Preparation method of polygonatum-sibiricum-sourced exosome-like nano vesicles and application of nano vesicles in antitumor drugs

Abstract

The invention discloses a preparation method of polygonatum-sibiricum-sourced exosome-like nano-vesicles and application of the nano-vesicles in antitumor drugs, and relates to the technical field of biological medicines. The preparation method comprises the following steps: crushing fresh rhizoma polygonati slices, filtering and collecting to obtain a first filtrate; carrying out gradient centrifugation treatment on the first filtrate to obtain supernate; carrying out first membrane filtration treatment on the supernate to obtain second filtrate with the particle size of less than 450 nm; centrifuging the second filtrate to obtain a precipitate, and resuspending the precipitate with a PBS buffer solution to obtain a rhizoma polygonati exosome crude extract; and performing secondary membrane filtration treatment on the rhizoma polygonati exosome crude extract to obtain a third filtrate with the particle size of less than 220nm, thereby obtaining the rhizoma polygonati-derived exosome-like nano-vesicles. The polygonatum-sibiricum-sourced exosome-like nano-vesicle has remarkable inhibitory activity on tumors, provides a new strategy and means for tumor treatment, and has important scientific value and clinical application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing exosome-like nanovesicles derived from polygonatum and an application thereof in anti-tumor drugs. Background Art

[0002] In the field of biomedical research, cancer has always been a major problem that needs to be solved urgently, and it seriously threatens human health and life. Surgery and radiotherapy and chemotherapy are commonly used cancer treatment methods in clinical practice, but each has its own limitations. For example, surgical treatment is mostly used in the early stages of cancer and has poor effects on patients in the late stages. Radiotherapy and chemotherapy are often accompanied by serious adverse reactions, and patients are prone to drug resistance.

[0003] Plant-derived exosome-like nanovesicles (PELNs) have great potential in drug development because they are naturally rich in bioactive ingredients, including proteins, lipids, RNA and small molecules. PELNs not only have a natural nanoscale structure that is easily taken up by cells, but also have the pharmacological activity of the plant itself and a wide range of sources, providing a sufficient material basis for clinical application, and showing great potential in anti-inflammatory, anti-viral, anti-fibrosis, regulating intestinal flora and anti-tumor.

[0004] Polygonatum sibiricum belongs to the genus Polygonatum, Liliaceae, Liliales. As a typical plant with medicinal and edible homology, it has a long history of application. At present, the research on Polygonatum sibiricum-derived exosome-like nanoparticles (PSELNs) is still in its infancy. It is of great significance to develop an extraction method for PSELNs derived from Polygonatum sibiricum, clarify its specific components, and deeply explore its pharmacological activity. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing exosome-like nanovesicles derived from polygonatum and its application in anti-tumor drugs to solve the problems existing in the above-mentioned prior art. The exosome-like nanovesicles derived from polygonatum have significant inhibitory activity on tumors (such as lung cancer and colorectal cancer).

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing exosome-like nanovesicles derived from polygonatum, comprising the following steps:

[0008] After crushing the fresh polygonatum, filtering and collecting to obtain a first filtrate;

[0009] The supernatant is obtained after subjecting the first filtrate to gradient centrifugation;

[0010] The first membrane filtration treatment is performed on the supernatant to obtain a second filtrate with a particle size less than 450 nm;

[0011] Centrifugation is performed on the second filtrate to obtain a precipitate, which is resuspended with PBS buffer to obtain a crude extract of polygonatum sibiricum exosomes;

[0012] The second membrane filtration treatment is performed on the crude extract of polygonatum sibiricum exosomes to obtain a third filtrate with a particle size less than 220 nm, and the polygonatum sibiricum-derived exosome-like nanovesicles are obtained.

[0013] Furthermore, the gradient centrifugation treatment is centrifugation at 4000 rpm, 10000 rpm, and 12000 rpm for 90 min respectively.

[0014] Furthermore, the centrifugation of the second filtrate is carried out at 100000 g for 2 h.

[0015] The present invention also provides polygonatum sibiricum-derived exosome-like nanovesicles prepared by the above preparation method.

[0016] The present invention also provides the application of the above-mentioned polygonatum sibiricum-derived exosome-like nanovesicles in the preparation of anti-tumor drugs.

