Tripterygium wilfordii exosome, preparation method and application of tripterygium wilfordii exosome in preparation of medicine for treating cervical tumor

Abstract

The invention discloses application of a tripterygium wilfordii exosome in preparation of an anti-cervical cancer medicine and a tumor angiogenesis inhibiting medicine, and relates to the field of biological medicine. The medicinal plant exosome derived from tripterygium wilfordii is successfully separated, and it is proved that the medicinal plant exosome can regulate related pathways through delivery of functional nucleic acid molecules to inhibit proliferation and migration of cervical cancer cells and promote active oxygen related apoptosis so as to be used for tumor treatment. The tripterygium wilfordii exosome can further act on vascular endothelial cells in a tumor microenvironment, the migration function of Hela cells is inhibited, and then the effect of resisting cell proliferation in the tumor microenvironment is achieved. The tripterygium wilfordii exosome has the advantages of being simple in preparation method, remarkable in tumor growth resistance and tumor cell migration inhibition effect, good in biocompatibility, high in delivery efficiency and the like, and can be used as a novel nano-drug for tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Tripterygium wilfordii exosome, its preparation method, and its application in the preparation of drugs for treating cervical tumors. Background Technology

[0002] Cervical cancer (CC), also known as cervical cancer, is a malignant tumor that commonly occurs in the cervix and is caused by human papillomavirus (HPV) infection. It is currently a major threat to women's reproductive health. Many factors contribute to the development of cervical cancer, resulting in a variety of treatment methods, including radiotherapy, chemotherapy, surgery, targeted therapy, and preventative treatment. However, these treatments often cause serious side effects and are only effective in early stages, with limited efficacy in advanced or recurrent cases. Therefore, there is an urgent need to develop new and more effective treatment strategies to improve the later stages of cervical cancer treatment.

[0003] Tripterygium wilfordii ( Tripterygium wilfordii Tripterygium wilfordii (also known as Purple Gold Bark), belonging to the genus Tripterygium in the family Celastraceae, possesses properties such as promoting blood circulation, killing parasites, and reducing swelling and detoxifying. In recent years, Tripterygium wilfordii and its active substances have shown remarkable potential in the field of anti-tumor treatment, while also exhibiting significant effects in anti-inflammatory and immunosuppressive effects. However, while bringing positive therapeutic effects, it can also lead to certain adverse reactions, mainly affecting the cardiovascular, digestive, reproductive, and urinary systems, thus limiting its clinical application.

[0004] Exosomes are spherical or cup-shaped extracellular vesicles (EVs) with a double membrane structure, approximately 30-150 nm in diameter. Mammals, plants, microorganisms, and parasites can all produce EVs, mediating intercellular communication by delivering their contents such as proteins, microRNAs, and lipids. Studies have shown that proteins and small RNAs in plant exosomes can participate in various physiological and pathological mechanisms by regulating immune responses, angiogenesis, and cell proliferation and apoptosis. Plant-derived exosomes, in addition to possessing functional characteristics similar to animal exosomes, also offer feasibility for industrial production, excellent biocompatibility, and low immunogenicity, enabling them to efficiently load therapeutic molecules such as drugs and nucleic acids. In recent years, plant-derived exosomes have attracted attention due to their wide availability and high safety; however, there are currently no reports on the extraction of Tripterygium wilfordii exosomes and their application in the treatment of cervical cancer. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide an application of Tripterygium wilfordii exosomes in the preparation of drugs for inhibiting HeLa cells and treating cervical cancer, so as to solve the problems of toxic side effects and poor targeting of existing Tripterygium wilfordii active substances in treatment.

[0006] Another objective of this invention is to provide a method for preparing the above-mentioned Tripterygium wilfordii exosomes.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows: This invention provides the application of Tripterygium wilfordii exosomes in the preparation of drugs that inhibit HeLa cell activity, treat cervical cancer, and inhibit tumor angiogenesis.

[0008] Preferably, the Tripterygium wilfordii exosomes are cup-shaped or saucer-shaped, with a particle size range of 40-150 nm.

[0009] Preferably, the concentration of the Tripterygium wilfordii exosomes used is 50-150 μg / ml.

