Pharmaceutical composition for synergistically resisting breast cancer and improving myocardial injury

Abstract

The invention belongs to the technical field of biological medicines, and discloses a pharmaceutical composition for synergistically resisting breast cancer and improving myocardial injury, which comprises paris polyphylla extracellular vesicles and adriamycin, the combined application medicine composition has a synergistic anti-tumor effect, the inhibition effect on tumor cells can be greatly enhanced, and cardiotoxicity caused by adriamycin can be effectively relieved; in addition, the PPEVs has excellent oxidation resistance, and can effectively relieve oxidative stress and toxic damage to myocardial cells during DOX tumor treatment, obviously reduce the apoptosis rate of the myocardial cells, and further reduce DOX-related cardiotoxicity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition based on the combined use of extracellular vesicles (PPEVs) extracted from the natural plant Paris polyphylla var. Yunnanensis and doxorubicin (DOX), and its application in anti-tumor treatment and reducing the cardiotoxicity of doxorubicin. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women worldwide, and its incidence continues to rise worldwide. The main treatments for breast cancer include radiotherapy, surgical resection, and chemotherapy. However, these traditional treatments have certain limitations. For example, chemotherapy is often limited in clinical application due to its significant toxic side effects. Therefore, the development of new drug treatment strategies with high efficiency and fewer side effects has become an important problem that needs to be solved urgently.

[0003] Doxorubicin is an anthracycline antibiotic widely used in the treatment of various malignant tumors, but its serious adverse reactions limit its clinical use. Doxorubicin may also cause damage to the hematopoietic system, manifested as bone marrow suppression, leading to leukopenia, erythrocytes and thrombocytopenia, thereby increasing the risk of infection, anemia and bleeding tendency. However, the most challenging side effect is cardiotoxicity, which is mainly manifested as cumulative dose-related myocardial damage, which can lead to congestive heart failure, arrhythmia and myocardial fibrosis. Its mechanism of action involves oxidative stress, mitochondrial damage, DNA fragmentation and cell apoptosis, and cardiotoxicity is usually irreversible and may even continue to develop many years after the end of treatment. Therefore, how to reduce its cardiotoxicity has become a research hotspot.

[0004] In recent years, plant-derived extracellular vesicles (PDEVs) have attracted widespread attention as a new type of biotherapeutic agent, especially showing great application potential in the field of wound healing. PDEVs are widely available and easy to separate. They are rich in lipids, proteins and nucleic acids and have biochemical properties that mediate intercellular communication. Compared with traditional drug delivery systems, PDEVs have many advantages, such as small size, negative surface charge, and high stability, which can be efficiently absorbed by cells. In addition, compared with animal-derived extracellular vesicles, PDEVs have lower immunogenicity, which further improves their safety. Studies have shown that PDEVs not only retain the chemical composition and pharmacological properties of the plants from which they are derived, but also outperform animal-derived vesicles in terms of biological activity.

[0005] Paris polyphylla var. yunnanensis was first recorded in "Shennong Ben Cao Jing" and occupies an important position in traditional medicine due to its excellent effects of clearing heat and detoxifying, dissipating swelling and nodules, cooling the liver and calming the mind. As one of the top ten traditional Chinese medicines in Yunnan, Paris polyphylla var. yunnanensis is widely used in clinical practice, especially showing significant curative effects in the treatment of diseases such as scrofula, herpes, and uterine bleeding. In recent years, with the in-depth study of modern pharmacology, the chemical components and biological activities of Paris polyphylla var. yunnanensis have been gradually revealed, and its potential value in anti-inflammatory, anti-tumor, immune regulation and other aspects has attracted much attention. Summary of the Invention

[0006] The purpose of the present invention is to provide a drug combination that can efficiently anti-tumor and reduce myocardial damage caused by chemotherapeutic drugs. This drug combination uses extracellular vesicles of Paris vietnamensis and doxorubicin for combined medication, and this combined medication has a significant anti-angiogenic effect. In addition, the drug combination can significantly inhibit the cell survival rate, cell migration rate, cell invasion rate and cell apoptosis rate of 4T1 breast cancer cells, and can reduce myocardial damage caused by doxorubicin by improving cardiomyocyte apoptosis, cytoskeleton, scavenging ROS and nuclear morphology.

[0007] Therefore, the technical solution provided by the present invention is as follows:

[0008] A drug combination for synergistically anti-breast cancer and improving myocardial damage, comprising extracellular vesicles of Paris vietnamensis and doxorubicin.