[0017] Furthermore, the tumor is lung cancer.

[0018] Furthermore, the tumor is colorectal cancer.

[0019] The present invention also provides an anti-tumor drug, the active ingredient of which includes the above-mentioned polygonatum sibiricum-derived exosome-like nanovesicles.

[0020] Furthermore, the tumor is lung cancer or colorectal cancer.

[0021] Furthermore, the anti-tumor drug further includes pharmaceutically acceptable excipients.

[0022] The present invention discloses the following technical effects:

[0023] The present invention develops a preparation method of polygonatum sibiricum-derived exosome-like nanovesicles, and analyzes the components of polygonatum sibiricum-derived exosome-like nanovesicles through metabolomics and proteomics, and obtains the specific metabolic components and protein composition of polygonatum sibiricum-derived exosome-like nanovesicles, which is of great significance for further exploring its pharmacological activity.

[0024] The pharmacological activities of exosome-like nanovesicles derived from Polygonatum sibiricum were studied in this invention. The results showed that they had significant inhibitory activities against tumors (such as lung cancer and colorectal cancer), providing new strategies and means for tumor treatment and having important scientific value and clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 It is the transmission electron microscope observation image of PSELNs; the scale bar is 100 nm;

[0027] Figure 2 It is the Zeta potential detection image of PSELNs;

[0028] Figure 3 It is the particle size detection image of PSELNs;

[0029] Figure 4 It is the classification diagram of metabolites of PSELNs;

[0030] Figure 5 It is the detection result diagram of the cell viability of A549 cells under the action of different concentrations of PSELNs (N = 6, ***: P < 0.001);

[0031] Figure 6 It is for the IC 50 detection result image of PSELNs on A549 cells;

[0032] Figure 7 It is the detection result diagram of the cell viability of PC-9 cells under the action of different concentrations of PSELNs (N = 6, ***: P < 0.001);

[0033] Figure 8 It is for the IC 50 detection result image of PSELNs on PC-9 cells;

[0034] Figure 9 It is the detection result diagram of the cell viability of HCT-15 cells under the action of different concentrations of PSELNs (N = 6, ***: P < 0.001);

[0035] Figure 10 It is for the IC 50 detection result image of PSELNs on HCT-15 cells. DETAILED DESCRIPTION OF THE INVENTION

[0036] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms used in the present invention are merely for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0040] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] I. Experimental Method

[0043] 1. Extraction of PSELNs

[0044] (1) After slicing fresh polygonatum sibiricum that has been washed clean, add PBS to extract juice, and collect the first filtrate through filtration with double-layer gauze.

[0045] (2) Continuously centrifuge the first filtrate obtained in step (1) at 4 °C, centrifuge at 4000 rpm, 10000 rpm, and 12000 rpm for 90 min respectively; discard the precipitate and take the supernatant.

[0046] (3) Filter the supernatant obtained in step (2) through a 450 nm microporous filter membrane to obtain a second filtrate.

[0047] (4) The second filtrate obtained in step (3) was centrifuged in an ultrahigh-speed refrigerated centrifuge at 4°C, 100,000 g for 2 h, the supernatant was discarded, and the precipitate was resuspended in 1 mL of PBS buffer to obtain a crude extract of Polygonatum sibiricum exosomes.

[0048] (5) Add the crude extract of Polygonatum sibiricum exosomes obtained in step (4) into a syringe pump, filter with a 220 nm microporous filter membrane, set the syringe pump flow rate to 100 μL / min, and filter to obtain a third filtrate, which is the purified PSELNs.

[0049] 2. Characterization of PSELNs

[0050] (1) Morphological observation

[0051] The extracted PSELNs were fixed on a copper grid, and after standing for 5 min, the copper grid was taken out and photographed using a transmission electron microscope (TEM).

[0052] (2) Particle size measurement

[0053] Take 50 μL PSELNs and add PBS to dilute to 1 mL. First, wash the sample pool with deionized water and then with PBS. Then, add the diluted PSELNs to the sample pool and use Nanosight NS300 (Malvern) to measure the concentration and particle size of PSELNs.

[0054] (3) Zeta potential analysis

[0055] Take 50 μL of PSELNs and dilute to 1 mL with deionized water. Then wash the sample pool with deionized water, add the diluted PSELNs into the sample pool, and measure the Zeta potential of PSELNs at 25°C using a particle size and potential measuring instrument (Malvern, Nano ZS90).

