Preparation method and application of baikal skullcap root exosome

Abstract

The invention discloses a preparation method and application of scutellaria baicalensis exosomes, and relates to the technical field of Chinese herbal medicine, and the preparation method comprises the following steps: juicing fresh scutellaria baicalensis, filtering, collecting filtrate, carrying out primary centrifugal impurity removal, transferring the collected precipitate into a sucrose gradient solution after impurity removal, and carrying out secondary centrifugation, and finally, collecting the exosome in the solution in the set sucrose concentration interval, and carrying out third centrifugation to obtain the scutellaria baicalensis exosome. The baikal skullcap root exosome provided by the invention can play a role in resisting colon cancer by regulating and controlling the expression quantity of PD-L1, and the baikal skullcap root exosome is derived from plants, relatively low in production cost and has the advantages of relatively good stability and easiness in large-scale production, so that the baikal skullcap root exosome has relatively low treatment cost and provides a more economic choice for treatment of colon cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of Chinese herbal medicines, and more particularly, to a method for preparing Scutellaria baicalensis exosomes and their applications. Background Art

[0002] Colorectal cancer is one of the malignant tumors with relatively high incidence and mortality rates globally, seriously threatening human health. Although modern medicine has made remarkable progress in early diagnosis and treatment regimens, the treatment of patients with advanced or metastatic colorectal cancer still faces huge challenges, and the treatment effects and prognoses are not ideal. During the occurrence and development of tumors, the immune escape mechanism plays a key role, among which the immune suppression pathway mediated by programmed death ligand 1 (PD-L1) is particularly important. As an immune checkpoint molecule expressed on the surface of tumor cells, PD-L1 can specifically bind to programmed death receptor 1 (PD-1) on the surface of T cells, inhibiting the activation and proliferation of T cells, thereby helping tumor cells escape the recognition and clearance of the immune system. A large number of clinical studies have shown that the overexpression of PD-L1 is closely related to immune escape, disease progression, and poor prognosis of various malignant tumors, making it an important target for tumor immunotherapy.

[0003] In the field of traditional Chinese medicine, the flavonoids contained in Scutellaria baicalensis have been proven to have significant anti-inflammatory, antioxidant, and anti-tumor activities. Recent studies have further found that Scutellaria baicalensis extracts can not only directly inhibit the proliferation of various tumor cells but also improve the tumor microenvironment by regulating the functions of immune cells. Meanwhile, exosomes, as important mediators of intercellular communication, play a key role in tumor immune regulation and are involved in regulating the activation and functions of immune cells. Exosomes derived from plants have become a research hotspot due to their unique immune regulatory properties, and there is evidence indicating that they can enhance the anti-tumor immune response of the body. However, there is currently no report on whether Scutellaria baicalensis exosomes can affect tumor immune escape by regulating PD-L1 expression, and the exploration of this scientific issue has important theoretical and application values.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a Scutellaria baicalensis exosome that can play an anti-colorectal cancer role by regulating PD-L1 expression. The Scutellaria baicalensis exosome is derived from plants, has a relatively low production cost, and has the advantages of good stability and being easy to produce on a large scale. Therefore, it has a relatively low treatment cost and provides a more economical option for the treatment of colorectal cancer.

[0006] The present invention is realized as follows:

[0007] The present invention provides a method for preparing Scutellaria baicalensis Georgi exosomes, comprising the following steps: juicing fresh Scutellaria baicalensis Georgi, filtering to collect the filtrate, performing a first centrifugation for impurity removal, transferring the collected precipitate to a sucrose gradient solution after the impurity removal is completed for a second centrifugation, and finally collecting the exosomes in a solution within a set sucrose concentration range, and obtaining Scutellaria baicalensis Georgi exosomes after a third centrifugation.

[0008] In some preferred embodiments, the first centrifugation for impurity removal is to centrifuge the filtrate at 2 - 6 °C at 500 - 1000×g for 20 - 45 min, at 2500 - 4000×g for 30 min - 1 h, and at 8000 - 12000×g for 1 - 2 h, then collect the supernatant and centrifuge it again at 150000 - 250000×g for 1 - 2 h and resuspend the precipitate.

