Preparation method and application of exosome-like nano vesicles derived from houttuynia cordata

Abstract

The invention discloses a preparation method and application of exosome-like nano-vesicles derived from herba houttuyniae, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: centrifuging herba houttuyniae filtrate for 40 minutes at the speed of 4000 rpm and centrifuging for 60 minutes at the speed of 10000 rpm in sequence. The invention also provides an application of the cordate houttuynia-derived exosome-like nano-vesicle prepared by the preparation method in preparation of anti-tumor drugs. The cordate houttuynia-derived exosome-like nano-vesicle prepared by the preparation method disclosed by the invention has the advantages of safety, economy and low toxicity, also has good biocompatibility and a nano-scale structure, and brings new hope for overcoming the cancer problem. According to the extraction method, the key active ingredients of the herba houttuyniae are extracted, so that the biological activity of the exosome in the anti-tumor aspect is enhanced, and therefore, the extraction method of the HELNs and application research of the HELNs have important significance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to a preparation method and application of exosome-like nanovesicles derived from Houttuynia cordata Thunb. Background Art

[0002] Cancer is a disease that seriously threatens human health, and lung cancer is the main cause of cancer death. At present, the effective treatment methods for cancer mainly include surgical resection, radiotherapy, and chemotherapy. However, they all have their own limitations: Surgical resection is likely to cause relatively large trauma and often leads to tumor recurrence or metastasis due to incomplete resection; Radiotherapy not only has poor efficacy because some tumors are insensitive to radiation, but also may damage the normal tissues around cancer cells, resulting in serious sequelae such as organ failure; As the most commonly used and relatively most effective cancer treatment method, chemotherapy eliminates tumors through cytotoxicity, but small molecule drugs often accumulate non-specifically in tumor tissues, leading to very serious toxic side effects. Therefore, it is crucial to explore safer and more effective cancer treatment methods.

[0003] Exosomes are nanoscale membrane vesicles actively secreted by cells, with diameters usually between 30 - 150 nm. They contain rich bioactive substances such as proteins, nucleic acids, and lipids, and play an important role in cell-to-cell communication, like the "information couriers" of cells. They are mainly distributed in various body fluids, have functions such as cell communication, cell migration, promoting angiogenesis, and anti-tumor immunity, are closely related to the occurrence and progression of various diseases, and can not only be used as biological indicators for diagnosing various diseases, but also as treatment means.

[0004] Houttuynia cordata Thunb. is the fresh whole herb or dried aerial part of Houttuynia cordata, a plant of the Saururaceae family. It is mainly produced in the areas south of the Yangtze River in China. It is slightly cold in nature and pungent in taste, mainly entering the lung meridian, and has the effects of clearing heat and detoxifying, eliminating carbuncles and discharging pus, and promoting diuresis and relieving stranguria. It is commonly used to treat lung abscess with expectoration of pus and blood, phlegm-heat cough and asthma, dysentery, stranguria, carbuncles and sores. Existing research has shown that Houttuynia cordata extracts and their active ingredients have significant anti-inflammatory effects and can effectively relieve the symptoms of various inflammatory diseases. In addition, Houttuynia cordata has a good therapeutic effect on lung diseases; Another study shows that Houttuynia cordata extracts have obvious inhibitory effects on colon cancer, leukemia, lung cancer, and liver cancer. Its potential anti-tumor mechanism may involve regulating cell signaling pathways, inducing apoptosis of tumor cells, inhibiting the proliferation and migration of tumor cells, etc., bringing new hope for solving the cancer problem. Therefore, it is of great significance to study the extraction method of Houttuynia cordata exosome-like nanovesicles and their application research. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing exosome-like nanovesicles derived from Houttuynia cordata and its application, so as to solve the problems existing in the above-mentioned prior art. According to the plant characteristics of Houttuynia cordata, the present invention extracts and applies exosomes from it, and proves that it has an anti-tumor effect.

