role in cancer treatment. Overall, this invention is the first to reveal the application prospects of *Fernonia cinerea*-derived exosome-like vesicles in the inhibition of prostate and bladder cancer, providing new insights for developing cancer therapies based on plant-derived extracellular vesicles.

[0017] Therefore, the present invention also provides the use of the aforementioned Pteris vittata exosome-like vesicles in the preparation of medicaments for treating prostate cancer and / or bladder cancer.

[0018] Furthermore, the drug treats cancer by inhibiting the growth of cancer cells.

[0019] Specifically, inhibiting cancer cell growth means inhibiting the proliferation, migration, and invasion of cancer cells, and blocking the cancer cell cycle.

[0020] The present invention also provides a therapeutic drug containing the above-mentioned *Pteris vittata* exosome-like vesicles.

[0021] The drug can be prepared into dosage forms such as pills, capsules, granules, oral liquids, powders, tablets, lozenges, and injections. For different dosage forms, a suitable drug carrier in this field can be selected.

[0022] The drug carrier used can be solid, liquid, or gaseous. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0023] In preparing oral dosage forms of drugs, any convenient drug medium can be used. For example, water, ethanol, oil, alcohol, flavoring agents, preservatives, coloring agents, etc., are used to form oral liquid dosage forms, such as suspensions, capsules, and solutions; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulators, emulsifiers, lubricants, binders, and disintegrants, can be used to form oral solid dosage forms, such as powders, capsules, and tablets. Due to their ease of administration, tablets and capsules are the preferred oral dosage units using solid drug carriers, and tablets can be coated using standard aqueous or non-aqueous techniques.

[0024] Tablet formulations containing *Pteris vittata*-derived plant exosome-like vesicles prepared according to the present invention can be prepared by compression or molding, optionally using one or more excipients or adjuvants. The active ingredient can also be compressed in a suitable machine in a free-flowing form (e.g., powder or granules), optionally mixed with binders, lubricants, inert diluents, surfactants, or dispersants. Molded tablets can be molded in a suitable machine, i.e., a mixture of powdered compounds moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of active ingredient. For example, formulations intended for oral administration to humans may contain about 0.05 mg to about 5 g of active drug, mixed with a suitable and convenient carrier material, which may comprise about 5% to 95% of the total composition. Unit dosage forms typically contain approximately 1 mg to approximately 2 g of active ingredient, usually in doses of 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0025] The drugs suitable for parenteral administration in this invention can be prepared as aqueous solutions or suspensions. Suitable surfactants, such as hydroxypropyl cellulose, may be included, and dispersions can also be prepared in mixtures of glycerol, liquid polyethylene glycol, and their oils. Furthermore, preservatives may be added to prevent harmful microbial growth.

[0026] The pharmaceutical products suitable for injection in this invention include sterile aqueous solutions or dispersions. Furthermore, the pharmaceutical product may be in the form of a sterile powder for the provisional preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile for the provisional preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be an effective liquid so that the injectable pharmaceutical ingredient must remain stable under the production and storage conditions; therefore, it is best to preserve it to prevent contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, such as containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0027] The medicaments of the present invention can be in forms suitable for topical use, such as aerosols, creams, ointments, lotions, powders, or the like. Furthermore, the medicaments can be in suitable forms for transdermal drug delivery devices, and these formulations can be prepared using the medicaments of the present invention through conventional processing methods. For example, a cream or ointment with a desired consistency can be prepared by mixing a hydrophilic material and water, and about 5 wt% to about 10 wt% of a compound.

[0028] The medicament of the present invention can be in a form suitable for rectal administration, wherein the carrier is solid. It is preferable to formulate the mixture into a unit dosage form suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be made by first forming a mixture containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0029] In addition to the carrier components mentioned above, the above-mentioned drugs may include one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Furthermore, other excipients, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, colorants, and flavoring agents, may be added to make the formulation isotonic with the blood of the intended recipient. Components containing plant exosome-like vesicles derived from *Pteris vittata* (the present invention) can also be prepared in powder or concentrated form.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention provides, for the first time, a *Pteris vittata* exosome-like vesicle, prepared by differential ultracentrifugation combined with membrane filtration. The anticancer efficacy of this *Pteris vittata* exosome-like vesicle was further tested in multiple prostate cancer and bladder cancer cell lines. Results showed that the *Pteris vittata* exosome-like vesicle effectively inhibited the proliferation, migration, and invasion of prostate cancer cells, arrested the cell cycle, and thus inhibited cancer cell growth, playing a therapeutic role in prostate cancer. Furthermore, the *Pteris vittata* exosome-like vesicle also inhibited the proliferation of bladder cancer cells. This indicates that the *Pteris vittata* exosome-like vesicle can have therapeutic effects on prostate cancer and / or bladder cancer, providing new strategies and ideas for the treatment of prostate and bladder cancer. Attached Figure Description

[0031] Figure 1 The results represent the characterization of exosome-like vesicles from *Pteris vittata*. Among them, Figure 1 In the diagram, A represents NTA; B represents TEM results; C represents particle size results; and D represents zeta potential results.

