Application of nanovesicles from viola yedoensis makino in preparation of anti-tumor drugs

Abstract

This invention provides the application of *Viola yedoensis*-derived nanovesicles in the preparation of antitumor drugs, belonging to the field of biomedical technology. The method for preparing *Viola yedoensis*-derived nanovesicles includes the following steps: mixing whole *Viola yedoensis* plants with PBS buffer, homogenizing, and then centrifuging at a gradient to obtain vesicles with a particle size of 100-200 nm. The *Viola yedoensis*-derived nanovesicles of this invention exhibit good biocompatibility and, by inducing mitochondrial-dependent cell death pathways in tumor cells, demonstrate highly efficient and broad-spectrum inhibitory effects on pancreatic cancer, colorectal cancer, and liver cancer, providing a new strategy for achieving highly efficient, low-toxicity, and highly targeted tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Viola yedoensis-derived nanovesicles in the preparation of antitumor drugs. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human health. Currently, the main methods of clinical tumor treatment still include surgical resection, chemotherapy and radiotherapy. However, radiotherapy and chemotherapy can also cause serious damage to the body's normal proliferating cells and produce a series of serious toxic side effects, which greatly affect the patient's quality of life and treatment compliance.

[0003] In recent years, plant-derived vesicle-like nanoparticles (PDVLNs) have attracted widespread attention due to their good biocompatibility, low immunogenicity, and ability to carry various active ingredients (such as proteins, lipids, and RNA). Violsse herba is a traditional Chinese medicine commonly used for clearing heat and detoxifying, reducing swelling and dissipating nodules. However, traditional Chinese medicine extracts suffer from low bioavailability and lack of targeting in clinical applications, making standardized treatment difficult. Currently, research on Violsse herba-derived nanoparticles is still lacking. Therefore, there is an urgent need to provide a novel treatment strategy that is highly efficient, low in toxicity, and highly targeted to achieve safe and effective tumor treatment. Summary of the Invention

[0004] To address the aforementioned technical problems, the primary objective of this invention is to provide the application of Viola yedoensis-derived nanovesicles in the preparation of antitumor drugs. The Viola yedoensis-derived nanovesicles (VDVLNs) of this invention possess good biocompatibility and exhibit highly efficient and broad-spectrum inhibitory effects against pancreatic cancer, colorectal cancer, and liver cancer.

[0005] The second objective of this invention is to provide an antitumor drug.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of Viola yedoensis-derived nanovesicles in the preparation of antitumor drugs.

[0007] In one implementation, the tumor includes pancreatic cancer, colorectal cancer, and liver cancer.

[0008] As one embodiment, the preparation method of the Viola yedoensis-derived nanovesicles includes the following steps: mixing whole Viola yedoensis with PBS buffer, homogenizing, and then centrifuging according to gradient to obtain vesicles with a particle size of 100~200 nm.

[0009] In one embodiment, the gradient centrifugation includes the following steps: collecting the first filtrate after centrifugation at 400~600×g for 8~15 min; collecting the second filtrate after centrifugation at 1500~3500×g for 25~35 min; collecting the third filtrate after centrifugation at 8000~12000×g for 50~70 min; and obtaining a precipitate after centrifugation at 80000~120000×g for 100~140 min.

[0010] As one implementation method, the ratio of the whole Viola yedoensis plant to PBS buffer is 1:5~7 g / mL.

[0011] The present invention also provides an antitumor drug comprising the above-mentioned Viola yedoensis-derived nanovesicles.

[0012] In one embodiment, the concentration of the Viola yedoensis-derived nanovesicles is 10. 10 ~10 11 Particle count / mL.

[0013] In one implementation, the tumor includes pancreatic cancer, colorectal cancer, and liver cancer.

[0014] As one implementation, the antitumor drug also includes pharmaceutically acceptable excipients.

[0015] In one embodiment, the excipients include one or more of the following: diluent, binder, disintegrant, anti-adhesive, flow aid, preservative, and emulsifier.