[0010] The present invention also provides a method for preparing the above-mentioned Tripterygium wilfordii exosomes, comprising the following steps: (1) After washing the fresh Tripterygium wilfordii root, add PBS buffer solution, and pulp to obtain the original pulp; (2) Filter the raw pulp and centrifuge it using differential centrifugation. Collect the supernatant from the last centrifugation. (3) The supernatant was centrifuged at high speed, the supernatant was discarded, the precipitate was resuspended in PBS, and the purified Tripterygium wilfordii exosome-like nanovesicles (PELNVs) were collected.

[0011] Preferably, in step (1), the mass-to-volume ratio of the Tripterygium wilfordii root and the PBS buffer solution is 100g:300mL.

[0012] Preferably, in step (2), the parameters of the differential centrifugation are: first, centrifuge at 3000g for 10-15min at 4℃ and take the supernatant; then, centrifuge at 5000g for 30-35min at 4℃ and take the supernatant; finally, centrifuge at 10000g for 30-35min at 4℃.

[0013] Preferably, in step (3), the parameters for ultracentrifugation are: centrifugation at 110000g for 95-100min at 4℃.

[0014] This invention successfully isolated exosomes from the medicinal plant Tripterygium wilfordii, which can inhibit the proliferation and migration of cervical cancer cells and promote reactive oxygen species-related apoptosis by delivering functional nucleic acid molecules to regulate related pathways for tumor treatment. The Tripterygium wilfordii exosomes described in this invention can further act on vascular endothelial cells in the tumor microenvironment, inhibiting the migration function of HeLa cells, thereby exerting an anti-tumor microenvironment cell proliferation effect. The Tripterygium wilfordii exosomes described in this invention have advantages such as simple preparation method, significant anti-tumor growth and tumor cell migration inhibition effects, good biocompatibility, and high delivery efficiency, and can be used as a novel nanomedicine for tumor treatment.

[0015] The beneficial effects of this invention are as follows: (1) The preparation method provided by the present invention is simple, fast, highly reproducible and suitable for large-scale preparation of Tripterygium wilfordii exosomes.

[0016] (2) The Tripterygium wilfordii exosomes used in this invention are of natural origin, have low toxicity and good biocompatibility, and overcome the toxic side effects of traditional Tripterygium wilfordii extracts.

[0017] (3) This invention is the first to report that Tripterygium wilfordii exosomes can play a good role in treating cervical cancer by inhibiting HeLa cell activity and HeLa cell migration. Attached Figure Description

[0018] Figure 1 Electron micrograph of Tripterygium wilfordii exosomes; Figure 2 This is a diagram showing the particle size distribution of exosomes. Figure 3 Two-photon confocal image of Dio-labeled Tripterygium wilfordii exosomes co-cultured with HeLa cells for 4 h; Figure 4 This is a diagram showing the inhibitory effect of Tripterygium wilfordii exosomes on HeLa cell activity. Figure 5 Figure showing the results of the scratch assay to detect the effect of Tripterygium wilfordii exosomes on HeLa cell migration. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in more detail below with reference to specific embodiments. However, it should be understood that the content of this invention is not limited to this invention.

[0020] Example 1: Preparation of Tripterygium wilfordii exosomes (1) Wash fresh Tripterygium wilfordii root three times with ultrapure water, cut the root into 0.5cm wide pieces with garden shears, add the root to a juicer at a mass-volume ratio of 100g:300mL, and crush it in the juicer to obtain the pulp. (2) Extraction of Tripterygium wilfordii exosomes: The raw pulp was filtered through 8 layers of gauze and centrifuged at 3000g for 10 min at 4℃, and the supernatant was collected; then the supernatant from the previous step was centrifuged again at 5000g for 30 min at 4℃, and the supernatant was collected; then the supernatant from the previous step was centrifuged again at 10000g for 30 min at 4℃) to remove tuber fragments, dead cells and cell debris; (3) Exosome purification: The supernatant from the last round was transferred to an ultracentrifuge tube and centrifuged at 100,000g for 90 min at 4°C. The supernatant was discarded, and the precipitate was the exosome. The precipitate was gently pipetted with PBS to form an exosome suspension, which could be used directly for the next experiment or transferred to a -80°C freezer for storage.

[0021] Example 2: Physicochemical characteristics of Tripterygium wilfordii exosomes 1. Morphological observation of Tripterygium wilfordii exosomes using transmission electron microscopy The exosome suspension was suspended as droplets on a copper grid using a 10 μL pipette. The copper grid containing the droplets was allowed to stand for 10 minutes, and then the droplets were absorbed with absorbent paper. 1% phosphotungstic acid staining solution was then added to the copper grid as droplets again, and allowed to stand for 1.5 minutes. The staining solution was then absorbed with absorbent paper again. The copper grid was allowed to dry overnight, and then observed using a transmission microscope.