[0009] In some preferred embodiments, the sucrose gradient solution is a continuous gradient solution with a sucrose concentration of 15% - 60%.

[0010] In some preferred embodiments, the second centrifugation is to ultra - centrifuge the sucrose solution at 2 - 6 °C at 150000 - 250000×g for 1 - 2 h.

[0011] In some preferred embodiments, the set sucrose concentration range is a sucrose concentration range of 30% - 45%.

[0012] In some preferred embodiments, the third centrifugation is to ultra - centrifuge the collected exosomes at 150000 - 250000×g for 1 - 2 h.

[0013] In some preferred embodiments, the nano - diameter of the Scutellaria baicalensis Georgi exosomes is 50 - 80 nm.

[0014] The present invention also provides an application of Scutellaria baicalensis Georgi exosomes in anti - cancer drugs.

[0015] In some preferred embodiments, the anti - cancer drug is a drug that inhibits cancer cell proliferation and / or metastasis.

[0016] In some preferred embodiments, the anti - cancer drug is a drug that inhibits the expression of PD - L1.

[0017] The present invention has the following beneficial effects:

[0018] The present invention firstly proposes that Scutellaria baicalensis exosomes can play an anti - colon cancer role by regulating the expression of PD - L1. As exosomes derived from natural plants, they have good biocompatibility and low cytotoxicity, can effectively regulate the immune system, and inhibit immune escape. By regulating the expression of PD - L1, they inhibit the immune escape of cancer cells, showing the therapeutic advantages of natural sources, with potential low toxicity and relatively wide adaptability. Scutellaria baicalensis exosomes are derived from plants, with relatively low production costs, and have the advantages of good stability and easy large - scale production. Therefore, they have lower treatment costs, providing a more economical option for the treatment of colon cancer. Brief Description of the Drawings

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0020] Figure 1 It is a diagram showing the sucrose density gradient centrifugation of Scutellaria baicalensis exosomes in the embodiments of this application;

[0021] Figure 2 It is a transmission electron microscopy (TEM) result diagram of Scutellaria baicalensis exosomes in the embodiments of this application;

[0022] Figure 3 It is a nanoparticle tracking analysis (NTA) result diagram of Scutellaria baicalensis exosomes in the embodiments of this application;

[0023] Figure 4 It is the protein concentration quantification of Scutellaria baicalensis exosomes in the embodiments of this application;

[0024] Figure 5 It is the protein molecular weight distribution diagram of Scutellaria baicalensis exosomes in the embodiments of this application;

[0025] Figure 6 It is a liquid chromatography detection result diagram of Scutellaria baicalensis exosomes in the embodiments of this application;

[0026] Figure 7 It is a result diagram showing the inhibition of the viability of colon cancer cells by Scutellaria baicalensis exosomes in the embodiments of this application;

[0027] Figure 8 It is an anti - inflammatory effect diagram of Scutellaria baicalensis exosomes on macrophages in the embodiments of this application;

[0028] Figure 9 It is the regulatory effect of Scutellaria baicalensis exosomes on the expression of PD - L1 protein in colon cancer cells in the embodiments of this application. Detailed Embodiments

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0030] A method for preparing Scutellaria baicalensis Georgi exosomes and its application proposed in the present application will be specifically described below.