[0016] The advantages of this invention compared to existing technologies are as follows: This invention provides the application of Viola yedoensis-derived nanovesicles in the preparation of antitumor drugs. The invention obtains Viola yedoensis-derived nanovesicles (VDVLNs) through a mild and non-toxic preparation process, making them suitable for clinical translation. Furthermore, the prepared VDVLNs exhibit good biocompatibility. By inducing ROS accumulation, decreased mitochondrial membrane potential, and mitochondrial morphological damage in tumor cells, they trigger the mitochondrial-dependent cell death pathway in tumor cells, demonstrating a highly efficient and broad-spectrum inhibitory effect on tumor cells. This invention applies Viola yedoensis, a traditional plant-derived nanovesicle, to the development of antitumor drugs, integrating traditional Chinese medicine resources with nanotechnology, providing a new technical route for developing novel natural tumor treatment platforms. Attached Figure Description

[0017] Figure 1 This is a flowchart of the preparation process of VDVLNs.

[0018] Figure 2The morphology and physical properties of VDVLNs are characterized as follows: A is the vesicle morphology of VDVLNs observed by TEM; B is the NTA particle size distribution; C is the zeta potential detection; D is the standard curve of protein content detection by BCA method; E is the protein content of VDVLNs; and F is the SDS-PAGE protein electrophoresis analysis.

[0019] Figure 3 The inhibitory effect of VDVLNs on the proliferation of PANC-1, HCT116, and Huh-7 tumor cells (WST-1 experiment).

[0020] Figure 4 The results of VDVLNs inhibiting the clonogenic ability of three types of tumor cells (clonal formation experiment).

[0021] Figure 5 The WST-1 assay was used to detect the effects of nanovesicles from different plant sources on tumor cell proliferation. In the WST-1 assay, A represents the absorbance value of HCT116 cells after treatment with nanovesicles from different plant sources, B represents the inhibition rate of HCT116 cells by nanovesicles from different plant sources, C represents the absorbance value of Huh-7 cells after treatment with nanovesicles from different plant sources, and D represents the inhibition rate of Huh-7 cells by nanovesicles from different plant sources.

[0022] Figure 6 The graph shows the changes in ROS levels in PANC-1, HCT116, and Huh-7 cells induced by VDVLNs treatment.

[0023] Figure 7 The diagram shows the changes in mitochondrial membrane potential induced by VDVLNs treatment in PANC-1, HCT116, and Huh-7 cells.

[0024] Figure 8 Mitotracker staining was used to observe the morphological fragmentation and aspect ratio changes of mitochondria in VDVLNs-induced PANC-1, HCT116, and Huh-7 cells. Detailed Implementation

[0025] This invention provides the application of Viola yedoensis-derived nanovesicles in the preparation of antitumor drugs.

[0026] The preparation method of the violet-derived nanovesicles in this invention includes the following steps: mixing the whole violet plant with 1×PBS buffer and homogenizing to obtain a homogenate, and then centrifuging according to a gradient to obtain violet-derived vesicles with a particle size of 100~200 nm.

[0027] In this invention, fresh whole Viola yedoensis plants are collected, washed, and then mixed with PBS buffer at a ratio of 1:5~7 g / mL, preferably 1:6 g / mL. The PBS buffer is 1×PBS buffer with a pH of 7.0~8.0. After mixing, homogenization is performed at a speed of 8000~12000 rpm, preferably 9000 rpm, 10000 rpm, or 11000 rpm. The homogenization temperature is 4~8℃, and the homogenization is performed intermittently, with each cycle consisting of 10~20 s homogenization followed by an 8~10 s interval, and the homogenization cycle is repeated 3~5 times. The whole Viola yedoensis plants used in this invention are commercially available products.