[0056] (4) BCA protein concentration determination

[0057] Take 1.2mL of protein standard solution and add it to the protein standard. After fully dissolving, prepare a final concentration of 0.5mg / mL protein standard solution. Prepare BCA working solution, take PSELNs and add an equal amount of protein lysis solution, place on ice to fully lyse, and mix. Then add the standard and PSELNs samples to the sample wells of the 96-well plate. Add 200μL of BCA working solution to each well and place at 37℃ for 25min. Measure A with an enzyme reader 562 The protein concentration of the sample was calculated based on the standard curve and the PSELNs sample volume.

[0058] 3. PSELNs Component Analysis

[0059] (1) PSELNs Metabolite Analysis

[0060] First, slowly thaw 0.5 mL of PSELNs at 4 °C and place it in a centrifuge tube. Add 2 volumes of extraction solution (methanol / acetonitrile, 1:1, V / V), vortex for 60 s, extract ultrasonically at low temperature for 30 min, centrifuge at 12,000 rpm for 10 min at 4 °C, and take the supernatant. Place it at -20 °C for 1 h to precipitate proteins, continue to centrifuge at 12,000 rpm for 10 min at 4 °C, take the supernatant for vacuum drying, add 100 μL of 50% acetonitrile solution for reconstitution, vortex, centrifuge at 12,000 rpm for 10 min at 4 °C, and take the supernatant for on-machine detection. The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometer (QExactive HFX, Thermo, USA). The Q Exactive HFX high-resolution mass spectrometry system is used to collect the first-level and second-level spectra. The original data is first pre-processed using Progenesis QI (Waters Corporation, Milford, USA) software for baseline filtering, peak identification, peak matching, retention time correction, peak alignment, etc., to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity. The peaks containing second-level mass spectrometry data are identified using a self-built traditional Chinese medicine second-level mass spectrometry database and corresponding fragmentation rules to obtain the non-target metabolome of PSELNs.

[0061] (2) PSELNs Protein Analysis

[0062] Lysis buffer was added to PSELNs, and the mixture was sonicated and centrifuged at 12,000 g for 5 min. Protein reduction and alkylation were performed with TCEP and CAA at 60 °C. After digesting the sample, it was centrifuged at 12,000 g, and the supernatant was taken for peptide purification using a self-made SDB-RPS desalting column. The peptide eluate was dried in vacuo and stored at -20 °C for later use. All PSELNs samples were analyzed on an Ultimate 3000 RSLCnano system, and MS was operated in data-dependent acquisition (DDA) top 20 mode with a full scan range of 350 - 1500 m / z. Fragment ion scans were recorded at a resolution of 15,000, an AGC of 100,000, and a maximum fill time of 50 ms. The MS raw data of PSELNs were analyzed using the Andromeda database search algorithm with MaxQuant (1.6.6.0). Proteins that could not be distinguished based on unique peptides were merged into a single proteome by MaxQuant, and the search results were filtered at a 1% FDR at the peptide and protein levels. The protein composition of PSELNs was obtained.

[0063] II. Experimental Results

[0064] 1. Identification of PSELNs

[0065] As Figure 1 shown, the morphology of PSELNs observed under a transmission electron microscope was cup-shaped, disk-shaped, or oval, with a clear bilayer lipid structure, a bright outer layer, and a relatively dim inner layer. The average Zeta potential of PSELNs measured by a particle size and zeta potential analyzer was -26.6 mV (potential distribution as Figure 2 shown), and the average particle size of PSELNs measured by Nanosight was 142.4 nm (particle size distribution as Figure 3 shown). The protein concentration of PSELNs measured by BCA protein concentration assay was 2.29 mg / mL.

[0066] 2. Active metabolites and proteins in PSELNs

[0067] The active metabolites of PSELNs are shown in Table 1, and the classification proportion of metabolites is as Figure 4 shown. A total of 23 active metabolites were listed with an oral bioavailability (OB) ≥ 30. The protein composition of PSELNs is shown in Table 2, and a total of 18 proteins were analyzed.