[0009] Further, in the above-mentioned drug combination for synergistically anti-breast cancer and improving myocardial damage, the mass ratio of doxorubicin to extracellular vesicles of Paris vietnamensis is (1-10):(1-10).

[0010] Further, in the above-mentioned drug combination for synergistically anti-breast cancer and improving myocardial damage, the mass ratio of doxorubicin to extracellular vesicles of Paris vietnamensis is (1-3):(1-3).

[0011] Further, in the above-mentioned drug combination for synergistically anti-breast cancer and improving myocardial damage, the mass ratio of doxorubicin to extracellular vesicles of Paris vietnamensis is 1:1 or 2:1 or 1:2 or 1:3 or 3:1 or 2:3 or 3:2.

[0012] Further, in the above-mentioned drug combination for synergistically anti-breast cancer and improving myocardial damage, the mass ratio of doxorubicin to extracellular vesicles of Paris vietnamensis is 1:1.

[0013] Further, the above-mentioned drug combination for synergistically anti-breast cancer and improving myocardial damage is an injection.

[0014] The second technical solution provided by the present invention is the above-mentioned pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, and the application of the pharmaceutical composition as a medicine for treating breast cancer and reducing cardiotoxicity.

[0015] Furthermore, for the above-mentioned pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, the application of the pharmaceutical composition as a medicine for inhibiting the proliferation, migration and invasion of breast cancer cells and inhibiting angiogenesis of human umbilical vein endothelial cells.

[0016] Furthermore, for the above-mentioned pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, the application of the pharmaceutical composition as a medicine for improving myocardial injury caused by doxorubicin.

[0017] Furthermore, for the above-mentioned pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, the application of the pharmaceutical composition as a medicine for scavenging reactive oxygen species generated by oxidative stress in cardiomyocytes, repairing the cytoskeleton, inhibiting doxorubicin uptake, and reducing doxorubicin-induced apoptosis of cardiomyocytes.

[0018] Compared with the prior art, the present invention has the following beneficial effects;

[0019] 1) The technical solution provided by the invention uses 4T1 cells in breast cancer as the research object, and combines extracellular vesicles and doxorubicin for co-drug use, and conducts in vitro anti-cancer activity tests on 4T1 cells. The results show that the combined use of Paris vietnamensis extracellular vesicles and doxorubicin has a very obvious effect on the cell survival rate of 4T1 breast cancer cells, greatly reducing its survival rate, increasing the apoptosis rate of 4T1 cells, and significantly reducing the cell migration and invasion rates, indicating that the combination drug has significant inhibitory effects on the growth and proliferation of 4T1 breast cancer cells. The present invention also finds that Paris vietnamensis extracellular vesicles can significantly inhibit the tube formation effect of human umbilical vein epithelial cells, so the pharmaceutical composition has the potential to become a new type of anti-tumor preparation.

[0020] 2) The technical solution provided by the invention combines Paris vietnamensis extracellular vesicles with the broad-spectrum anti-tumor drug doxorubicin for synergistically treating breast cancer. Since Paris vietnamensis extracellular vesicles can inhibit angiogenesis and promote apoptosis of tumor cells, the two drugs in combination can enhance the sensitivity of breast cancer cells to doxorubicin. In addition, Paris vietnamensis can improve myocardial injury caused by doxorubicin. We used H9C2 cardiomyocytes as the research object and conducted in vitro doxorubicin injury activity tests on H9C2 cardiomyocytes. The results show that Paris vietnamensis extracellular vesicles can improve H9C2 cardiomyocyte apoptosis, cytoskeleton, scavenge ROS, and nuclear morphological changes caused by doxorubicin, and reduce myocardial injury caused by doxorubicin. Compared with traditional chemotherapy drug treatment, the pharmaceutical composition has multiple advantages, such as enhancing anti-tumor activity and reducing cardiotoxic effects caused by chemotherapy drugs.

[0021] 3) The drug combination of the present invention synergistically inhibits breast cancer and can suppress the migration of 4T1 cells. Compared with the single use of doxorubicin, the cell migration rate decreased by about 15% and the cell invasion rate decreased by about 10% after the combined use of drugs, while the cell apoptosis rate increased by about 22%.

[0022] 4) The extracellular vesicles of Paris vietnamensis in the drug combination of the present invention can improve the myocardial cell damage caused by doxorubicin. Under the action of Paris vietnamensis cells, the apoptosis rate of H9C2 cells caused by doxorubicin decreased by about 10%. In addition, it can improve the disorder of the cytoskeleton and mitochondrial structure of H9C2 cells caused by doxorubicin, and can also scavenge ROS in H9C2 cells to reduce the myocardial damage caused by doxorubicin. Description of the Drawings

[0023] Figure 1 It shows the tube formation of HUVCE cells under the action of extracellular vesicles of Paris vietnamensis (PPEVs) at different concentrations.