[0032] Figure 2The effects of *Pteris vittata* exosome-like vesicles on cell proliferation and IC50 of prostate cancer cell lines (DU145, RM-1, PC-3) and bladder cancer cell lines (UMUC, J82, TCCSUP, 5637) after 48 hours were investigated, along with the IC50 values ​​for each cell type. 50 Value. Among them, Figure 2 A shows the effect of different concentrations of *Gynostemma pentaphyllum* exosome-like vesicles on the proliferation curves of various prostate cancer cells; B shows the IC50 of *Gynostemma pentaphyllum* exosome-like vesicles on different prostate cancer cells. 50 Value; C represents the effect of different concentrations of *Gynostemma pentaphyllum* exosome-like vesicles on the proliferation curves of various bladder cancer cells; D represents the IC50 value of *Gynostemma pentaphyllum* exosome-like vesicles on different bladder cancer cells. 50 value.

[0033] Figure 3 The image shows the results of flow cytometry analysis of the uptake of prostate cancer DU145 cells by exosome-like vesicles from *Pteris vittata*. Figure 3 Figure A shows the results of flow cytometry detection of DU145 cells uptake of *Pteris vittata* exosome-like vesicles; Figure B shows the quantitative assessment of the average fluorescence intensity of DU145 cells uptake of *Pteris vittata* exosome-like vesicles at different time points; Figure C shows the quantitative assessment of the positive uptake rate of DU145 cells of *Pteris vittata* exosome-like vesicles (DiD label) at different time points.

[0034] Figure 4 The figure shows the effect of *Pteris vittata* exosome-like vesicles on DU145 cell clones.

[0035] Figure 5 The figure shows the results of treating DU145 cells with *Pteris vittata* exosome-like vesicles and their migration and invasion abilities. Among them, Figure 5 In Figure A, the Transwell assay was used to evaluate the ability of *Pteris vittata* exosome-like vesicles to inhibit DU145 cell migration and invasion; in Figure B, the quantitative evaluation of the inhibitory effect of *Pteris vittata* exosome-like vesicles on DU145 cell migration was performed; and in Figure C, the quantitative evaluation of the inhibitory effect of *Pteris vittata* exosome-like vesicles on DU145 cell invasion was performed.

[0036] Figure 6 To investigate the effect of *Pteris vittata* exosome-like vesicles on the DU145 cell cycle using flow cytometry. Figure 6 In Figure A, flow cytometry was used to detect the cell cycle arrest effect of *Pteris vittata* exosome-like vesicles on DU145 cells; in Figure B, the effect of *Pteris vittata* exosome-like vesicles on different cell cycles of DU145 cells was quantitatively assessed.

[0037] Figure 7 To investigate the effect of *Pteris vittata* exosome-like vesicles on the apoptosis capacity of DU145 cells using flow cytometry. Figure 7In Figure A, flow cytometry was used to detect the effect of *Pteris vittata* exosome-like vesicles on DU145 cell apoptosis; in Figure B, the effect of *Pteris vittata* exosome-like vesicles on the apoptosis rate of DU145 cells was quantitatively assessed. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0040] All data in the following examples are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software (version 9.0). Comparisons between two groups were performed using Student's t-test, and intergroup comparisons were conducted using one-way ANOVA followed by Tukey post-hoc tests for multiple comparisons. A p-value less than 0.05 was considered statistically significant.

[0041] Example 1: Preparation and characterization of exosome-like vesicles from *Pteris vittata* 1. Preparation of exosome-like vesicles from Pteris vittata (1) Use pre-cooled PBS or PBS ice cubes (3-5 ℃) to mix with fresh whole Pteris multifida (each gram of fresh whole Pteris multifida tissue is mixed with 1 mL of PBS solution), juice the fresh whole Pteris multifida using a juicer (2-5 minutes) and filter through sterile gauze to obtain the filtrate; (2) Centrifuge the filtrate at 4000 rpm for 30 min, then centrifuge at 12000 rpm for 30 min. The centrifuged liquid is filtered through 0.8 μm, 0.45 μm and 0.22 μm filter membranes in sequence to remove the precipitate and obtain the supernatant. (3) After the supernatant was subjected to ultracentrifugation (150,000 g for 90 min), the precipitate was resuspended with an appropriate amount of PBS to obtain Pteris vittata exosome-like vesicles.