[0031] The inventors innovatively discovered that Scutellaria baicalensis Georgi exosomes do not contain the two key components, baicalein and baicalin, which are traditionally considered to have anti-cancer activity, but still have good performance in anti-tumor aspects. Through systematic in vitro and in vivo experiments, the inventors found that Scutellaria baicalensis Georgi exosomes can significantly down-regulate the expression level of PD-L1 protein on the surface of tumor cells. As an immune checkpoint protein, the overexpression of PD-L1 will lead to the inhibition of T cell function and is an important mechanism for tumor immune escape. By regulating this key immune checkpoint, Scutellaria baicalensis Georgi exosomes can effectively relieve the inhibition of the tumor on the immune system and restore the killing function of T cells against tumors. This discovery provides a new idea for cancer immunotherapy. Compared with traditional small molecule drugs or antibody drugs, Scutellaria baicalensis Georgi exosomes have the advantages of simple preparation process, low cost, and small toxic and side effects, and are expected to become an affordable new option for cancer treatment. More importantly, this natural exosome-based treatment strategy avoids the complexity of genetic engineering modification and has better clinical translation potential, opening up an innovative path for the development of new tumor immunotherapy drugs.

[0032] A method for preparing Scutellaria baicalensis Georgi exosomes includes the following steps: juicing fresh Scutellaria baicalensis Georgi, filtering to collect the filtrate, then performing the first centrifugation for impurity removal, transferring the collected precipitate to a sucrose gradient solution after the impurity removal is completed for the second centrifugation, and finally collecting exosomes in a solution within a set sucrose concentration range, and obtaining Scutellaria baicalensis Georgi exosomes after the third centrifugation.

[0033] In some preferred embodiments, the first centrifugation for impurity removal is to centrifuge the filtrate at 2-6°C at 500-1000×g for 20-45 min, 2500-4000×g for 30 min-1 h, and 8000-12000×g for 1-2 h, then collect the supernatant and centrifuge it again at 150000-250000×g for 1-2 h and resuspend the precipitate.

[0034] Among them, the preparation of Scutellaria baicalensis Georgi exosomes by gradient centrifugation not only improves the purity of exosomes, but also ensures their biological activity and the reliability of subsequent experiments. First, initial low-speed centrifugation can limitedly remove cell debris and large particle impurities. Then, medium-speed centrifugation can further remove smaller organelles and some apoptotic bodies. Subsequently, high-speed centrifugation can remove larger vesicle structures such as microvesicles. This strategy of gradually increasing the centrifugal force can maximize the retention of target exosomes while systematically removing impurities of different particle sizes. Finally, ultra-high-speed centrifugation can efficiently precipitate exosomes, while impurities such as soluble proteins are retained in the supernatant and removed. The whole process is carried out in a low-temperature environment of 2-6 °C, which can effectively maintain the integrity and biological activity of the exosome membrane structure.

[0035] In some preferred embodiments, the sucrose gradient solution is a continuous gradient solution with a sucrose concentration of 15%-60%. In the embodiments of the present application, it is set as a continuous gradient of four sucrose concentrations of 15%, 30%, 45%, and 60% to facilitate observing the sucrose concentration range in which the final Scutellaria baicalensis Georgi exosomes are distributed.

[0036] In some preferred embodiments, the second centrifugation is to ultracentrifuge the sucrose solution at 150000-250000×g for 1-2 h at 2-6 °C. During the purification process of Scutellaria baicalensis Georgi exosomes, the second ultracentrifugation combined with sucrose density gradient centrifugation can achieve high-resolution separation based on the buoyant density of exosomes, effectively removing impurities such as residual protein aggregates, lipoprotein complexes, and other non-exosome vesicles. Sucrose gradient centrifugation uses the selective sedimentation of different density zones to enrich exosomes in a specific density range, thereby significantly improving the sample purity. The low-temperature environment can not only maintain the stability of the exosome membrane structure, but also inhibit protease activity and prevent the degradation of exosome proteins.

[0037] Among them, the specific density range in which Scutellaria baicalensis Georgi exosomes are concentrated is the sucrose concentration range of 30%-45%, preferably the sucrose concentration range of 30%-40%.

[0038] In some preferred embodiments, the nano-diameter of the Scutellaria baicalensis Georgi exosomes is 50-80 nm.

[0039] The present application also provides an application of Scutellaria baicalensis Georgi exosomes in anti-cancer drugs.

[0040] In some preferred embodiments, the drug can be applied to inhibit cancer cell proliferation and / or metastasis and inhibit the expression of PD-L1.