[0024] Figure 2 It is a screening diagram of the combined ratio of doxorubicin and PPEVs drugs;

[0025] Among them: (A) Histogram of cytotoxicity at different concentration ratios; (B) Drug combination curve at different ratios.

[0026] Figure 3 It is a microscopic image of the cell morphology of 4T1 cell migration after treatment with extracellular vesicles of Paris vietnamensis (PPEVs), doxorubicin (DOX), and the combined use of drugs for 0 hours and 24 hours.

[0027] Figure 4 It is a quantitative histogram of the cell migration rate of 4T1 cells after treatment with extracellular vesicles of Paris vietnamensis (PPEVs), doxorubicin (DOX), and the combined use of drugs for 0 hours and 24 hours.

[0028] Figure 5 It is a microscopic image of the cell morphology of 4T1 cell invasion after treatment with extracellular vesicles of Paris vietnamensis (PPEVs), doxorubicin (DOX), and the combined use of drugs.

[0029] Figure 6 It is a quantitative histogram of the cell invasion rate of 4T1 cells after treatment with extracellular vesicles of Paris vietnamensis (PPEVs), doxorubicin (DOX), and the combined use of drugs.

[0030] Figure 7 It is an apoptosis analysis diagram of 4T1 cells after treatment with extracellular vesicles of Paris vietnamensis (PPEVs), doxorubicin (DOX), and the combined use of drugs.

[0031] Figure 8It is a bar graph of cell survival rate of H9C2 cells treated with doxorubicin and doxorubicin (DOX) + Paris polyphylla extracellular vesicles (PPEVs) for 24 hours.

[0032] Figure 9 This is a cell apoptosis analysis diagram of H9C2 cells treated with doxorubicin and doxorubicin (DOX) + Paris polyphylla extracellular vesicles (PPEVs) for 24 hours.

[0033] Figure 10 These are fluorescence micrographs of different concentrations of Paris polyphylla extracellular vesicles (PPEVs) clearing ROS from H9C2 cells.

[0034] Figure 11 This is a quantitative fluorescence bar graph of ROS clearance in H9C2 cells by different concentrations of Paris polyphylla extracellular vesicles (PPEVs).

[0035] Figure 12 These are the actin fluorescence images of H9C2 cells treated with Paris polyphylla extracellular vesicles (PPEVs), doxorubicin (DOX), and Paris polyphylla extracellular vesicles (PPEVs) + doxorubicin (DOX) for 24 hours. DETAILED DESCRIPTION

[0036] The embodiments of the present invention will be described in detail below in conjunction with experimental examples. The following experimental examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If no specific conditions are specified in the experimental examples, they are carried out under conventional conditions or under conditions recommended by the manufacturer. If the specific manufacturers of the reagents and instruments used in the experimental examples are not specified, they represent conventional products that can be purchased commercially.

[0037] The reagents and equipment used in the specific experimental examples were Thiazolyl blue (MTT) and doxorubicin (DOX) purchased from Aladdin Biochemical Technology Co., Ltd.;

[0038] In the specific experimental example, the complete medium used to culture 4T1 cells consisted of RPMI 1640 basal medium (Beijing Solebow Technology Co., Ltd., 31800-500), 10% fetal bovine serum (ExCell Bio, FSD500) and 1% penicillin-streptomycin (Biosharp, BL505A).

[0039] Human umbilical vein epithelial cells (HUVCE) were cultured at 37°C and 5% CO 2 Cultured in an incubator under the following conditions;

[0040] The complete medium used was high-glucose DMEM medium (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., ZQ-121), supplemented with 10% fetal bovine serum (ExCell Bio, FSD500) and 1% penicillin-streptomycin (Biosharp, BL505A); the Matrigel matrix was used as the matrix glue;

[0041] The cell apoptosis detection kit, ROS detection kit, phalloidin actin, and DAPI nuclear dye were all purchased from Beyotime Biotechnology Co., Ltd.;

[0042] 4750E carbon dioxide incubator (NUAIRE, USA); MF52-N inverted fluorescence microscope (Guangzhou Mingmei Optoelectronic Technology Co., Ltd.); LSM800 laser confocal microscope (Carl Zeiss, Germany), FACS Celesta flow cytometer (BD), SpectraMax M4 multi-functional microplate reader (Molecular Devices, USA), 5430R centrifuge (Eppendorf Centrifuge).

