Application of Brucea javanica exosomes in the preparation of anti-breast cancer drugs and drugs for inhibiting tumor angiogenesis

Abstract

The present invention discloses an application of Brucea javanica exosomes in the preparation of anti-breast cancer drugs and drugs for inhibiting tumor angiogenesis, and relates to the field of biomedicine. The present invention successfully isolated the medicinal plant exosomes derived from Brucea javanica, and proved that it can inhibit breast cancer cell proliferation, migration and promote reactive oxygen-related cell apoptosis by delivering effective nucleic acid molecules to regulate related pathways for tumor treatment. The Brucea javanica exosomes described in the present invention can further act on vascular endothelial cells in the tumor microenvironment, inhibit the secretion of vascular growth factors and endothelial cell function, and then play an anti-angiogenesis effect in the tumor microenvironment. The Brucea javanica exosomes described in the present invention have the advantages of simple preparation method, significant anti-tumor growth and anti-tumor angiogenesis efficacy, good biocompatibility, high delivery efficiency, etc., and can be used as a new type of nanomedicine for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicines, and specifically relates to an application of Brucea javanica exosomes in the preparation of anti-breast cancer drugs and drugs for inhibiting tumor angiogenesis. Background Art

[0002] Malignant tumors are a problem that threatens human health and needs to be solved urgently. Most clinical treatment options, such as surgical resection and chemoradiotherapy, have obvious bottlenecks such as large side effects, chemotherapy resistance, and high recurrence rate. Since single-drug therapy cannot achieve the expected tumor treatment effect, people have begun to seek multimodal combined treatments, such as simultaneously addressing tumor cells and the microenvironment on which they depend for survival. Angiogenesis is a key player in the microenvironment of solid tumors, promoting the supply of oxygen and nutrients required for tumor progression. Vascular endothelial growth factor VEGF, which regulates tumor angiogenesis, is abnormally highly expressed in a variety of solid tumors and is closely related to the occurrence and metastasis of tumors, suggesting that combined anti-tumor and inhibition of angiogenesis in the tumor microenvironment may be a promising therapy.

[0003] Exosomes are a type of extracellular vesicles with a diameter of about 30-150nm and a lipid bilayer structure formed during the cell secretion process. They contain a variety of biologically active substances such as proteins, nucleic acids, and lipids derived from mother cells, and play an important regulatory role in a variety of biological processes and disease treatments. Not only can they be efficiently taken up by cells to improve the in vivo bioavailability of biological macromolecular active substances, but they can also further carry other exogenous drugs or functional molecules for a variety of uses such as drug co-delivery and treatment.

[0004] In recent years, exosome vesicles secreted by medicinal plants have also been shown to have significant therapeutic effects and are expected to develop into a new type of natural nanoformulation. Compared with animal and bacterial exosomes, they have significant advantages such as biocompatibility, higher stability, longer biological half-life, and higher cell and tissue penetration. This makes them important clinical application value in the fields of tumors, inflammation, and immunotherapy as natural nanoformulation targeted delivery carriers, and has great development prospects in the field of innovative drug preparations. However, there is a lack of research on plant exosomes, and the specific mechanisms involved are still unclear, which greatly limits their application potential. Summary of the invention

[0005] In view of the shortcomings of existing drugs for tumor treatment, the present invention aims to provide a novel plant exosome nanomedicine for anti-tumor and anti-angiogenesis in tumor microenvironment with high efficiency and safety. The present invention successfully prepared and separated exosomes from the medicinal plant Brucea javanica for the first time, proving that it can inhibit tumor cell proliferation, migration and promote reactive oxygen-related cell apoptosis by delivering effective nucleic acid molecules to regulate related pathways for tumor treatment. The Brucea javanica exosomes in the present invention can further act on vascular endothelial cells in the tumor microenvironment, inhibit the secretion of vascular growth factors and endothelial cell function, and then play the role of anti-angiogenesis in the tumor microenvironment. The Brucea javanica exosomes in the present invention have the advantages of simple preparation method, significant anti-tumor and angiogenesis effects, good biocompatibility, and high delivery efficiency. The present invention will provide new reference significance and value for the further development of various medicinal plant exosomes and their application in tumor treatment.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] The present invention proposes the use of Brucea javanica exosomes in the preparation of anti-breast cancer drugs.