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

[0007] Technical solution one: A method for preparing exosome-like nanovesicles derived from Houttuynia cordata, which includes the steps of centrifuging the Houttuynia cordata juice at 4000 rpm for 40 min and then at 10000 rpm for 60 min in sequence.

[0008] Further, the above steps specifically include:

[0009] After juicing Houttuynia cordata and filtering, Houttuynia cordata juice is obtained. The Houttuynia cordata juice is centrifuged at 4000 rpm for 40 min and then at 10000 rpm for 60 min in sequence. After collecting the supernatant, it is filtered through a 450 nm microporous filter membrane to obtain a filtrate. The filtrate is mixed with an equal volume of PEG 6000 solution, allowed to stand, and then centrifuged at a low speed to obtain a precipitate. The precipitate is resuspended with PBS and filtered to obtain the Houttuynia cordata-derived exosome-like nanovesicles.

[0010] Further, the Houttuynia cordata juice is obtained by juicing the washed Houttuynia cordata with PBS.

[0011] The preparation of Houttuynia cordata juice includes: washing Houttuynia cordata, adding pre-cooled PBS to a juicer, crushing with the juicer, stopping for 1 min every 1 min of crushing, repeating 5 times, filtering out the Houttuynia cordata residue with a gauze, and collecting the Houttuynia cordata juice.

[0012] Further, the standing is carried out at 4 °C for 24 h.

[0013] Further, the low-speed centrifugation is carried out at 4 °C and 10000 rpm for 60 min.

[0014] Further, the filtering after resuspending with PBS is filtering with a 220 nm microporous filter membrane.

[0015] Technical solution two: The Houttuynia cordata-derived exosome-like nanovesicles prepared by the above-mentioned preparation method.

[0016] Technical solution three: The application of the Houttuynia cordata-derived exosome-like nanovesicles in the preparation of anti-tumor drugs.

[0017] The main metabolites prepared by the present invention are shown in Table 1. Among them, the active metabolites in the Houttuynia cordata-derived exosome-like nanovesicles can also be used to prepare anti-tumor drugs, and anti-tumor includes inhibiting the proliferation of lung cancer cells.

[0018] The present invention discloses the following technical effects:

[0019] The present invention extracts and isolates Houttuynia cordata Thunb-derived exosome-like nanovesicles (HELNs), and uses TEM and Nanosight to identify and characterize the morphology, size, concentration and Zeta potential of the extracted HELNs. It is observed under a transmission electron microscope that the extracted HELNs are oval vesicles, the Zeta potential is -35.8 mV, and the concentration is 2.63×10 9 particles / mL, and the particle size is concentrated at 114 nm, proving that the HELNs are successfully extracted. The metabolite analysis results of HELNs show that carboxylic acids and their derivatives account for 23.29%; fatty acyl groups account for 10.96%; isoprenoid lipids account for 10.27%; flavonoids account for 8.22%. The results of the CCK-8 experiment show that the IC 50 of A549 cells is 44.94 μg / mL, and the IC 50 of PC-9 cells is 39.15 μg / mL. The results of the HELNs uptake experiment show that the HELNs are successfully taken up by A549 cells and PC-9 cells. In addition, the present invention uses the results of network pharmacology analysis to find a total of 46 HELNs targets acting on lung cancer. The GO and KEGG results show that HELNs inhibit lung cancer mainly by regulating biological processes such as phosphatidylinositol 3-kinase / protein kinase B signal transduction, miRNA transcription, humoral immune response, angiogenesis, endothelial cell migration, gene expression, apoptosis process, and regulation of transcription by RNA polymerase II. The main affected signaling pathways are: proteoglycans in cancer, pathways in cancer, P13-Akt signaling pathway, HIF-1 signaling pathway, Rap signaling pathway, microRNA in cancer, VEGF signaling pathway, etc.