[0028] Then, the present invention performs gradient centrifugation on the homogenate, the gradient centrifugation comprising the following steps: after centrifuging the homogenate at 400~600×g for 8~15 min, a first filtrate is collected. The centrifugation speed is preferably 500×g, and the centrifugation time is preferably 9 min, 11 min, or 13 min. The present invention utilizes low-speed centrifugation to remove large tissue fragments from the homogenate. Then, the first filtrate is centrifuged at 1500~3500×g for 25~35 min, and a second filtrate is collected. The centrifugation speed is preferably 2000×g, 2500×g, or 3000×g, and the centrifugation time is preferably 28 min, 31 min, or 34 min. The present invention utilizes medium-speed centrifugation to remove impurities such as fibers from the first filtrate. Then, the second filtrate is centrifuged at 8000-12000×g for 50-70 min to collect the third filtrate. The preferred centrifugation speed is 9000×g, 1000×g, or 11000×g, and the preferred centrifugation time is 55 min, 60 min, or 65 min. This invention utilizes high-speed centrifugation to remove cell debris from the second filtrate. Then, the third filtrate is centrifuged at 80000-120000×g for 100-140 min. The preferred centrifugation speed is 90000×g, 100000×g, or 110000×g, and the preferred centrifugation time is 110 min, 120 min, or 130 min. To fully collect the nanovesicle precipitate, this invention performs 2-3 rounds of centrifugation on the third filtrate. This invention utilizes ultra-high-speed centrifugation to extract the nanovesicle precipitate from the third filtrate.

[0029] In this invention, the tumors include pancreatic cancer, colorectal cancer, and liver cancer. This invention verifies that nanovesicles derived from Viola yedoensis have a highly efficient inhibitory effect on the proliferation of PANC-1, HCT116, and Huh-7 tumor cells. The present invention provides an antitumor drug comprising the above-mentioned Viola yedoensis-derived nanovesicles.

[0030] In the antitumor drug of this invention, the concentration of the Viola yedoensis-derived nanovesicles is 10. 10 ~10 11 Particle count / mL, the preferred concentration is 3 × 10⁻⁶. 10 Particle count / mL, 5×10 10 Particle count / mL, 7×10 10 Particle count / mL or 9×10 10 Particle count / mL. The nanovesicles derived from Viola yedoensis in this invention are from 1×10⁻⁶ 10 The particle count / mL indicates that the cell proliferation of PANC-1, HCT116, and Huh-7 tumor cells is significantly inhibited.

[0031] In this invention, the tumor includes pancreatic cancer, colorectal cancer, and liver cancer; the antitumor drug further includes pharmaceutically acceptable excipients. In this invention, the excipients include one or more of diluents, binders, disintegrants, anti-adhesives, flow aids, preservatives, and emulsifiers.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or under conditions recommended by the company.

[0034] Example 1 Preparation method of Viola yedoensis-derived nanovesicles (VDVLNs) (preparation process see [link]). Figure 1 ): Fresh whole Viola yedoensis plants were collected, washed, and then 1×PBS (pH 7.4) was added at a ratio of 1:6 g / mL. Homogenization was performed at 4℃ for 10,000 rpm for 15 seconds followed by a 10-second interval, for a total of 3 cycles. Vesicles were then extracted by gradient centrifugation as follows: the homogenate was centrifuged at 500×g for 10 min to collect the first filtrate, which removed large tissue fragments; the first filtrate was then centrifuged at 2,000×g for 30 min to collect the second filtrate, which removed impurities such as fibers; the second filtrate was then centrifuged at 10,000×g for 60 min to collect the third filtrate, which removed cell debris; the third filtrate was then centrifuged at 100,000×g for 120 min to collect VDVLNs precipitate and the fourth filtrate; the fourth filtrate was then centrifuged at 100,000×g for 120 min to collect the VDVLNs precipitate.

[0035] Example 2 Preparation method of Viola yedoensis-derived nanovesicles (VDVLNs): Fresh whole Viola yedoensis plants were collected, washed, and then 1×PBS (pH 7.4) was added at a ratio of 1:7 g / mL. Homogenization was performed at 4℃ for 15 seconds at 9000 rpm with a 10-second interval as one cycle, for a total of 3 cycles. Vesicles were then extracted by gradient centrifugation as follows: the homogenate was centrifuged at 600×g for 8 min to collect the first filtrate, which removed large tissue fragments; the first filtrate was then centrifuged at 3000×g for 25 min to collect the second filtrate, which removed impurities such as fibers; the second filtrate was then centrifuged at 11000×g for 65 min to collect the third filtrate, which removed cell debris; and the third filtrate was centrifuged at 90000×g for 120 min to collect the VDVLNs precipitate.