[0008] The present invention also proposes the use of Brucea javanica exosomes in the preparation of drugs for inhibiting tumor angiogenesis.

[0009] Furthermore, the tumor is a breast tumor.

[0010] Furthermore, the Brucea javanica exosomes are prepared by the following method:

[0011] 1) Mixing fresh active parts of Brucea javanica with phosphate buffer and squeezing the juice for 5-15 times, each squeezing time for 15-60 seconds, to obtain a slurry containing Brucea javanica exosomes;

[0012] 2) filtering out the residue in the slurry obtained in step 1) and collecting the juice, and obtaining a precipitate containing Brucea javanica exosomes by differential centrifugation of the juice;

[0013] 3) resuspending the precipitate obtained in step 2) with PBS, and purifying to obtain the Brucea javanica exosomes;

[0014] Furthermore, in the step 2), the differential centrifugation method is: centrifuging the filtered juice at 1000×g for 5-20 min, 4000×g for 20-60 min, and 10000×g for 60-120 min at 4°C to obtain a supernatant; then centrifuging the supernatant at 100000×g-150000×g for 60-150 min at 4°C to obtain a precipitate containing Brucea javanica exosomes.

[0015] Furthermore, in step 3), the purification is performed by filtering through a 0.22 μm membrane filter.

[0016] Furthermore, in step 3), the purification is performed by sucrose density gradient centrifugation.

[0017] Furthermore, the brucea javanica exosomes contain active substances, and the active substances are derived from one or more of proteins, lipids, mRNA, and small RNA contained in the brucea javanica exosomes; the small RNA is one or more of miRNA, lncRNA, and circleRNA.

[0018] The reagents and raw materials used in the present invention are commercially available.

[0019] The present invention is beneficial in that:

[0020] (1) The Brucea javanica exosomes provided by the present invention are cheap, easy to obtain, safe, low-toxic, and highly biocompatible, and can overcome the problems of high cost, poor efficacy, easy drug resistance, high toxicity and adverse reactions, and low absorption and delivery efficiency of tumor treatment drugs.

[0021] (2) The Brucea javanica exosomes provided by the present invention can significantly reduce the expression of PI3K / Akt / mTOR pathway proteins in tumor cells, inhibit tumor cell proliferation, migration and invasion, and efficiently induce tumor cell apoptosis through the ROS / Caspase pathway, thereby exerting the therapeutic effect of anti-tumor growth and metastasis.

[0022] (3) The brucea javanica exosomes provided by the present invention can significantly reduce the secretion of VEGF vascular growth factor in vascular endothelial cells, inhibit endothelial cell proliferation, migration and tube formation, thereby exerting an anti-tumor angiogenesis therapeutic effect.

[0023] (4) The Brucea javanica exosomes provided by the present invention can be used as a biomacromolecule delivery nanocarrier, which can be efficiently taken up by cells and deliver the active substances contained inside, cross-border regulating the expression of multiple genes and proteins in mammalian cells to exert therapeutic effects, providing a new strategy for cross-border medical treatment of animals and plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below in conjunction with the accompanying drawings:

[0025] Figure 1 This is a particle size distribution diagram of the Brucea javanica exosomes obtained in one embodiment of the present invention;

[0026] Figure 2 This is a TEM image of the Brucea javanica exosomes obtained in one embodiment of the present invention;

[0027] Figure 3 This is a particle size distribution diagram of Brucea javanica exosomes of different density bands obtained in an embodiment of the present invention;

[0028] Figure 4 This is the effect of different concentrations of Brucea javanica exosomes on 4T1 cell proliferation in one embodiment of the present invention;

[0029] Figure 5 This is the effect of different concentrations of Brucea javanica exosomes on MCF-7 cell proliferation in one embodiment of the present invention;

[0030] Figure 6 This is the effect of different concentrations of Brucea javanica exosomes on L02 cell proliferation in one embodiment of the present invention;

[0031] Figure 7 This is the effect of different concentrations of Brucea javanica exosomes on the proliferation of HUVECs in one embodiment of the present invention;