[0022] The transmission electron microscopy morphological observation results of the exosomes of the present invention (prepared according to Example 1) are shown in the figure below. Figure 1 The extracted material exhibits a uniform cup-shaped vesicle structure with a clearly visible central depression, and most of the vesicles within the field of view are morphologically intact. These morphological characteristics are consistent with those of exosomes.

[0023] 2. Determination of particle size of Tripterygium wilfordii exosomes using a nanoparticle size and Zeta potential analyzer. The particle size of the prepared Tripterygium wilfordii exosomes was analyzed using a nanoparticle size and Zeta potential analyzer. A certain amount of exosome suspension was diluted to a final volume of about 1.5 ml and added to a quartz cuvette, and the particle size was measured using the instrument.

[0024] The particle size analysis results of the exosomes of this invention (prepared using Example 1) are shown below. Figure 2 The extracted material has a particle size that is mostly between 40-150 nm, which is consistent with the particle size range of exosomes.

[0025] Efficacy Verification: The Use of Tripterygium wilfordii Exosomes in Inhibiting HeLa Cell Activity I. Tripterygium wilfordii exosomes can enter HeLa cells and exert their effects. 1 μl of Dio dye solution was added to 1 ml of exosome suspension obtained by ultracentrifugation of Tripterygium wilfordii exosomes. After incubation at 37°C for 20 min, the mixture was ultracentrifuged and resuspended in PBS to complete the labeling of Tripterygium wilfordii exosomes (prepared in Example 1) with Dio dye. HeLa cells were cultured at 3 × 10⁶ cells per well. 5 Cells were seeded at a density suitable for six-well plates. After 24 hours, 100 μl of Tripterygium wilfordii exosomes (prepared using Example 1) at a concentration of 3.5 mg / ml were added to each well. After co-culturing in the dark for 4 hours, cells were fixed with 4% paraformaldehyde fixative. Cell nuclei were labeled with DAPI staining solution, and then observed using a two-photon confocal microscope. The results are shown in the figure. Figure 3 The presence of abundant green fluorescence around the cell nucleus, i.e., within the cytoplasm, indicated that Tripterygium wilfordii exosomes could enter HeLa cells and exert their effects.

[0026] II. Cell viability was detected using the CCK-8 assay, which included the following steps: 1. The concentration of extracted exosomes was determined by BCA protein quantitative analysis.

[0027] Take out the 1.5mL centrifuge tube labeled "Protein Standard Preparation Solution" and "Protein Standard (BSA)" from the Beyotime BCA Protein Concentration Assay Kit (P0010, 500 tests / kit). Take 1mL of liquid from the protein standard preparation solution and add it to the 1.5mL centrifuge tube labeled "Protein Standard (BSA)" and dissolve it thoroughly.

[0028] Dilute the protein standard BSA solution to a concentration of 2 mg / mL as a stock solution. Note that during dilution, try to ensure that the diluent for the protein standard solution is the same as the solvent for the protein sample being tested.

[0029] Dilute the 2 mg / mL protein standard BSA stock solution prepared in the previous step into a test tube. Similarly, make sure that the diluent is as similar as possible to the solvent of the protein sample to be tested. The dilution range is 0.025-2 mg / mL.

[0030] Mix reagent A and reagent B thoroughly and evenly according to a volume ratio of 50:1, and prepare fresh for each use.

[0031] Take 20 μL of the protein standard solutions of different concentrations diluted in the previous step and the protein samples to be tested, and add them to the 96-well microplate in sequence.

[0032] Add 200 μL of BCA working solution to each well and shake the plate for 30 seconds using the microplate reader's shaker function to mix the solution. Incubate at 37°C for 30 minutes.

[0033] The absorbance at 562 nm was measured using a microplate reader. A standard curve was plotted with the protein standard concentration on the x-axis and the absorbance on the y-axis, yielding the linear equation: y = 0.6476x + 0.0302 (R²). 2 =0.9995). After sample dilution, the protein content in the extracted exosomes was calculated to be 3.2 μg / μL using a standard curve.