[0036] Example 3 Preparation method of Viola yedoensis-derived nanovesicles (VDVLNs): Fresh whole Viola yedoensis plants were collected, washed, and then 1×PBS (pH 7.4) was added at a ratio of 1:5 g / mL. Homogenization was performed at 4℃ for 10,000 rpm for 15 seconds followed by a 10-second interval for 3 cycles. Vesicles were extracted by gradient centrifugation as follows: the homogenate was centrifuged at 450×g for 15 min to collect the first filtrate, which removed large tissue fragments; the first filtrate was then centrifuged at 1,500×g for 35 min to collect the second filtrate, which removed impurities such as fibers; the second filtrate was centrifuged at 9,000×g for 70 min to collect the third filtrate, which removed cell debris; the third filtrate was centrifuged at 110,000×g for 130 min to collect VDVLNs precipitate and the fourth filtrate; the fourth filtrate was then centrifuged at 110,000×g for 130 min to collect the VDVLNs precipitate.

[0037] Comparative Example 1 Preparation method of ginseng-derived nanovesicles (GDVLNs): The difference from Example 1 is that Viola yedoensis is replaced with ginseng, while the other steps are the same as in Example 1.

[0038] Comparative Example 2 Preparation method of HDVLNs derived from Hedyotis diffusa: The difference from Example 1 is that Viola yedoensis is replaced with Hedyotis diffusa, while the other steps are the same as in Example 1.

[0039] Physicochemical characterization of Viola yedoensis-derived nanovesicles (VDVLNs) in Experiment Example 1 1. Structure and morphology The VDVLNs prepared in Example 1 were diluted with 1×PBS (pH 7.4) to approximately 1×10⁻⁶.8 ~1×10 9 Particle count / mL: 10 µL was added to a carbon film copper grid and allowed to stand at room temperature for 2 min. After aspirating excess liquid, 10 µL of 2% phosphotungstic acid was added for negative staining and stained at room temperature for 1 min. After staining, excess staining solution was aspirated and the mixture was allowed to air dry at room temperature. The vesicle structure and morphology were then observed using transmission electron microscopy (TEM). Figure 2 The TEM images shown in Figure A reveal that VDVLNs exhibit a typical bilayer membrane structure with complete membrane proteins and contents, indicating that the extraction method of this invention can maintain the integrity of the Viola yedoensis nanovesicle structure.

[0040] 2. Particle size and concentration measurement The VDVLNs prepared in Example 1 were added to PBS, and the particle size distribution and particle concentration were detected using NTA. The detection results are shown below. Figure 2 As shown in Figure B, the particle size distribution is concentrated in the range of 100–200 nm, and the concentration can reach 10. 10 Particle count / mL.

[0041] 3. Zeta potential analysis The VDVLNs prepared in Example 1 were diluted with 1×PBS (pH 7.4) to approximately 1×10⁻⁶. 8 ~1×10 9 Particle count / mL, zeta potential analysis was performed using a particle size and potential measuring instrument. Detection results are shown below. Figure 2 As shown in Figure C, the surface negative charge of VDVLNs is approximately -30 mV.

[0042] 4. Protein content analysis The VDVLNs prepared in Example 1 were lysed with an appropriate amount of RIPA lysis buffer to extract total protein from the nanovesicles. The lysis was performed by incubating on ice for 30 min with intermittent shaking. After lysis, the lysate was collected as the protein sample for analysis. Subsequently, BCA working solution was prepared according to the BCA protein quantification kit instructions, and standard solutions of different concentration gradients were prepared using bovine serum albumin (BSA) standards. These solutions were added to 96-well plates to plot a standard curve (see [link to kit]). Figure 2 (D). Add an appropriate amount of the protein sample to be tested to a 96-well plate, with 3 replicates per group. Then add BCA working solution to each well, mix gently, and incubate at 37°C in the dark for 30 min. After incubation, measure the absorbance of each well at 562 nm using a microplate reader. Calculate the protein concentration of the sample according to the BSA standard curve to obtain the protein content of VDVLNs. Experimental results are as follows: Figure 2 As shown in Figure E, the results indicate that the protein content in the Viola yedoensis nanovesicles extracted by this invention can reach 1200 µg / mL.