[0068] Table 1 Active metabolites of PSELNs

[0069]

[0070]

[0071] Table 2 Protein components of PSELNs

[0072]

[0073] Example 2

[0074] 1. Experimental method

[0075] Verify the anti-cancer effect of the PSELNs prepared in Example 1:

[0076] The anti-cancer effect of the PSELNs of the present invention was verified by three cancer cell lines. The verified cell lines included human lung cancer cells (A549 cells and PC-9 cells) and human colorectal cancer cells (HCT-15 cells), and the verification process was as follows:

[0077] Seed cells (1×10 4 cells / well) in a 96-well plate and incubate in a 37°C incubator for 24 h; set a control group and an experimental group, with 6 replicates in each group, and add the corresponding concentration of PSELNs prepared to each well. Then continue to incubate the cells at a constant temperature for 24 h, and then add 100 μL of the pre-prepared CCK-8 mixture (preparation method: 90 μL of complete medium + 10 μL of CCK-8 reagent) to each well. Subsequently, incubate in a 37°C environment and measure the absorbance value at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader; measure once every half hour until a suitable absorbance value is obtained, record the data and analyze it.

[0078] 2. Experimental results

[0079] As Figure 5 shown, when A549 cells were given PSELNs at a concentration of 20 μg / mL, a significant difference was shown compared with the control group (P<0.001), presenting the ability of PSELNs to inhibit the viability of A549 cells. Finally, the IC 50 of PSELNs against A549 cells was calculated to be 31.03 μg / mL (see Figure 6 ).

[0080] When PC-9 cells were given PSELNs at a concentration of 20 μg / mL, a significant difference was also shown compared with the control group (P<0.001), presenting the ability of PSELNs to inhibit the viability of PC-9 cells (see Figure 7 ), and the IC 50 of PSELNs against PC-9 cells was calculated to be 39.80 μg / mL (see Figure 8 ).

[0081] The CCK-8 experimental results of A549 cells and PC-9 cells confirmed the anti-lung cancer ability of PSELNs.

[0082] When HCT-15 cells were treated with PSELNs at a concentration of 20 μg / mL, significant differences were also shown compared with the control group (P<0.001), indicating that PSELNs inhibited the viability of HCT-15 cells (see Figure 9 ), and the IC 50 of PSELNs against HCT-15 cells was calculated to be 38.44 μg / mL (see Figure 10 ).

[0083] The CCK-8 assay results of HCT-15 cells confirmed the anti-colorectal cancer ability of PSELNs.

[0084] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing exosome-like nanovesicles derived from Polygonatum sibiricum, characterized in that, Comprising the following steps: After pulverizing fresh polygonatum sibiricum, filtering and collecting to obtain a first filtrate; Performing gradient centrifugation on the first filtrate to obtain a supernatant; Performing a first membrane filtration on the supernatant to obtain a second filtrate with a particle size less than 450 nm; Performing centrifugation on the second filtrate to obtain a precipitate, resuspending with PBS buffer to obtain a crude extract of polygonatum sibiricum exosomes; Performing a second membrane filtration on the crude extract of polygonatum sibiricum exosomes to obtain a third filtrate with a particle size less than 220 nm, obtaining the exosome-like nanovesicles derived from polygonatum sibiricum.

2. The preparation method according to claim 1, characterized in that, The gradient centrifugation is centrifuging at 4000 rpm, 10000 rpm and 12000 rpm for 90 min respectively.

3. The preparation method according to claim 1, characterized in that, Performing centrifugation on the second filtrate is centrifuging at 100000 g for 2 h.

4. An exosome-like nanovesicle derived from polygonatum sibiricum prepared by the preparation method according to any one of claims 1-3.

5. Use of an exosome-like nanovesicle derived from polygonatum sibiricum as claimed in claim 4 in the preparation of an anti-tumor drug.

6. The application according to claim 5, wherein The tumor is lung cancer.

7. The application according to claim 5, characterized in that, The tumor is colorectal cancer.

8. An anti-tumor drug, characterized in that, The active ingredient comprises the exosome-like nanovesicle derived from polygonatum sibiricum as claimed in claim 4.

9. The anti-tumor drug according to claim 8, wherein The tumor is lung cancer or colorectal cancer.

10. The anti-tumor drug according to claim 8, characterized in that, The anti-tumor drug further comprises a pharmaceutically acceptable excipient.

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