[0034] 2. CCK-8 assay for cell viability HeLa cells were seeded into 96-well plates and cultured adherently for 24 h. Then, three concentration gradients of 50 μg / mL, 100 μg / mL, and 150 μg / mL were set up, and the corresponding concentrations of Tripterygium wilfordii exosomes were added to the 96-well plates. After incubation for 24 h, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for another 2 h. The absorbance was measured at 570 nm using a microplate reader, and the cell viability was calculated.

[0035] See results Figure 4 The results showed that when the concentration of Tripterygium wilfordii exosomes was 50 μg / mL, it did not significantly inhibit the activity of HeLa cells compared with the control group. However, at concentrations above 100 μg / mL, Tripterygium wilfordii had a highly significant inhibitory effect on the activity of HeLa cells. The inhibitory effect on cell activity became more pronounced with the increase of exosome concentration.

[0036] III. Scratch assay to detect the effect of Tripterygium wilfordii exosomes on the migration ability of HeLa cells HeLa cells were seeded in 6-well plates and cultured adherently for 24 hours. The culture medium was discarded, and the cells were washed once with 1 mL of PBS. 2 mL of PBS buffer was added to each well. Using a 100 µL pipette tip, fine, straight scratches were made perpendicular to the bottom of the 6-well plate. Floating cells were washed away with PBS, and fresh PBS was added. Images were taken, recording the position and scratch spacing for each image. After taking images, the PBS was discarded, and 2 mL of fresh culture medium containing 50 μg / mL, 100 μg / mL, and 150 μg / mL of Tripterygium wilfordii exosomes were added to each well. The cells were returned to the incubator and cultured for another 12 hours. The culture medium was discarded, and the cells were washed once with 1 mL of PBS. 2 mL of PBS was added to each well, and images were taken again from the same position, recording the scratch spacing for each image. After taking images, the PBS was discarded, and 2 mL of fresh culture medium containing a specific concentration of Tripterygium wilfordii exosomes was added. The cells were cultured for another 12 hours, and 2 mL of PBS was added to each well. Images were taken again from the same position, recording the scratch spacing for each image.

[0037] See results Figure 5The results showed that after 12 hours of treatment with Tripterygium wilfordii exosomes, the migration rate of the control group was approximately 7.41%. There was no significant difference between the control and 50 μg / ml exosome concentrations after 12 hours. However, when the concentration was increased to 100 μg / ml and 150 μg / ml, the migration rate decreased significantly after 12 hours, to 4.66% and 1.41%, respectively. After 24 hours of treatment, the migration rate of the control group was 22.81%. At this point, all three concentrations of Tripterygium wilfordii exosomes showed a highly significant inhibitory effect on HeLa cell migration, at 18.99%, 16.74%, and 5.60%, respectively, indicating that Tripterygium wilfordii exosomes can inhibit the migration ability of HeLa cells.

Claims

1. The application of a Tripterygium wilfordii exosome in the preparation of drugs for inhibiting HeLa cell activity, treating cervical cancer, and inhibiting tumor angiogenesis.

2. The application according to claim 1, characterized in that, The Tripterygium wilfordii exosomes are cup-shaped or saucer-shaped, with a particle size ranging from 40 to 150 nm.

3. The application according to claim 1 or 2, characterized in that, The concentration of the Tripterygium wilfordii exosomes used is 50-150 μg / ml.

4. A method for preparing Tripterygium wilfordii exosomes, characterized in that, Includes the following steps: (1) After washing the fresh Tripterygium wilfordii root, add PBS buffer solution, and pulp to obtain the original pulp; (2) Filter the raw pulp and centrifuge it using differential centrifugation. Collect the supernatant from the last centrifugation. (3) The supernatant was centrifuged at high speed, the supernatant was discarded, the precipitate was resuspended in PBS, and the purified Tripterygium wilfordii exosome-like nanovesicles (PELNVs) were collected.

5. The preparation method according to claim 4, characterized in that, In step (1), the mass-to-volume ratio of the Tripterygium wilfordii root and the PBS buffer solution is 100g:300mL.

6. The preparation method according to claim 4 or 5, characterized in that, In step (2), the parameters of differential centrifugation are as follows: first, centrifuge at 3000g for 10-15 min at 4℃ and take the supernatant; then, centrifuge at 5000g for 30-35 min at 4℃ and take the supernatant; finally, centrifuge at 10000g for 30-35 min at 4℃.

7. The preparation method according to claim 4 or 6, characterized in that, In step (3), the parameters for ultracentrifugation are: centrifugation at 110000g for 95-100 min at 4℃.

Images