[0032] Figure 8 This is the effect of different concentrations of exosomes from other plants on the proliferation of 4T1 cells in a pair of ratios of the present invention;

[0033] Fig. 9 This is the effect of different concentrations of exosomes from other plants on the proliferation of HUVECs in a pair of ratios of the present invention;

[0034] Fig.10 This is the effect of 50 μg / mL Brucea javanica exosomes on the apoptosis level of 4T1 cells in one embodiment of the present invention;

[0035] Fig.11 This is a Western blot analysis diagram of the effect of Brucea javanica exosomes on apoptotic protein CleavedCaspase3 / 9 in 4T1 cells in one embodiment of the present invention;

[0036] Fig.12 This is a Western blot expression analysis diagram of the effect of Brucea javanica exosomes on PI3K / Akt / mTOR pathway proteins in 4T1 cells in one embodiment of the present invention;

[0037] Fig.13 This is the effect of Brucea javanica exosomes on the VEGF secretion content in 4T1 cells in one embodiment of the present invention;

[0038] Fig.14 This is the effect of Brucea javanica exosomes on the VEGF secretion content in HUVECs in one embodiment of the present invention;

[0039] Fig.15 This is the effect of Brucea javanica exosomes on HUVECs migration in one embodiment of the present invention;

[0040] Fig.16 This is the effect of Brucea javanica exosomes on HUVECs tube formation in one embodiment of the present invention;

[0041] Fig.17 This is the inhibition of the size of subcutaneous breast cancer transplanted tumors in mice by Brucea javanica exosomes in one embodiment of the present invention;

[0042] Fig.18 This is a Western blot expression analysis diagram of the effect of Brucea javanica exosomes on PI3K / Akt / mTOR pathway proteins, Caspase apoptosis proteins, and MMP transfer proteins in subcutaneous breast cancer transplanted tumors in mice in one embodiment of the present invention;

[0043] Fig.19 This is an immunofluorescence staining image of Brucea javanica exosomes on angiogenesis-related proteins VEGF and CD31 in subcutaneous breast cancer transplanted tumors in mice in one embodiment of the present invention. DETAILED DESCRIPTION

[0044] The present invention is further illustrated below by way of examples.

[0045] The mouse breast cancer cells (4T1), human breast cancer cells (MCF-7), human umbilical vein endothelial cells (HUVECs), and human normal liver cells (L02) used in this example were cultured in complete culture medium containing 10% fetal bovine serum, penicillin (50 U / mL), and streptomycin (50 U / mL) at 37°C and 5% CO2.

[0046] Example 1 Extraction of Brucea javanica exosomes by ultracentrifugation and purification using membrane filter

[0047] Take fresh Brucea javanica fruit, wash it with deionized water, and squeeze the juice in a juicer with PBS buffer. After filtering with a strainer to remove the residue, centrifuge the juice in a desktop refrigerated centrifuge at 4°C 1000×g for 10 min, 4000×g for 20 min, and 10000×g for 60 min to remove large particles and cell debris. Then centrifuge the supernatant in a floor-standing ultracentrifuge at 4°C 150000×g for 120 min, take the bottom precipitate, resuspend it with an appropriate amount of PBS, and filter it through a 0.22μm membrane filter to obtain Brucea javanica exosomes. Use a Malvern particle size analyzer to measure the particle size and potential of the exosomes, use a transmission electron microscope to photograph the morphology of the exosomes, and use a BCA kit to quantify the protein concentration of the exosomes. Figure 1 As shown in Figure 1, the particle size of the exosomes obtained from the leaves of Brucea javanica was 131.76 nm. Figure 2 As shown, the exosomes of Brucea javanica cotyledons exhibit a teacup-shaped structure under transmission electron microscopy, which is consistent with the classic structural characteristics of exosomes.