[0042] 2. Characterization of exosome-like vesicles from *Pteris vittata* (1) Determination of vesicle particle number using nanoflow cytometry: The extracted Pteris vittata exosome vesicles were diluted appropriately with PBS, and the particle concentration of the diluted sample was determined using a NanoFCM Flow NanoAnalyzer U30E nanoflow cytometer. The results are as follows: Figure 1 As shown in Figure A, the concentration of the extracted Pteris vittata exosome-like vesicles was 1.21 × 10^12 particles / mL.

[0043] (2) Morphological observation using transmission electron microscopy: 10 μL of the *Pteris vittata* exosome vesicles prepared in Example 1 was aspirated using a pipette and dropped onto a copper grid to precipitate for several minutes. The floating liquid was then absorbed by filter paper. 20 μL of uranium acetate was dropped onto the copper grid to precipitate for several minutes, and the floating liquid was then absorbed by filter paper. After drying at room temperature for 5 minutes, the images were examined and imaged under a JEM1400 electron microscope. The imaging results are as follows: Figure 1 As shown in Figure B, the obtained *Pteris vittata* exosome-like vesicles exhibit a vesicle-like structure under electron microscopy.

[0044] (3) Particle size and zeta potential were determined using a dynamic light scattering (DLS) instrument: 1 μL of *Pteris vittata* exosome vesicles from Example 1 was pipetted and diluted with 999 μL of PBS. The diluted *Pteris vittata* exosome vesicle samples were then placed in particle size distribution dishes and zeta potential distribution dishes for analysis using a DLS instrument. The results are as follows: Figure 1 C and Figure 1 As shown in Figure D, the size of the exosome-like vesicles of *Pteris vittata* is 156.6 ± 7.87 nm, and the zeta potential is -17.45 ± 0.78 mV.

[0045] Example 2: Effects of Pteris vittata exosome-like vesicles on the viability of prostate and bladder cancer cells. The cytotoxic effects of *Pteris vittata* exosome-like vesicles on prostate cancer cell lines (DU145, RM-1, PC-3) and bladder cancer cell lines (UMUC, J82, TCCSUP, 5637) were verified using the CCK8 assay. The specific procedure is as follows: (1) Seeding: Prostate cancer cells or bladder cancer cells in the logarithmic growth phase were selected and seeded in 96-well plates (5*10^9 cells / well). 3 / well). Incubate for 24 hours to ensure full cell adhesion. Set up 5 replicates per group.

[0046] (2) Adding drugs: Prepare solutions of appropriate concentrations according to experimental requirements. Take out the 96-well plate from the incubator, carefully remove the supernatant from the well plate with a pipette, and then add fresh culture medium containing different concentrations of Pteris vittata exosome-like vesicles (0 μg / mL (control), 0.39 μg / mL, 0.78 μg / mL, 1.56 μg / mL, 3.13 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) for 48 hours of stimulation treatment.

[0047] (3) Color development: Place the 96-well plate in a dark environment, remove the original culture medium and wash gently several times with PBS. Then add 100 μL of CCK-8 solution (containing 90% blank culture medium) to each well and mix well. Immediately place the plate in an incubator and continue incubation for 1 hour. After incubation, remove the plate and measure the color at 450 nm using a microplate reader.

[0048] (4) Results analysis: Cell proliferation activity (%) = (average OD value of each drug-treated group - average OD value of the blank group) / (average OD value of the control group - average OD value of the blank group) * 100%. The results were plotted using GraphPad Prism 10.1.2.