[0043] In this application, the Paris vietnamensis cell-derived extracellular vesicles were prepared on September 10, 2023, with the patent number 202311169659.5 and the application publication number CN 117210384A. The technical solutions provided in the examples were preferably used for preparation.

[0044] Example 1

[0045] This example provides a pharmaceutical composition for synergistically anti-breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis cell-derived extracellular vesicles is 1:1.

[0046] Example 2

[0047] This example provides a pharmaceutical composition for synergistically anti-breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis cell-derived extracellular vesicles is 1:10.

[0048] Example 3

[0049] This example provides a pharmaceutical composition for synergistically anti-breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis cell-derived extracellular vesicles is 10:1.

[0050] Example 4

[0051] This example provides a pharmaceutical composition for synergistically anti-breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis cell-derived extracellular vesicles is 2:1.

[0052] Example 5

[0053] This example provides a pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis extracellular vesicles is 1:2.

[0054] Example 6

[0055] This example provides a pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis extracellular vesicles is 1:3.

[0056] Example 7

[0057] This example provides a pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis extracellular vesicles is 3:1.

[0058] Example 8

[0059] This example provides a pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis extracellular vesicles is 2:3.

[0060] Example 9

[0061] This example provides a pharmaceutical composition for synergistically treating breast cancer and improving myocardial injury, and the mass ratio of doxorubicin to Paris vietnamensis extracellular vesicles is 3:2.

[0062] Experimental example 1 The in vitro tube formation experiment of HUVCE cells was used to evaluate the angiogenesis-promoting or anti-angiogenic effects of the drugs to be evaluated.

[0063] Thaw the Matrigel in a 4°C refrigerator one night in advance, and pre-cool all experimental consumables. The next day, rinse the 96-well plate with pre-cooled PBS, add Matrigel (DMEM:Matrigel = 2:1), add 50 μL to each well, and place it in a 37°C incubator for 30 minutes. After digesting the HUVEC cells, inoculate them on the Matrigel at a density of 5×105 cells per well. Set up a blank group, low (5 μg / mL), medium (10 μg / mL), and high (20 μg / mL) PPEVs groups. The blank group was added with 100 μL of complete medium, and the low, medium, and high PPEVs groups were added with 100 μL of complete medium containing their respective concentrations of PPEVs. After culturing in the incubator for 8 hours, stain with a cell viability staining kit and take pictures for recording. The experimental results are as Figure 1 shown. The experimental results show that PPEVs can significantly inhibit blood vessel formation, and this inhibitory effect shows a concentration dependence.

[0064] Experimental example 2 The MTT colorimetric method is often used to detect the relative cell viability.

[0065] Digest, centrifuge and resuspend 4T1 cells from the culture flask in complete medium. After counting, inoculate them into a 96-well plate at a density of 5000 cells per well and culture them in an incubator for 24 hours. The blank control group is added with 100 μL of complete medium. The PPEVs group is added with complete medium containing PPEVs at concentration gradients of 5, 10, 20, 40, 80, and 160 μg / mL. After culturing for 24 hours, discard the medium, add 100 μL of 1640 medium containing 0.5 mg / mL MTT, and continue culturing for 4 hours. After discarding the medium, add 150 μL of DMSO and shake for 10 minutes, then measure the absorbance value at a wavelength of 492 nm on an enzyme-linked immunosorbent assay (ELISA) reader. The experimental results are as Figure 2 shown in A. PPEVs can promote the apoptosis of 4T1 cells, and its IC50 is 98.65 μg / mL.

[0066] Experimental Example 3

[0067] Digest, centrifuge and resuspend 4T1 cells from the culture flask in medium. After counting, inoculate them into a 96-well plate at a density of 5000 cells per well and culture them in an incubator for 24 hours. The blank control group is added with 100 μL of complete medium. The DOX group is added with complete medium containing DOX at concentration gradients of 5, 10, 20, 40, 80, and 160 μg / mL. After culturing for 24 hours, discard the medium, add 100 μL of 1640 medium containing 0.5 mg / mL MTT, and continue culturing for 4 hours. After discarding the medium, add 150 μL of DMSO and shake for 10 minutes, then measure the absorbance value at a wavelength of 492 nm on an ELISA reader. The experimental results are as Figure 2 shown in A. DOX can promote the apoptosis of 4T1 cells, and its IC50 is 25.65 μg / mL.