[0020] The Houttuynia cordata Thunb-derived exosome-like nanovesicles prepared by the present invention are safe, economical, and low-toxic, and also have good biocompatibility and a nanoscale structure, bringing new hope for overcoming cancer problems. The present invention extracts the key active ingredients of Houttuynia cordata Thunb, thereby enhancing the biological activity of exosomes in anti-tumor. Therefore, the extraction method of HELNs provided by the present invention and its application research have important significance. Brief Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings described below 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.

[0022] Figure 1 It is the TEM image (500nm) of HELNs;

[0023] Figure 2 It is the particle size characterization diagram of HELNs;

[0024] Figure 3 It is the potential characterization diagram of HELNs;

[0025] Figure 4 It is the proportion of metabolic components of HELNs;

[0026] Figure 5 It is the CCK-8 result of A549 cells;

[0027] Figure 6 It is the IC 50 result of A549 cells;

[0028] Figure 7 It is the CCK-8 result of PC-9 cells;

[0029] Figure 8 It is the IC 50 result of PC-9 cells;

[0030] Figure 9 It is the diagram of HELNs being taken up by A549 cells;

[0031] Figure 10 It is the diagram of HELNs being taken up by PC-9 cells;

[0032] Figure 11 It is the diagram of HELNs acting on lung cancer targets;

[0033] Figure 12 It is the GO analysis result;

[0034] Figure 13 It is the KEGG analysis result. Detailed implementation manners

[0035] Now, the various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0036] It should be understood that the terms used in this invention are only for describing specific embodiments and are not intended to limit the invention. Additionally, for the numerical ranges in this 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 this invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this 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.

[0038] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0039] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] 1. Extraction of Houttuynia cordata - derived exosome - like nanovesicles (HELNs)

[0042] (1) Wash the roots of Houttuynia cordata, add pre - cooled PBS (Houttuynia cordata:PBS = 100 g / 300 ml) to a juicer, break it with the juicer, stop for 1 minute every 1 minute of breaking, repeat 5 times, then filter out the Houttuynia cordata residue with a gauze, and dispense the collected Houttuynia cordata juice into centrifuge tubes, and temporarily place it on ice;

[0043] (2) Continuously centrifuge the Houttuynia cordata juice filtered in step (1): Centrifuge at 4000 rpm for 40 minutes at 4 °C; centrifuge at 10000 rpm for 60 minutes; discard the precipitate and take the supernatant;

[0044] (3) Filter the obtained supernatant with a 450 nm microporous membrane to obtain a filtrate;

[0045] (4) Prepare a 20% PEG 6000 solution, mix the filtrate obtained in step (3) with the PEG 6000 solution in equal volume, place it at 4 °C for 24 h, then centrifuge the co-precipitated solution at 4 °C and 10000 rpm for 60 min, discard the supernatant, resuspend the precipitate with PBS, and then filter it through a 220 nm microporous membrane to obtain HELNs.

[0046] 2. Characterization of HELNs

[0047] (1) Morphological observation

[0048] Dilute the HELNs sample to an appropriate multiple for particle size detection. Place the copper grid on the filter paper, add 10 μL of the HELNs suspension dropwise onto the copper grid, let it stand for 10 min, then add 1 drop of PBS solution, and after the liquid dries slightly, add 4% paraformaldehyde solution, let it stand, and air dry at room temperature. Observe the morphology and size of HELNs under a transmission electron microscope.

[0049] (2) Particle size measurement

[0050] Take 1 mL of the diluted HELNs solution, equilibrate it at room temperature for 2 min, and then detect the particle size of HELNs with a nanoparticle size analyzer.

[0051] (3) Zeta potential analysis

[0052] Take 50 μL of HELNs, dilute it to 1 mL with deionized water. Then wash the sample cell with deionized water, and then add the diluted HELNs to the sample cell. Measure the Zeta potential of HELNs at 25 °C using a particle size and potential measuring instrument (Malvern, Nano ZS90).