[0049] The results are as follows Figure 2 As shown, compared with the control group, different concentrations of *Pteris vittata* exosome-like vesicles significantly inhibited the proliferation of prostate cancer cells and bladder cancer cells after 48 hours, and this inhibitory effect was dose-dependent. Figure 2 China A, Figure 2 (C). The half-maximal inhibitory concentration (IC50) of *Pteris vittata* exosome-like vesicles after 48 hours of treatment with DU145, RM-1, and PC-3 cells was calculated. 50 The concentrations were 2.1 μg / mL, 6.99 μg / mL, and 12.31 μg / mL, respectively. Figure 2 (Middle B). Meanwhile, the IC50 values ​​of *Pteris vittata* exosome-like vesicles after 48 hours of treatment with UMUC, J82, TCCSUP, and 5637 cells were... 50 The concentrations were 0.41 μg / mL, 0.59 μg / mL, 0.31 μg / mL, and 1.34 μg / mL, respectively. Figure 2 (D). This result also indicates that *Pteris vittata* exosome-like vesicles have a significant inhibitory effect on the proliferation of prostate cancer and bladder cancer cells.

[0050] Example 3: The uptake effect of Pteris vittata exosome-like vesicles on DU145 cells To detect the uptake of *Pteris vittata* exosome-like vesicles, 0.5 mg / mL of *Pteris vittata* exosome-like vesicles were first labeled in PBS with 1 μM DiD fluorescent dye under light-protected conditions and incubated overnight at 4 °C with shaking in the dark. After labeling, unbound free dye was removed by ultracentrifugation. The labeled *Pteris vittata* exosome-like vesicles were resuspended in serum-free cell culture medium, and DU145 cells were treated with a concentration of 2 μg / mL *Pteris vittata* exosome-like vesicles per well. DU145 cells were incubated at a concentration of 3*10^9 cells / well. 5Cells were seeded at a density of [number] cells / well in 6-well plates and incubated at 37 °C with 5% CO2 until adherence. Fluorescently labeled *Pteris vittata* exosome-like vesicles were added, and incubation continued for the appropriate time. After incubation, untaken *Pteris vittata* exosome-like vesicles were removed by washing several times with PBS. The cell pellet was then collected by centrifugation and resuspended in fresh PBS. Finally, the distribution of intracellular fluorescence signals was recorded using flow cytometry to analyze the uptake of *Pteris vittata* exosome-like vesicles in detail.

[0051] The results are as follows Figure 3 As shown, exosome-like vesicles from *Pteris vittata* can be phagocytosed by DU145 cells, and their number increases in a time-dependent manner.

[0052] Example 4 Cell Cloning Experiment DU145 prostate cancer cells in logarithmic growth phase were selected and seeded into 6-well plates (1000 cells / well) and cultured until fully adherent. After culture, culture medium containing different concentrations of *Pteris vittata* exosome-like vesicles (0 μg / mL (control), 0.5 μg / mL, 1 μg / mL, 2 μg / mL) was added to the wells for 48 h of intervention. Once cell colonies formed, the 6-well plates were removed, the supernatant was aspirated, washed with PBS, fixed with paraformaldehyde, stained with crystal violet, and then dried.

[0053] The results are as follows Figure 4 As shown, compared with the control group, the number of cell clones gradually decreased with increasing drug concentration after treatment with different concentrations of *Pteris vittata* exosome-like vesicles. These results further demonstrate that *Pteris vittata* exosome-like vesicles can inhibit the colony formation of DU145 cells.

[0054] Example 5 Cell migration and invasion experiment Cell migration and invasion abilities were both detected using Transwell chambers with an 8 μm pore size. The difference between the two assays is that the invasion assay pre-coated the Transwell membrane with a layer of matrix gel (0.5 mg / mL) to simulate the extracellular matrix barrier, while this step was not required for the migration assay. The experimental procedure was as follows: DU145 prostate cancer cells in logarithmic growth phase were harvested and the cell density was adjusted to 2 × 10⁻⁶ cells / mL. 5Cells were added at a concentration of 1,000 μL / mL serum-free to the upper chamber of a Transwell apparatus. The lower chamber was added with culture medium containing 20% ​​fetal bovine serum (FBS). Simultaneously, different concentrations of *Pteris vittata* exosome-like vesicles were added to the upper and lower chambers, respectively, with intervention concentrations set at 0 μg / mL (control), 0.5 μg / mL, 1 μg / mL, and 2 μg / mL. The mixture was incubated for 24 h. After incubation, the culture medium in both chambers was aspirated. Paraformaldehyde was then added to the lower chamber for fixation. After fixation, the fixative was discarded, and crystal violet staining solution was added. The cells were stained in the dark for 30 min. After staining, the cells were washed with PBS and air-dried in a fume hood. Finally, the upper chamber of the Transwell apparatus was observed under a microscope, and multiple fields of view were randomly selected for photographic recording.