[0068] Experimental Example 4

[0069] Digest, centrifuge and resuspend 4T1 cells from the culture flask in complete medium. After counting, inoculate them into a 96-well plate at a density of 5000 cells per well and culture them in an incubator for 24 hours. The blank control group is added with 100 μL of complete medium. Set 5 different mass ratios of doxorubicin to extracellular vesicles (1:1, 2:1, 1:2, 1:3, 3:1, 2:3, 3:2). The groups with different mass ratios of doxorubicin to extracellular vesicles are added with complete medium containing their respective concentration gradients of 5, 10, 20, 40, 80, and 160 μg / mL. After culturing for 24 hours, discard the medium, add 100 μL of 1640 medium containing 0.5 mg / ml MTT, and continue culturing for 4 hours. After discarding the medium, add 150 μL of DMSO and shake for 10 minutes, and measure the absorbance value at a wavelength of 492 nm on an ELISA reader. The experimental results are as Figure 2As shown in , the IC50 values of doxorubicin (DOX)∶Paris vietnamensis extracellular vesicles (PPEVs) at different combined mass ratios (1:1, 2:1, 1:2, 1:3, 3:1, 2:3, 3:2) were 1.67, 1.84, 2.97, 6.92, 14.84, 4.62, and 7.62 μg / mL, respectively. It was demonstrated that regardless of the ratio, the cell inhibition rate of the drug combination was lower than that of the individual drugs used alone.

[0070] Experimental Example 5

[0071] Based on the cell viability results of PPEVs, DOX administered alone, and in combination detected in Experimental Examples 2 - 4, the cell viability data of PPEVs and DOX at different concentrations were input into GraphPad 8.0.2 software to calculate the IC50 values and their regression equations for single and combined drug administrations. Meanwhile, using CompuSyn 1.0 software, the combination index (CI50) for the combined use of the drugs was further calculated. We plotted the relationship curve of the CI values and the effect (fa) of doxorubicin and PPEVs at different combined ratios ( Figure 2 B). According to the changing trend of the CI values, we found that when the CI value was greater than 1, it indicated an antagonistic effect between the two drugs; when the CI value was less than 1, it showed a synergistic effect between the two drugs, and the smaller the CI value, the stronger the synergistic effect. Therefore, based on Experimental Example 4, we preferred the drug combination with a synergistic ratio of 1∶1 to have the strongest inhibitory effect on 4T1 cells.

[0072] Experimental Example 6

[0073] 4T1 cell migration experiment: The 4T1 cells were digested from the culture flask, centrifuged, and resuspended in the culture medium. After counting, they were seeded into a 24-well plate at a density of 1.5×105 cells per well. The blank group, PPEVs group, DOX group, and combined drug administration group were set up, with 3 replicates in each group. After culturing for 24 hours until the cells covered the well plate, a scratch was made using a 200 μL pipette tip, ensuring consistent scratching force. The blank group was added with 0.5 mL of 1640 medium with a serum concentration ratio of 2%, the DOX group was added with 0.5 mL of 2% serum medium containing 2.5 μg / mL doxorubicin, and the combined drug administration group was added with 0.5 mL of 2% serum medium containing 2.5 μg doxorubicin and 2.5 μg PPEVs. Subsequently, the cells were placed in an incubator and cultured for 12 hours and 24 hours. The relative migration distance of the cells was observed using an inverted microscope and quantitatively analyzed using Image J software. The results of the cell scratch experiment ( Figure 3 、 Figure 4)It was shown that the cell migration rate in the PPEVs intervention group (84.86 ± 4.67%) was significantly decreased compared with that in the blank group (93.27 ± 1.32%), indicating that PPEVs could effectively inhibit the migration of tumor cells. In addition, the cell migration rate in the combined drug group (57.01 ± 6.67%) showed a more significant inhibitory effect compared with that in the doxorubicin single drug group (72.62 ± 4.81%), further demonstrating that the drug combination of PPEVs and doxorubicin could synergistically inhibit the migration of tumor cells.

[0074] Experimental Example 7 4T1 Cell Invasion Assay

[0075] ① Starvation treatment of cells: Replace the culture medium with serum-free basal medium, and continue to place the cells in the cell incubator (37 °C, 5% CO 2 ) for 12 hours to complete the starvation treatment of the cells.

[0076] ② Preparation of Transwell chambers: Melt Matrigel matrix glue overnight at 4 °C, and mix it evenly on ice according to the volume ratio of matrix glue: serum-free DMEM basal medium = 1:8. Place the Transwell chamber in a 24-well plate, and use a pre-cooled pipette tip to take 50 μL of the matrix glue dilution and add it evenly into the chamber (avoid generating air bubbles), then place it in the cell incubator (37 °C, 5% CO 2 ) overnight for solidification.