[0053] (4) Analysis of HELNs metabolites

[0054] Take 1 mL of HELNs, add an equal volume of extract methanol-acetonitrile solution (1:1, v / v), vortex for 60 s, low-temperature ultrasonic for 30 min, twice, place at -20 °C for 1 h to precipitate protein, centrifuge at 12000 × g, 4 °C for 20 min, freeze-dry the supernatant, re-dissolve in 200 μL 30% ACN, vortex, centrifuge at 14000 × g at 4 °C for 15 min, and take the supernatant for microcentrifuge. According to the liquid phase: chromatographic column: Waters HSST3 (100×2.1mm, 1.8μm); mobile phase: phase A is ultrapure water solution (containing 0.1% formic acid), phase B is acetonitrile solution (containing 0.1% formic acid); flow rate 0.3mL / min; column temperature 40℃; injection volume 2μL; elution gradient: 0minA phase / B phase (100:0, v / v), 1minA phase / B phase (100:0, v / v), 12minA phase / B phase (5:95, v / v), 13minA phase / B phase (5:95, v / v), 13.1minA phase / B phase (100:0, v / v), 17minA phase / B phase (100:0, v / v) parameter detection, after the detection is completed, data preprocessing is performed. The obtained HELNs metabolite data were pre-processed by baseline filtering, peak identification, peak matching, retention time correction and peak alignment to obtain a data matrix containing retention time, mass-to-charge ratio and peak intensity. The peaks containing secondary mass spectrometry data were identified using the self-built Chinese herbal medicine secondary mass spectrometry database and the corresponding fragmentation rules to obtain the non-target metabolites of HELNs.

[0055] (5) Verification of the anticancer effect of HELNs

[0056] The anticancer effect of the HELNs of the present invention was verified by human lung cancer cells (A549 cells and PC-9 cells), and the verification process was as follows:

[0057] 1) BCA protein concentration determination

[0058] Take 1.0mL 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 an appropriate amount of BCA working solution at a ratio of 50:1 and mix thoroughly. Take an appropriate amount of HELNs and add an equal amount of protein lysis solution, place on ice to fully lyse and mix. Then add the standard and HELNs 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 the absorbance of A562 with an enzyme reader. Calculate the protein concentration of the sample based on the standard curve and the volume of the HELNs sample.

[0059] 2) CCK-8 experiment

[0060] A549 cells and PC-9 cells (1×10 4Cells / well), incubated in an incubator at 37 °C for 24 h; set a control group and an experimental group, with 6 replicate wells in each group, and add the prepared HELNs with corresponding concentrations to each well. Then continue to incubate for 24 h, and then add 100 μL of the pre-prepared CCK-8 mixture to each well (preparation method: 90 μL of complete medium + 10 μL of CCK-8 reagent). Subsequently, incubate in an environment at 37 °C, and measure the absorbance value at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader; measure once every half hour, record the data and analyze it.

[0061] 3) Network pharmacology research

[0062] Import the PubChem numbers of the top 20 metabolites identified by non-target metabolomics into the BATMAN 2.0 database, screen for target genes with a confidence level > 0.6 (LR = 13.26) and P < 0.05 as conditions, and the target genes with higher confidence are the main targets of HELNs. Then search for the target genes of lung cancer in the GeneCards database and the OMIM database, merge them, remove duplicates, and take the intersection with the targets of HELNs. The intersection genes are the targets of HELNs acting on lung cancer. Then draw a protein-protein interaction (PPI) network of these targets, and perform Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Ontology (GO) analysis to find the related signaling pathways and biological processes of HELNs acting on lung cancer.

[0063] 3. Experimental results

[0064] (1) Identification of HELNs

[0065] Extract and isolate HELNs, and use transmission electron microscopy (TEM) and Nanosight to identify and characterize the morphology, size, concentration, and Zeta potential of the extracted HELNs, as Figures 1-3 shown. Observed under a transmission electron microscope, the extracted HELNs are oval vesicles, with a Zeta potential of -35.8 mV, a concentration of 2.63×10 9 particles / mL, and the particle size is concentrated at 114 nm, proving that HELNs have been successfully extracted.