[0041] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0042] Example 1

[0043] This example provides a method for preparing Scutellaria baicalensis Georgi exosomes, and the steps include:

[0044] After washing the fresh Scutellaria baicalensis Georgi purchased from Weifang, Shandong, it was ground into Scutellaria baicalensis Georgi juice using a juicer, and then filtered. Subsequently, the filtered Scutellaria baicalensis Georgi juice was centrifuged at 1000×g for 30 min, 3000×g for 30 min, and 10000×g for 60 min at 4°C respectively to remove cell debris and large particulate matter. The obtained supernatant was then ultracentrifuged, and the precipitate was resuspended in PBS after centrifugation at 200000×g for 1.5 h. Then the precipitate was transferred to sucrose density gradient solutions with different concentrations (15%, 30%, 45% and 60%) and centrifuged at 200000×g for 1.5 h below 4°C. The centrifugation results are as Figure 1 shown. The layer of Scutellaria baicalensis Georgi exosomes is between 30-40% sucrose concentration, and yellow-green suspended exosomes can be seen. Finally, exosomes were collected from the 30%-40% sucrose junction and the excess sucrose was removed by ultracentrifugation at 200000×g for 1 h, namely, Scutellaria baicalensis Georgi exosomes were prepared.

[0045] Characterization and detection of Scutellaria baicalensis Georgi exosomes:

[0046] Take out 10 μL of exosomes. Pipette 10 m of the sample and drop it on the copper grid for 1 min, and then suck off the floating liquid with filter paper. Pipette 10 μL of uranyl acetate and drop it on the copper grid for 1 min, and then suck off the floating liquid with filter paper. Dry it at room temperature for several minutes, and then perform electron microscopy detection and imaging at 100 kv. The transmission electron microscopy imaging results are as Figure 2 shown. Scutellaria baicalensis Georgi exosomes are round or oval, with a typical bilayer membrane structure, and the appearance of the membrane is smooth or slightly wrinkled.

[0047] Nanoparticle tracking analysis (NTA) was adopted: The exosomes were diluted to an appropriate concentration with PBS, and the NanoSight NS300 was used to detect the particle size distribution to ensure the main distribution range. The results are as Figure 3 shown. The nanometer diameter of Scutellaria baicalensis Georgi exosomes is between 50-80 nm, and the concentration of exosomes is 8 to the 10th power / mL.

[0048] Detection of exosome protein concentration: The results are as Figure 4 shown. According to the results of BCA protein quantification, the protein concentration of Scutellaria baicalensis Georgi exosomes is about 3 mg / mL; Western blot was used to detect exosome proteins: Take 40 μg of exosome protein samples, mix them with 4×Loading buffer and denature at 95°C for 5 min, and separate them by 10% SDS-PAGE gel electrophoresis. The - results are as Figure 5 shown. According to the results of agarose gel electrophoresis, the molecular weight of Scutellaria baicalensis Georgi exosome proteins is about 55-70 kDa.

[0049] Example 2

[0050] First, the Scutellaria baicalensis exosomes prepared in Example 1 were ultrasonically disrupted with methanol and then centrifuged to obtain the supernatant, which was filtered through a 0.22 μm filter membrane and reserved. Chromatographic separation was performed using an ACQUITY UPLC HSS T3 chromatographic column (1.8 μm, 2.1×100 mm), and the column temperature was maintained at 35 °C. The mobile phase consisted of 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B), and a gradient elution program was adopted: within 0 - 30 min, the proportion of phase B increased linearly from 40% to 100%, the flow rate was kept constant at 0.3 mL / min, and the detection wavelength was 275 nm. Baicalein and baicalin standard products were used as comparison samples and tested under the same chromatographic separation conditions. The results are as Figure 6 shown. Small molecule compounds in Scutellaria baicalensis exosomes were detected by UPLC. By comparing the retention times and ultraviolet spectral characteristics of baicalein and baicalin standard products, the analysis results showed that the chromatographic peaks of these two marker components were not detected in the Scutellaria baicalensis exosome samples.