[0077] ③ Hydration of the Transwell chamber basement membrane: Before use, add 50 μL of pre-warmed (37 °C) serum-free DMEM basal medium to each Transwell chamber, rinse the chamber, and let it stand at room temperature for 20 minutes to hydrate the matrix glue, then aspirate the excess medium.

[0078] ④ Cell preparation: Take out the tumor cells, digest and collect the cells with 0.25% trypsin / EDTA, resuspend the cells with an appropriate amount of serum-free DMEM medium, and adjust the cell density to 5×10 5 cells / mL.

[0079] ⑤ Invasion assay: Add 500 μL of DMEM complete medium containing 10% FBS to the lower chamber of the Transwell chamber, and add 200 μL of the cell suspension to the upper chamber. The experiment was divided into 4 groups: blank group, extracellular vesicle group, doxorubicin group, and combined drug administration group, and cultured for 24 hours.

[0080] ⑥ Fixation: Use forceps to take out the Transwell chamber, discard the medium in the lower chamber, wipe off the non-invasive cells in the upper chamber with a cotton swab, and wash 3 times with PBS. Fix the cells with 4% paraformaldehyde solution at 4 °C for 15 minutes, discard the fixative, and then wash 3 times with PBS.

[0081] ⑦ Staining and counting: Stain with 0.1% crystal violet staining solution for 5 minutes. After discarding the staining solution, wash with PBS 3 times and air dry. Observe under a microscope, randomly select 5 fields of view per well, and after taking pictures, dissolve the crystal violet with 33% glacial acetic acid and measure the absorbance at a wavelength of 570 nm on an enzyme-linked immunosorbent assay (ELISA) reader. The experimental results showed ( Figure 5 , Figure 6 ), the cell invasion rate of PPEVs was 82.06 ± 2.9%, the cell invasion rate of doxorubicin was 39.91 ± 1.18%, and the cell invasion rate of the combined administration group was 28.97 ± 3.09%. Both PPEVs and doxorubicin alone could significantly inhibit the invasion ability of tumor cells, and the inhibitory effect on invasion in the combined treatment group was more significant, indicating that the drug combination could synergistically enhance the anti-tumor invasion inhibitory effect.

[0082] Experimental Example 8

[0083] Apoptosis experiment of 4T1 cells: Digest 4T1 cells from the culture flask, centrifuge, and resuspend in complete medium. After counting, seed them in a 6-well plate at a density of 200,000 cells per well and culture in an incubator for 24 hours. Divide into a blank group, a DOX group, a PPEVs group, and a DOX + PPEVs group. The blank group was added with 1 mL of complete medium, the DOX group was added with 1 mL of complete medium containing DOX at a concentration of 2.5 μg / mL, the PPEVs group was added with 1 mL of complete medium containing PPEVs at a concentration of 2.5 μg / mL, and 1 mL of complete medium containing DOX and PPEVs at their respective concentrations of 2.5 μg / mL was added. Continue to culture in the incubator for 24 hours, collect the culture medium and wash the cells with PBS, then add trypsin without EDTA for digestion. Collect the cells and centrifuge at 1000 rpm for 5 minutes. After discarding the medium, wash the cells with 1 mL of PBS and centrifuge at 1000 rpm for 5 minutes, then discard the supernatant (n = 3). Then add 300 μL of apoptosis kit buffer to resuspend the cells. Stain with the dye in the kit 15 minutes before loading onto the machine, and then analyze using a flow cytometer. The experimental results ( Figure 7 ) showed that PPEVs could significantly promote the apoptosis of 4T1 cells, and the apoptosis rate was 27.6%. Among them, the proportion of early apoptotic cells was 11.6%, and the proportion of late apoptotic cells was 16.8%. Compared with the PPEVs group, the cell apoptosis rate of the doxorubicin single-drug group was higher, reaching 33.4%, with the proportion of early apoptosis being 13.2% and the proportion of late apoptosis being 20.2%. The combined administration group significantly enhanced the apoptosis of 4T1 cells. Whether it was the proportion of early apoptotic cells or late apoptotic cells, it increased significantly compared with the single-drug group. Specifically, the proportion of early apoptosis in the combined treatment group was 20.6%, and the proportion of late apoptosis was 35.3%, showing a stronger apoptosis-inducing effect than single-drug treatment.