[0066] (2) Active metabolites in HELNs

[0067] The analysis results of HELNs metabolites are as Figure 4 shown. The results show that the proportion of carboxylic acids and their derivatives is 23.29%; fatty acyl groups are 10.96%; isoprenoid lipids are 10.27%; flavonoids are 8.22%. The top 20 metabolites in the metabolites are shown in Table 1.

[0068] Table 1

[0069]

[0070] (3) CCK-8 assay results

[0071] The CCK-8 assay results are as Figures 5-8 shown. The results show that there was no significant difference between A549 cells and the control group when HELNs were given at a concentration of 5 μg / mL (P > 0.05); while when the concentration of HELNs was ≥10 μg / mL, there was a significant difference compared with the control group (P < 0.001). Finally, the IC 50 of A549 cells was calculated to be 44.94 μg / mL. When PC-9 cells were given HELNs at a concentration of 5 μg / mL, there was also no significant difference compared with the control group (P > 0.05); while when the concentration of HELNs was ≥10 μg / mL, there was a significant difference compared with the control group (P < 0.001), and the IC 50 of PC-9 cells was calculated to be 39.15 μg / mL.

[0072] (4) HELNs uptake assay results

[0073] The HELNs uptake assay results are as Figure 9 and Figure 10 shown. The fluorescence localization results show that HELNs were successfully taken up by A549 cells and PC-9 cells.

[0074] (5) Network pharmacology analysis results

[0075] A total of 46 targets of HELNs acting on lung cancer were found by network pharmacology analysis. After sorting them by degree, the results are as Figure 11 shown. As Figure 12 and 13 shown, the GO and KEGG results show that HELNs inhibit lung cancer mainly by regulating biological processes such as phosphatidylinositol 3-kinase / protein kinase B signal transduction, miRNA transcription, humoral immune response, angiogenesis, endothelial cell migration, gene expression, apoptosis process, and regulation of transcription by RNA polymerase II, and the main affected signaling pathways are: proteoglycans in cancer, pathways in cancer, P13-Akt signaling pathway, HIF-1 signaling pathway, Rap signaling pathway, microRNA in cancer, VEGF signaling pathway, etc. The results show that the main metabolites in HELNs can inhibit the proliferation of lung cancer cells and are used for the preparation of anti-tumor drugs.

[0076] 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 Houttuynia cordata, characterized in that: The method comprises the steps of centrifuging the houttuynia cordata juice at 4000 rpm for 40 minutes and at 10000 rpm for 60 minutes in sequence.

2. The preparation method according to claim 1, characterized in that: The steps specifically include: The Houttuynia cordata juice was squeezed and filtered to obtain Houttuynia cordata juice, the Houttuynia cordata juice was centrifuged at 4000 rpm for 40 min and 10000 rpm for 60 min, the supernatant was collected and filtered through a 450 nm microporous membrane to obtain a filtrate, the filtrate was mixed with an equal volume of PEG6000 solution, the mixture was allowed to stand and then centrifuged at a low speed to obtain a precipitate, the precipitate was resuspended in PBS and then filtered to obtain the Houttuynia cordata-derived exosome-like nanovesicles.

3. The preparation method according to claim 2, characterized in that: The houttuynia cordata juice is obtained by squeezing the washed houttuynia cordata with PBS.

4. The preparation method according to claim 2, characterized in that: The standing is placing at 4°C for 24 hours.

5. The preparation method according to claim 2, characterized in that: The low-speed centrifugation is carried out at 4°C and 10,000 rpm for 60 min.

6. The preparation method according to claim 2, characterized in that: The filtration after resuspending with PBS is performed by filtering with a 220 nm microporous filter membrane.

7. The Houttuynia cordata-derived exosome-like nanovesicles prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the Houttuynia cordata-derived exosome-like nanovesicles according to claim 7 in the preparation of anti-tumor drugs.

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