[0048] Example 2 Extraction of Brucea javanica exosomes by ultracentrifugation and purification by sucrose density gradient centrifugation

[0049] Take fresh Brucea javanica fruit, wash it with deionized water, and squeeze the juice in a juicer with PBS buffer. After filtering with a strainer to remove the residue, the juice was centrifuged in a desktop refrigerated centrifuge at 4°C 1000×g for 10min, 4000×g for 20min, and 10000×g for 60min to remove large particles and cell debris. Then the supernatant was centrifuged in a floor-standing ultracentrifuge at 4°C 150000×g for 120min, and the bottom precipitate was resuspended with an appropriate amount of PBS. The resuspended solution was purified by sucrose density gradient centrifugation. The specific purification method is: prepare sucrose density gradient solutions with four different concentrations of 8%, 30%, 45% and 60%, add the resuspended solution to the top of the density gradient solution through a syringe, and then centrifuge it in a floor-standing ultracentrifuge at 4°C 150000×g for 120min, and recover the exosomes enriched between different concentration interfaces. Then, the sucrose solution was removed by centrifugation at 150,000 × g for 60 min at 4°C in a desktop ultracentrifuge, and the bottom precipitate was resuspended with a small amount of PBS to obtain a solution of exosomes from brucea javanica leaves with different bands. Figure 3 The particle size distribution of exosomes from Brucea javanica leaves in different bands is shown.

[0050] Example 3 Characterization of Brucea javanica exosomes and qualitative and quantitative analysis of their contents

[0051] Separation of proteins contained in Brucea javanica exosomes: The proteins contained in Brucea javanica exosomes were separated and extracted with SDT (4% SDS, 100mMTris-HCl, pH7.6) buffer. 20μg of protein was taken from each sample, mixed with 5X loading buffer, boiled for 5min, separated with 4%-20% SDS-PAGE gel (constant pressure 180V, 45min), and stained with Coomassie blue R-250 to display protein bands.

[0052] Isolation and analysis of nucleic acids contained in brucea javanica exosomes: The total RNA of brucea javanica exosomes was isolated and purified using Trizol reagent, and the nucleic acids in brucea javanica exosomes, including mRNA and small RNA (miRNA, lncRNA, circle RNA), were isolated and analyzed through lysis, labeling, amplification, paired-end sequencing, and sequence mapping procedures.

[0053] Separation and analysis of lipids contained in Brucea javanica exosomes: Lipids were extracted using the MTBE method. Briefly, the sample (100 μL) was spiked with internal lipid standards, then separated using reversed-phase liquid chromatography, positive and negative ions were detected using electrospray ionization, and finally the lipid molecules contained in Brucea javanica exosomes and internal standard lipid molecules were peak extracted and identified.

[0054] Example 4 Brucea javanica exosomes specifically inhibit the survival rate of tumor and vascular endothelial cell models

[0055] The proliferation activity of cells was detected using the CCK-8 kit. 4T1, HUVECs, MCF-7, and L02 cells were seeded on 96-well plates at a cell density of 5000 cells / well and incubated overnight in an incubator at 37°C and 5% CO2. After the cells adhered to the wall, the medium was changed and 1, 10, 50, 100, and 200 μg / mL exosome solutions diluted with culture medium were given, respectively. Blank wells and control wells were also set up. After the administration, the cells were incubated in the incubator for 24 hours, and then replaced with 10 μL CCK-8 solution diluted with fresh serum-free culture medium. After continuing to incubate for 1 hour, the absorbance at 450 nm was detected with an enzyme reader. Figure 4 , 5 As shown in Figures 6 and 7, the proliferation rates of 4T1, HUVECs, and MCF-7 cells decreased with the increase in the concentration of Brucea brucea exosomes, indicating that the treatment of Brucea brucea exosomes effectively inhibited the growth and survival of tumor cells and vascular endothelial cells, and had a strong anti-tumor and anti-angiogenesis effect. Brucea brucea exosomes had no obvious toxicity to L02 cells, indicating that they had high biological safety.