[0084] Detection of relative viability of H9C2 cells

[0085] H9C2 cells were digested from the culture flask, centrifuged and resuspended in the medium. After counting, they were seeded in a 96-well plate at a density of 5000 cells per well and cultured in an incubator for 24 hours. In the model group, H9C2 cells were induced to apoptosis with different concentrations of DOX (0.5, 1, 2 μg / mL); in the drug administration group, 5 μg / mL PPEVs were added; and in the blank control group, 100 μL of complete medium was added. After 24 hours of culture, the medium was discarded, and 100 μL of medium containing 0.5 mg / mL MTT was added to each well and cultured for another 4 hours. Subsequently, the medium was discarded, 150 μL of DMSO was added, and it was gently shaken for 10 minutes to fully dissolve the crystals. Finally, the absorbance value was measured at a wavelength of 492 nm on an enzyme-linked immunosorbent assay (ELISA) reader. The experimental results showed ( Figure 8 ), PPEVs could significantly increase the viability of cells after apoptosis induced by different concentrations of doxorubicin. When the concentration of doxorubicin was 0.5 mg / mL, the cell viability was 53.17 ± 2.28%, while after intervention with PPEVs, the cell viability increased to 64.17 ± 4.46%; when the concentration of doxorubicin was 1 mg / mL, the cell viability was 47.37 ± 3.58%, while after PPEVs intervention, the cell viability was 60.94 ± 2.41%; when the concentration of doxorubicin was 2 mg / mL, the cell viability was 45.17 ± 1.73%, while after PPEVs intervention, the cell viability was 59.78 ± 3.39%. These results indicate that PPEVs can effectively improve the cytotoxicity of doxorubicin to H9C2 cells and enhance cell viability.

[0086] Experimental example 10 Detection of apoptosis of H9C2 cells

[0087] After digestion and centrifugation of H9C2 cells from the culture flask, they were resuspended in medium and counted, and then seeded in a 6-well plate at a density of 200,000 cells per well and cultured in an incubator for 24 hours. The experimental groups included a model group (DOX group) and low (2.5 μg / mL), medium (5 μg / mL), and high-dose (10 μg / mL) PPEVs groups. After culturing, for the H9C2 cells in the model group (DOX group), complete medium containing 2.5 μg / mL DOX was added; for the low-dose (2.5 μg / mL) PPEVs group, complete medium containing 2.5 μg / mL PPEVs was added; for the medium-dose (5 μg / mL) group, complete medium containing 5 μg / mL PPEVs was added; and for the high-dose (10 μg / mL) group, complete medium containing 10 μg / mL PPEVs was added. Then the cells were continued to be cultured in the incubator for 24 hours. After the culture ended, the culture medium and PBS washing solution were collected, and digestion was carried out using trypsin without EDTA. After collecting the cells, they were centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL PBS. They were centrifuged again at 1000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in 300 μL of the apoptosis kit buffer. 15 minutes before detection on the machine, the cells were stained with the fluorescent dye in the kit, and then flow cytometry analysis was performed. The results are shown in Figure (9). When the concentration of PPEVs was 10 μg / mL, compared with the model group, the apoptosis rate of the cells decreased by 11%. It can be seen from the apoptosis map that PPEVs mainly exerts its effect by reducing the proportion of late apoptotic cells.

[0088] Experimental Example 11 Detection of ROS Scavenging by PPEVs in H9C2 Cells

[0089] After digestion and centrifugation of H9C2 cells in the logarithmic growth phase from the culture flask, they were resuspended in medium and counted, and then seeded in a 48-well plate at a density of 20,000 cells per well and cultured in an incubator for 24 hours. After the culture ended, the medium was discarded, and a blank group, a positive drug group (H 2 O 2 group) (1 μg / mL), a positive drug (1 μg / mL) + low-dose exosome group (5 μg / mL), a positive drug (1 μg / mL) + medium-dose exosome group 10 μg / mL, and a positive drug (1 μg / mL) + high-dose exosome group 20 μg / mL were set up. 200 μL of complete medium was added to the blank group, and 200 μL of complete medium containing different drug concentrations was added to the other experimental groups. At 37 °C and 5% CO 2After culturing in an incubator for 6 h, a fluorescence probe of the kit was added and incubated at 37 °C for 20 min, and then visualized and photographed using an inverted fluorescence microscope. Meanwhile, the above experimental steps were repeated. The cells were seeded in a 6-well plate, and after the same drug administration and incubation treatments, flow cytometry quantitative analysis was performed. The experimental results are shown in Figures (10, 11). Compared with the positive drug group, the ROS level in H9C2 cells treated with PPEVs was significantly decreased and showed a certain concentration dependence. By quantitatively analyzing the fluorescence intensity of intracellular ROS using a flow cytometer, the results showed that the fluorescence intensity of the (positive group) was significantly higher than that of the PPEVs treatment group, and the difference was statistically significant (P < 0.0001). Under the action of PPEVs, the fluorescence intensity gradually decreased. When the concentration of PPEVs was 5 μg / mL, the fluorescence intensity decreased by 44% compared with the positive group; when the concentration of PPEVs was 20 μg / mL, the fluorescence intensity was almost the same as that of the blank group, indicating that PPEVs significantly inhibited the production of ROS.