[0051] Example 3

[0052] Colon cancer HT-29 (P:5) and HCT-116 (P:5) cells were placed in (McCoy's 5A + 10% fetal bovine serum + 1% penicillin-streptomycin double antibody solution medium) for culture. When the colon cancer HT-29 and HCT-116 cells grew to 80%, the cells were digested, centrifuged and resuspended, and counted (take 50 μL, add 5 μL trypan blue and mix well; cell suspension: trypan blue = 10:1).

[0053] The Scutellaria baicalensis exosomes prepared in Example 1 were used, and 7 drug concentrations (6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 400 μg / mL) were set, with 3 replicates for each concentration, and the sample addition volume was 100 μL;

[0054] Then, colon cancer HT-29 and HCT-116 cells were seeded in a 96-well plate, with the number of seeded cells being 5000 cells / well and the volume of cell suspension being 100 μL / well; 100 μL of the Scutellaria baicalensis exosome culture medium with the corresponding concentration was added to the 96-well plate, and the drug addition time was recorded (aspirate the old PBS in the top row and add an equal volume of drug-containing culture medium as the blank well to eliminate the influence of drug color). Observe whether the seeding is uniform under the microscope and then place it in the incubator.

[0055] Finally, the supernatant was aspirated and replaced with 100 μL of new medium, and 10 μL of CCK-8 was added in the dark, 10 μL per well. The detection results are as Figure 7 shown. The CCK-8 results showed that the IC50 of HT-29 was 116 μg / mL, and the IC50 of HCT-116 was 94.97 μg / mL; the LDH results showed that Scutellaria baicalensis exosomes were basically non-toxic to HT-29 and HCT-116 cells.

[0056] Example 4

[0057] An LPS-induced macrophage inflammation model was established. RAW264.7 (P:3) or primary macrophages were selected and stimulated with LPS (such as 1 μg / mL) for 24 h to induce an inflammatory response. Then, the exosomes of Scutellaria baicalensis were used for intervention experiments. Concentration gradients of the exosomes of Scutellaria baicalensis (6.25, 12.5, 25, 50, 100, and 200 μg / mL) were set, and the cells were co-treated with LPS for 24 h. Meanwhile, an LPS group and a blank control group were set. Then, the NO release was detected by the Griess method. The cell supernatant was collected, reacted with Griess reagent, and the absorbance at 540 nm was measured. The NO concentration was calculated by comparing the standard curve.

[0058] The results are as Figure 8 shown. Data analysis showed that the exosomes of Scutellaria baicalensis significantly inhibited NO release at >12.5 μg / mL (p<0.05), confirming its anti-inflammatory effect. It was thus speculated that the exosomes of Scutellaria baicalensis could change the tumor microenvironment, inhibit tumor growth and metastasis by regulating macrophage polarization, transforming it from the tumor-promoting M2 type to the anti-tumor M1 type. In addition, the anti-inflammatory effect may also reduce inflammation-mediated tumor cell immune escape and enhance the ability of the body's immune system to recognize and eliminate tumor cells.

[0059] Example 5

[0060] First, the colon cancer cell lines HT-29 and HCT-116 were cultured in McCoy's 5A medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution in a cell culture incubator at 37 °C and 5% CO₂ until the cells grew to 80% confluence. Then the cells were divided into three groups: a control group (Con), a low-dose treatment group (SDE-L), and a high-dose treatment group (SDE-H). After the cells were cultured to 80% confluence, the corresponding concentrations of the exosomes of Scutellaria baicalensis were added to the SDE-L and SDE-H groups, while the control group was added with an equal amount of medium, and the cells were cultured for another 24 h.