[0056] Comparative Example 1: Other plant-derived exosomes specifically inhibit the survival rate of tumor and vascular endothelial cell models

[0057] Similar to the preparation method of Brucea javanica exosomes, the medicinal plants Taxus fruit exosomes, Curcuma zedoaria leaf exosomes, Cephalotaxus chinensis exosomes, Salvia miltiorrhiza exosomes, Astragalus membranaceus exosomes and Bletilla striata exosomes were extracted by ultracentrifugation and purified using a membrane filter, and the same experimental method as in Example 4 was used to detect the effects of different concentrations of exosomes from other plants on the proliferation of 4T1 cells and HUVECs. Figure 8 , 9 As shown, the cell proliferation rate did not show a significant decrease with the increase of exosome administration concentration, indicating that Taxus fruit exosomes, Curcuma leaf exosomes, Cephalotaxus exosomes, Salvia miltiorrhiza exosomes, Astragalus exosomes and Bletilla striata exosomes did not have similar anti-tumor and anti-angiogenic effects.

[0058] Example 5 Brucea javanica exosomes promote apoptosis of tumor cells related to the ROS / Caspase pathway

[0059] Take a 6-well plate and plate 4T1 cells at a concentration of 2 × 10 5 / well were inoculated in a 6-well plate, and the amount of cell suspension added was 2mL per well. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for routine culture overnight. The next day, the original culture medium was discarded, and 10 and 50μg / mL BF-Exos culture medium were added to each well of the treatment group, respectively, and continued to be placed in the incubator for 24h. The cell apoptosis kit and Western blot experiment were used to perform qualitative and quantitative analysis of cells in different groups, and Image J 1.8.0 image processing software was used to analyze the optical density values ​​of the bands to determine the effect of Brucea javanica exosomes on the level of tumor cell apoptosis. The results are shown in Fig.10 , 11 The results showed that the proportion of apoptotic cells in the Brucea javanica exosome group was higher, and the protein expression level of Cleaved Caspase3 / 9 in the cells was significantly higher than that in the control group.

[0060] Example 6 Brucea javanica exosomes inhibit the expression of PI3K / Akt / mTOR pathway proteins in tumor cells

[0061] Take a 6-well plate and plate 4T1 cells at a concentration of 2 × 10 5 / well were inoculated into 6-well plates, and the amount of cell suspension added was 2mL per well. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for routine culture overnight. The next day, the original culture medium was discarded, and 10 and 50μg / mL BF-Exos culture medium were added to each well of the treatment group, respectively, and the plates were placed in the incubator for another 24h. Western blot experiments were used to perform qualitative and quantitative analysis of cells in different groups, and Image J 1.8.0 image processing software was used to analyze the optical density values ​​of the bands to determine the effect of Brucea javanica exosomes on the expression of PI3K / Akt / mTOR pathway proteins in tumor cells. Fig.12 As shown, the expression of key phosphorylated proteins p-PI3K, p-Akt and p-mTOR in the cellular pathway was significantly downregulated in the Brucea javanica exosome group, indicating that this pathway was inhibited.

[0062] Example 7 Brucea javanica exosomes inhibit the secretion of VEGF in tumor cells and vascular endothelial cells

[0063] 4T1 and HUVECs cells were cultured at 5×10 4 Cells were seeded at a density of 100 cells / well on a 24-well plate and incubated overnight in a 37°C, 5% CO2 incubator. After the cells adhered to the plate, the medium was changed and 1, 10, and 50 μg / mL BF-Exos solution diluted with culture medium was given. After 24 hours, the supernatant culture medium was collected and the VEGF content was analyzed using an ELISA kit. Fig.13 , 14 As shown, Brucea javanica exosomes can significantly inhibit the secretion of the key angiogenic factor VEGF by tumor cells and vascular endothelial cells.

[0064] Example 8 Brucea javanica exosomes inhibit HUVECs migration and tube formation

[0065] Take a 24-well plate and plate HUVECs at 1×10 5 / well were inoculated in a 24-well plate, and the amount of cell suspension added was 1 mL per well. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for routine culture overnight. The next day, the original culture medium was discarded, and a sterile 200μ gun tip was used to quickly draw a longitudinal line in the cell layer to form a scratch of uniform width to establish a cultured cell wound model. Serum-free culture medium containing 5 and 10μg / mL BF-Exos was added to each well, and the width of the wound at 0 o'clock after the scratch was observed and photographed under an optical microscope. Samples were taken and photographed at 6, 12, and 24 hours, and the wound healing of each group was observed and measured by relevant drawing software.