[0090] Experimental Example 12 Detection of Phalloidin Actin in H9C2 Cells

[0091] After digesting and centrifuging H9C2 cells from the culture flask, they were resuspended and counted with the culture medium, and then seeded in a confocal culture dish at a density of 10,000 cells per well. The experiment was divided into a blank group, a PPEVs group (2.5 μg / mL), a DOX group (2.5 μg / mL), and a PPEVs (2.5 μg / mL) + DOX (2.5 μg / mL) group. The blank group was added with 1 mL of complete culture medium, the DOX group was added with 1 mL of complete culture medium containing a DOX concentration of 2.5 μg / mL, the PPEVs group was added with 1 mL of complete culture medium containing a PPEVs concentration of 2.5 μg / mL, and 1 mL of complete culture medium containing 2.5 μg of DOX and 2.5 μg of PPEVs was added. After culturing in an incubator for 24 hours, the cells were fixed with paraformaldehyde for 15 min, the supernatant was discarded, and the cells were washed 3 times with pre-cooled PBS. Subsequently, pre-cooled acetone was added for permeabilization treatment for 10 min, the supernatant was discarded, and the cells were washed 3 times again with pre-cooled PBS. Then, the diluted phalloidin dye at a ratio of 1:200 was added to the confocal culture dish and incubated for 30 min in the dark at room temperature, and then washed 3 times with PBS. Subsequently, DAPI nuclear dye was added and incubated for 15 min in the dark at room temperature, and then washed 3 times again with PBS. Finally, fluorescence visualization analysis was performed using a laser confocal microscope. The experimental results are as in ( Figure 12) As shown, in the blank group, the cellular myofilaments were arranged in regular parallel, with clear structures and strong fluorescence signals, indicating a complete cytoskeleton and uniform distribution of actin. However, after doxorubicin treatment, obvious breaks occurred in the cellular myofilaments, the fiber structure was disordered, and the fluorescence signal was significantly weakened at the same time. This change may be due to the degradation of actin caused by doxorubicin-induced oxidative stress, or the loss of fluorescence signal caused by cell apoptosis or necrosis. It is worth noting that after PPEVs intervention, the myofilament fibers of the cells resumed regular parallel arrangement, and the fluorescence signal of actin increased, indicating that PPEVs repaired the cytoskeletal damage caused by doxorubicin to a certain extent and had a protective effect on cell structure.

[0092] Experimental Example 13 Statistical Analysis of Experimental Examples

[0093] All experimental data were expressed as mean ± standard deviation (SD), and Student's t-test was used to compare the means of the two groups. One-way analysis of variance was performed, and then Tukey's test was used to adjust for multiple comparisons. All analyses were considered statistically significant at p < 0.05.

Claims

1. A pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage, characterized in that: Contains Paris polyphylla extracellular vesicles and doxorubicin.

2. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 1, characterized in that: The mass ratio of doxorubicin to the Paris polyphylla extracellular vesicles is (1-10):(1-10).

3. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 2, characterized in that: The mass ratio of doxorubicin to the Paris polyphylla extracellular vesicles is (1-3):(1-3).

4. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 3, characterized in that: The mass ratio of doxorubicin to Paris polyphylla extracellular vesicles is 1:1 or 2:1 or 1:2 or 1:3 or 3:1 or 2:3 or 3:

2.

5. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 4, characterized in that: The mass ratio of doxorubicin to the Paris polyphylla extracellular vesicles is 1:

1.

6. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 1, characterized in that: The pharmaceutical composition is used for preparing a drug for resisting breast cancer and alleviating cardiac toxicity.

7. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 6, characterized in that: The pharmaceutical composition is used as a drug for preparing a drug for inhibiting the proliferation, migration and invasion of breast cancer cells and inhibiting angiogenesis of human umbilical vein endothelial cells.

8. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 6, characterized in that: The pharmaceutical composition is used for preparing a drug for improving myocardial injury caused by adriamycin.

9. The pharmaceutical composition for synergistically fighting breast cancer and improving myocardial damage according to claim 8, characterized in that: The pharmaceutical composition is used as a drug for preparing a drug for removing active oxygen generated by oxidative stress in myocardial cells, repairing the cytoskeleton, inhibiting the uptake of doxorubicin, and reducing myocardial cell apoptosis caused by doxorubicin.

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