[0043] 5. Protein composition analysis The VDVLNs prepared in Example 1 were lysed with an appropriate amount of RIPA lysis buffer to extract total protein from the nanovesicles. During lysis, the mixture was incubated on ice for 30 min with intermittent shaking. After lysis, the lysate was collected as a protein sample. 1×SDS-PAGE loading buffer was added to the protein sample, and after mixing, the sample was heated at 100°C for 10 min to denature the protein. Subsequently, SDS-PAGE was used to perform proteomic analysis on the VDVLNs prepared in Example 1. The results are shown in [Figure number missing]. Figure 2 As shown in Figure F, VDVLNs contain abundant protein bands. This indicates that the Viola yedoensis nanovesicles extracted in this invention possess intact protein bands.

[0044] Antitumor effect of VDVLNs in Experiment 2 To verify the antitumor effect of VDVLNs, this invention used three cancer cell lines: PANC-1 (human pancreatic cancer cells), HCT116 (human colorectal cancer cells), and Huh-7 (human liver cancer cells). The specific operational steps are as follows: (1) Establishment of different treatment groups: VDVLNs were prepared according to the preparation method in Example 1 and prepared into 1×10 groups with PBS. 9 Particle count / mL, 5×10 9 Particle count / mL, 1×10 10 Particle count / mL, 5×10 10 Particle count / mL, 1×10 11 Particle number / mL of VDVLNs at different concentrations were 1×10 9 Treatment group, 5×10 9 Treatment group, 1×10 10 Treatment group, 5×10 10 Treatment group and 1×10 11 The treatment group and the control group were PBS, totaling 6 groups.

[0045] (2) Cell proliferation assay: PANC-1, HCT116, and Huh-7 cells were seeded into 96-well cell culture plates, with six replicates for each cancer cell type. Cells were incubated at 37°C. After cell attachment, VDVLNs from the different treatment groups were added and co-cultured for 24 h. Then, WST-1 reagent was added according to the WST-1 assay kit (Sigma-Aldrich) instructions, and the absorbance at 440 nm was measured using a microplate reader. Monitoring was performed continuously for 4 days. Results are shown in […]. Figure 3 Statistical analysis was performed on the data. The proliferation experiment results showed that 10 10VDVLNs treatment for 72 h significantly inhibited tumor cell proliferation and demonstrated anti-tumor effects.

[0046] (3) Clonal formation: 1000 tumor cells were seeded into cell culture dishes containing DMEM cell culture medium, divided into two groups, and incubated in a 37°C incubator. After the cells adhered, PBS and 1×10⁻⁶ PBS were added to each group respectively. 10 VDVLNs in the treatment group were co-cultured, with fresh cell culture medium changed every 3 days. After 14 days, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, photographed, and observed. The experimental results are shown below. Figure 4 Cloning experiments showed that VDVLNs treatment significantly reduced the clone number of three tumor cell lines, indicating that the nanovesicles derived from Viola yedoensis in this invention have anti-tumor effects.

[0047] Experimental Example 3: Antitumor Effects of Nanovesicles from Different Plant Sources To further evaluate the antitumor effect of VDVLNs and compare the biological activity of nanovesicles from different Chinese herbal medicine sources, this invention selected HCT116 and Huh-7 cells as in vitro tumor models.

[0048] (1) Establishment of different treatment groups: VDVLNs, GDVLNs and BDVLNs were prepared according to the preparation methods of Example 1, Comparative Example 1 and Comparative Example 2, and were prepared into 1×10 groups with PBS. 10 Particle count / mL was used to determine the groups as VDVLNs, BDVLNs, and GDVLNs, with PBS serving as the control group, for a total of 4 groups.