[0066] Take a 48-well plate, add 80 μL of Matrigel gel to each well and place it in a cell culture incubator for 30 minutes until it solidifies. 4 / well were inoculated in a 48-well plate, and the amount of cell suspension added was 500μL per well. A blank culture medium group and a 10μg / mL Brucea javanica exosome group were set up. At 3, 6, and 9h time points, tube formation photos were taken under 4X and 10X objective lenses, and the tube length and node number were determined by relevant drawing software.

[0067] like Fig.15 , 16 As shown in the data, in the blank group, the scratch wound of HUVECs tended to heal over time, and a three-dimensional network structure could be formed in 9 hours under the action of Matrigel. However, the exosomes of Brucea javanica could inhibit the migration of HUVECs and destroy the tube formation at a lower concentration, indicating that the exosomes of Brucea javanica could damage the function of vascular endothelial cells in vitro and play a role in inhibiting angiogenesis.

[0068] Example 9 Evaluation of the anti-tumor and anti-angiogenic effects of Brucea javanica exosomes on subcutaneous breast cancer mouse model

[0069] The experimental animals were 6-8 week old female BALB / c mice, inoculated in the right axilla with 0.1 mL of 1×10 7 / mL4T1 cell suspension was used to establish a subcutaneous transplantation breast cancer model. When the average tumor volume reached 50-100mm 3 The tumor-bearing mice were randomly divided into two experimental groups, with 6 mice in each group. One group was given PBS every 2 days, and the other group was given 6 mg / kg exosome solution. The weight and tumor volume of the mice were monitored every other day. The mice were killed on the 14th day, and the tumors in each group were collected. The results are shown in Figure 2. Fig.17As shown in the figure, the tumor in the control group rapidly increased in size within 12 days, while the exosomes of Brucea javanica significantly delayed the growth trend of the tumor. Western blot experiments were used to determine the expression of PI3K / Akt / mTOR pathway proteins, Caspase apoptosis proteins, and MMP transfer proteins. Immunofluorescence staining was used to analyze the expression of angiogenesis-related proteins VEGF and CD31. Image J 1.8.0 was used for image processing. Fig.18 , 19 As shown, compared with the control group, the expression of Caspase3 / 9 in the Brucea javanica exosome group was higher, and the expressions of p-PI3K, p-Akt, p-mTOR, MMP2 / 9, VEGF and CD31 were lower, confirming that Brucea javanica exosomes can intervene in the PI3K / Akt / mTOR pathway in vivo to achieve tumor treatment and achieve the function of inhibiting breast cancer growth, metastasis and angiogenesis.

[0070] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. The use of Brucea javanica exosomes in the preparation of anti-breast cancer drugs, characterized in that: The Brucea javanica exosomes are prepared by the following method: 1) Mix fresh Brucea javanica fruit with phosphate buffer and squeeze the juice for 5-15 times, each time for 15-60 s, to obtain a slurry containing Brucea javanica exosomes; 2) filtering out the residue in the slurry obtained in step 1) and collecting the juice, and obtaining a precipitate containing Brucea javanica exosomes by differential centrifugation of the juice; 3) The precipitate obtained in step 2) is resuspended in PBS and purified to obtain the Brucea javanica exosomes.

2. The use according to claim 1, characterized in that: In the step 2), the differential centrifugation method is: centrifuging the filtered juice at 1000 × g for 5-20 min, 4000 × g for 20-60 min, and 10000 × g for 60-120 min at 4°C to obtain a supernatant; The supernatant was then centrifuged at 4°C, 100,000× g-150,000× g for 60-150 min to obtain a precipitate containing Brucea javanica exosomes.

3. The use according to claim 1, characterized in that: In step 3), the purification is performed by filtering through a 0.22 μm membrane filter.

4. The use according to claim 1, characterized in that: In step 3), the purification is performed by sucrose density gradient centrifugation.

5. The use according to claim 1, characterized in that The brucea javanica exosomes contain active substances, and the active substances are derived from one or more of proteins, lipids, mRNA, and small RNA contained in the brucea javanica exosomes; the small RNA is one or more of miRNA, lncRNA, and circle RNA.

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