[0055] The results are as follows Figure 5 As shown, compared with the control group, *Pteris vittata* exosome-like vesicles significantly inhibited the migration ability of DU145 cells. In the invasion assay, *Pteris vittata* exosome-like vesicles also significantly inhibited the invasive ability of DU145 cells. These results confirm that *Pteris vittata* exosome-like vesicles exhibit a significant inhibitory effect on the migration and invasion ability of DU145 cells, and this effect is positively correlated with drug concentration.

[0056] Example 6 Cell cycle experiment DU145 prostate cancer cells in the logarithmic growth phase were selected and their density adjusted to 3*10^ 5 Cells were seeded in 6-well plates and cultured until adherent. The original culture medium was removed, and a medium containing *Pteris vittata* exosome-like vesicles (0 μg / mL (control), 0.5 μg / mL, 1 μg / mL, 2 μg / mL) was added. After 48 hours of intervention, cells were collected after trypsin digestion. Cells were mixed with 1 mL of pre-chilled 70% ethanol and fixed overnight at 4 °C. After overnight centrifugation, cells were resuspended in PBS and collected. 25 μL of propidium iodide stock solution and 10 μL of RNase A solution were added to 0.5 mL of staining buffer. 0.5 mL of propidium iodide staining solution was added to each cell sample tube, and the cell pellet was slowly and thoroughly resuspended. The cells were incubated at 37 °C in the dark for 30 minutes and then analyzed by flow cytometry.

[0057] The results are as follows Figure 6 As shown, compared with the control group, treatment of DU145 cells with *Pteris vittata* exosome-like vesicles resulted in a dose-dependent decrease in the number of cells in the G0 / G1 phase, a dose-dependent increase in the number of cells in the S phase, and a dose-dependent increase in the number of cells in the G2 / M phase. These results indicate that *Pteris vittata* exosome-like vesicles can arrest the cell cycle progression of DU145 cells.

[0058] Example 7 Apoptosis Experiment DU145 prostate cancer cells in the logarithmic growth phase were selected and their density adjusted to 3*10^ 5 Cells were seeded in 6-well plates and cultured until adherent. Then, *Pteris vittata* exosome-like vesicles (0 μg / mL (control), 0.5 μg / mL, 1 μg / mL, 2 μg / mL) were added. After 24 hours of intervention, cells were collected after trypsin digestion. Cells were washed twice with pre-chilled PBS, resuspended in 100 μL of Binding Buffer, and then mixed with 2 μL of FITC and 2 μL of PI. The mixture was incubated at room temperature in the dark for 15 minutes before flow cytometry analysis.

[0059] The results are as follows Figure 7 As shown, the apoptosis rate of DU145 cells did not show a dose-dependent change with increasing drug concentration, suggesting that *Pteris vittata* exosome-like vesicles had no significant effect on apoptosis of prostate cancer cells.

Claims

1. A method for preparing exosome-like vesicles from *Pteris vittata*, characterized in that, The process includes the following steps: mixing and crushing *Pteris vittata* and buffer solution to form a homogenate and filtering it; centrifuging at 4000 rpm for 30 min; centrifuging the supernatant at 12000 rpm for 30 min; filtering the supernatant sequentially through 0.8 μm, 0.45 μm and 0.22 μm filter membranes; then ultracentrifuging at 150000 g for 90 min; and resuspending in buffer solution to obtain the final product. The size of the *Pteris vittata* exosome-like vesicles is 156.6 ± 7.87 nm, and the zeta potential is -17.45 ± 0.78 mV.

2. The preparation method according to claim 1, characterized in that, The buffer solution is PBS buffer.

3. The preparation method according to claim 1, characterized in that, The ratio of the pteris vittata to the buffer solution is 1:1 to 3 g / mL.

4. The *Pteris vittata* exosome-like vesicles prepared by any of the preparation methods described in claims 1 to 3.

5. The use of the *Pteris vittata* exosome-like vesicles of claim 4 in the preparation of a medicament for treating prostate cancer and / or bladder cancer.

6. The application according to claim 5, characterized in that, The drug treats cancer by inhibiting the growth of cancer cells.

7. The application according to claim 6, characterized in that, The inhibition of cancer cell growth refers to inhibiting the proliferation, migration, and invasion of cancer cells, and blocking the cancer cell cycle.

8. The application according to claim 5, characterized in that, The dosage form of the drug is selected from pills, capsules, granules, oral liquids, powders, tablets, lozenges, or injections.

9. A therapeutic drug, characterized in that, It contains the *Pteris vittata* exosome-like vesicles as described in claim 4.

Images