[0049] (2) Cell proliferation assay: HCT116 and Huh-7 were seeded at 1×10⁻⁶ cells per cell line, respectively. 3 Cells were randomly assigned to 96-well cell culture plates (6 replicates per cancer cell type) and incubated at 37°C. After cell attachment, the different treatment groups were added and co-cultured for 24 h. Then, WST-1 reagent was added according to the WST-1 assay kit (Sigma-Aldrich) instructions, and the absorbance at 440 nm was measured using a microplate reader. Monitoring was performed continuously for 4 days, and the data were statistically analyzed. The experimental results are shown below. Figure 5 Experimental results showed that nanovesicles from different plant sources all had varying degrees of influence on tumor cell proliferation, but their inhibitory effects differed significantly. Among them, VDVLNs treatment for 96 h resulted in inhibition rates of 28% and 52% against HCT116 and Huh-7 cells, respectively, significantly higher than other plant-derived nanovesicles, revealing that Viola yedoensis-derived nanovesicles possess source-specific antitumor biological activity.

[0050] Experimental Example 4: Mechanism of VDVLNs-induced mitochondrial dysfunction To further explore the anti-tumor mechanism of VDVLNs, this invention selected three human tumor cell lines (PANC-1, HCT116, and Huh-7) for experimental analysis.

[0051] PANC-1, HCT116, and Huh-7 cells were seeded at 1×10⁻⁶ cells respectively. 3 Cells were cultured in 96-well plates (6 replicates per well) for each cancer cell type, and incubated at 37°C. After cell attachment, the cells were divided into six groups: a control group (PANC-1, HCT116, Huh-7) and a VDVLNs treatment group (VDVLNs). PBS and 1×10⁻⁶ ppm of PBS were added to each group. 10 VDVLNs with particle count / mL were co-cultured for 72 h, with the treatment group consisting of VDVLNs prepared in Example 1 and the control group consisting of PBS. Then, WST-1 reagent was added and incubated for 2 h according to the WST-1 assay kit (Sigma-Aldrich) instructions.

[0052] (1) Detection of reactive oxygen species (ROS) levels: Intracellular ROS levels were detected using the specific fluorescent probe DCFH-DA from the ROS kit (Beyotime). Results were observed using confocal fluorescence microscopy (Plan-Apochromat 100× / 1.40 Oil DIC Ziss). Figure 6 As shown, A, B, and C correspond to pancreatic cancer cells PANC-1, colon cancer cells HCT116, and liver cancer cells Huh-7, respectively. In each group of images A, B, and C, the left image is a Hoechst-stained image, used to label cell nuclei, exhibiting blue fluorescence; the middle image is a ROS fluorescent probe-stained image, exhibiting green fluorescence, with fluorescence intensity reflecting intracellular ROS levels; the right image is a combined Hoechst and ROS signal image, used to visually demonstrate the distribution of ROS in cells. Figure 6It was observed that the green fluorescence signal in the control group cells was weak, indicating a low level of intracellular ROS. However, after treatment with VDVLNs, the green fluorescence in all cell lines was significantly enhanced and the fluorescence distribution was more widespread, indicating that VDVLNs treatment can significantly increase the intracellular ROS level. This result shows that VDVLNs can induce a significant oxidative stress response in tumor cells. Further quantitative analysis of ROS fluorescence intensity was performed, and the results for groups A, B, and C are presented in bar charts. The horizontal axis of the bar chart represents the different treatment groups (control group and VDVLNs treatment group), and the vertical axis represents the relative fluorescence intensity of intracellular ROS. Compared with the control group, the fluorescence intensity of the VDVLNs treatment group was significantly increased, indicating that VDVLNs promotes the accumulation of ROS in tumor cells, suggesting that VDVLNs can activate oxidative stress in tumor cells and induce tumor cell apoptosis or necrosis.