[0061] After the treatment, total proteins were extracted from the cells of each group, and the protein concentration was quantified using a BCA protein quantification kit. Subsequently, SDS-PAGE gel electrophoresis was performed. The protein samples were loaded onto the gel at a certain ratio and separated by electrophoresis. After electrophoresis was completed, the proteins were transferred onto a PVDF or nitrocellulose membrane. Then, primary antibody incubation was performed using anti-PD-L1 and anti-GAPDH antibodies, and then secondary antibody incubation was performed using the corresponding secondary antibodies. Finally, chemiluminescent substrate was used for development, and the image was captured using a gel imaging system.

[0062] The results are as Figure 9As shown, Western blot analysis revealed that in the HT-29 and HCT-116 cell lines, compared with the control group (Con), the protein expression levels of PD-L1 in the Scutellaria baicalensis exosome treatment groups (SDE-L and SDE-H) decreased, especially in the high-dose treatment group (SDE-H), where the expression of PD-L1 was significantly reduced. This indicates that Scutellaria baicalensis exosomes can inhibit the expression of PD-L1 in colon cancer cells, and this inhibitory effect shows a dose-dependent manner.

[0063] Further quantitative analysis showed that compared with the control group, the PD-L1 expression level slightly decreased in the low-dose Scutellaria baicalensis exosome (SDE-L) treatment group, but the difference was not significant. In the high-dose Scutellaria baicalensis exosome (SDE-H) treatment group, the PD-L1 expression level significantly decreased and was statistically significant compared with the control group. Scutellaria baicalensis exosomes can significantly inhibit the expression of PD-L1 at a high dose (100 μg / mL).

[0064] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of Scutellaria baicalensis Georgi exosomes, characterized in that, It includes the following steps: Juice fresh Scutellaria baicalensis, filter it, collect the filtrate, conduct the first centrifugation for impurity removal, transfer the collected precipitate to a sucrose gradient solution after the impurity removal is completed for the second centrifugation, and finally collect exosomes in a solution within the set sucrose concentration range, and obtain Scutellaria baicalensis exosomes after the third centrifugation.

2. The preparation method of a kind of Scutellaria baicalensis Georgi exosome according to claim 1, characterized in that, For the first centrifugation for impurity removal, the filtrate is centrifuged at 2 - 6°C at 500 - 1000×g for 20 - 45 min, 2500 - 4000×g for 30 min - 1 h, and 8000 - 12000×g for 1 - 2 h, then the supernatant is collected and centrifuged again at 150000 - 250000×g for 1 - 2 h, and the precipitate is resuspended.

3. The preparation method of a kind of Scutellaria baicalensis Georgi exosomes according to claim 1, characterized in that, The sucrose gradient solution is a continuous gradient solution with a sucrose concentration of 15% - 60%.

4. The preparation method of a kind of Scutellaria baicalensis Georgi exosome according to claim 1, characterized in that, For the second centrifugation, the sucrose solution is ultracentrifuged at 2 - 6°C at 150000 - 250000×g for 1 - 2 h.

5. The preparation method of a kind of Scutellaria baicalensis Georgi exosome according to claim 1, wherein, The set sucrose concentration range is the sucrose concentration range of 30% - 45%.

6. The preparation method of a kind of Scutellaria baicalensis Georgi exosome according to claim 1, characterized in that, For the third centrifugation, the collected exosomes are ultracentrifuged at 150000 - 250000×g for 1 - 2 h.

7. The preparation method of a kind of Scutellaria baicalensis Georgi exosome according to claim 1, wherein, The nano - diameter of the Scutellaria baicalensis exosomes is 50 - 80 nm.

8. Application of Scutellaria baicalensis exosomes obtained by the preparation method according to any one of claims 1 - 5 in anti - cancer drugs.

9. Use of a Scutellaria baicalensis exosome according to claim 8 in an anticancer drug, characterized in that, The anti - cancer drug is a drug that inhibits the proliferation and / or metastasis of cancer cells.

10. The application of a Scutellaria baicalensis Georgi exosome in an anti-cancer drug according to claim 8, wherein, The anti - cancer drug is a drug that inhibits the expression of PD - L1.

Images