[0053] (2) Detection of mitochondrial membrane potential (JC-1): Mitochondrial membrane potential changes were detected using the JC-1 probe staining kit (Beyotime). Confocal fluorescence microscopy observations are shown below. Figure 7 As shown, A, B, and C correspond to PANC-1, HCT116, and Huh-7 cells, respectively. In the images of groups A, B, and C, the left side shows the fluorescence signal of JC-1 aggregates, which is red and its intensity reflects a high mitochondrial membrane potential; the middle side shows the fluorescence signal of JC-1 monomers, which is green. When the mitochondrial membrane potential decreases, JC-1 exists in monomeric form and emits green fluorescence; the right side shows a combined image of red and green fluorescence, used to comprehensively observe the mitochondrial functional status. Further quantitative analysis of the ratio of red to green fluorescence of JC-1 was performed, and the results of groups A, B, and C are presented in bar charts. The horizontal axis of the bar chart represents different treatment groups, and the vertical axis represents the ratio of red to green fluorescence intensity of JC-1. Compared with the control group, the ratio in the VDVLNs treatment group was significantly lower, indicating a significant decrease in mitochondrial membrane potential, suggesting that VDVLNs inhibit tumor cell proliferation by impairing mitochondrial function.

[0054] (3) Mitochondrial morphology analysis: After staining cell mitochondria with the MitoTracker red fluorescent probe (Thermo), confocal microscopy was performed for observation and image analysis. The results are as follows: Figure 8As shown in the images, A, B, and C correspond to PANC-1, HCT116, and Huh-7 cells, respectively. In the images of groups A, B, and C, the mitochondria in PANC-1, HCT116, and Huh-7 tumor cells treated with VDVLNs exhibited a broken, swollen, and fragmented morphology, with a significantly reduced average mitochondrial length-to-width ratio. This indicates an imbalance in mitochondrial dynamics and excessive fragmentation, suggesting that VDVLNs inhibit tumor growth by altering mitochondrial morphology and impairing mitochondrial function.

[0055] In summary, the nanovesicles derived from Viola yedoensis of this invention can specifically act on tumor cells, induce mitochondrial dysfunction, and enhance oxidative stress response, thereby achieving anti-tumor effects.

[0056] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. Application of Viola yedoensis-derived nanovesicles in the preparation of antitumor drugs.

2. The application according to claim 1, characterized in that, The tumors include pancreatic cancer, colorectal cancer, and liver cancer.

3. The application according to claim 1 or 2, characterized in that, The preparation method of the nanovesicles derived from Viola yedoensis includes the following steps: mixing whole Viola yedoensis with PBS buffer, homogenizing, and then centrifuging according to gradient to obtain vesicles with a particle size of 100~200 nm.

4. The application according to claim 3, characterized in that, The gradient centrifugation includes the following steps: collecting the first filtrate after centrifugation at 400~600×g for 8~15 min; collecting the second filtrate after centrifugation at 1500~3500×g for 25~35 min; collecting the third filtrate after centrifugation at 8000~12000×g for 50~70 min; and obtaining a precipitate after centrifugation at 80000~120000×g for 100~140 min.

5. The application according to claim 3, characterized in that, The ratio of the whole Viola yedoensis plant to PBS buffer is 1:5~7 g / mL.

6. An antitumor drug, characterized in that, This includes the Viola yedoensis-derived nanovesicles in the application described in claim 1.

7. The antitumor drug according to claim 6, characterized in that, The concentration of the Viola yedoensis-derived nanovesicles was 10. 10 ~10 11 Particle count / mL.

8. The antitumor drug according to claim 6 or 7, characterized in that, The tumors include pancreatic cancer, colorectal cancer, and liver cancer.

9. The antitumor drug according to claim 6 or 7, characterized in that, The antitumor drugs also include pharmaceutically acceptable excipients.

10. The antitumor drug according to claim 9, characterized in that, The excipients include one or more of the following: diluents, binders, disintegrants, anti-adhesion agents, flow aids, preservatives, and emulsifiers.

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