Preparation method and application of hedyotis diffusa exosome-like nanoparticles

Abstract

The invention discloses a preparation method and application of hedyotis diffusa exosome-like nanoparticles, and belongs to the technical field of biology. The invention provides a preparation method of hedyotis diffusa exosome-like nanoparticles. The preparation method comprises the following steps: extracting exosomes by utilizing ultracentrifugation treatment, and performing ultracentrifugation separation and purification on the exosomes by utilizing sucrose gradients with different concentrations. The invention also provides application of the hedyotis diffusa exosome-like nanoparticles prepared by the preparation method in preparation of medicines for treating intestinal inflammation. The prepared oldenlandia diffusa source exosome-like nanoparticles can overcome the barrier of gastrointestinal tracts, can still preserve good biological activity in a gastrointestinal variable environment, has a relatively high internalization rate, and is non-toxic. A new thought is expected to be provided for preparing the medicine for improving the intestinal inflammation in the future.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method of Hedyotis diffusa exosome-like nanoparticles and applications thereof. Background Art

[0002] Colitis refers to inflammatory lesions of the colon caused by various reasons. Fungi, bacteria and viruses can all cause colitis. If it is not controlled and treated, it will develop into colon cancer. There are many drugs for treating colitis on the market, but there are problems such as the high price of Western medicine and the slow efficacy of traditional Chinese medicine.

[0003] According to the position statement of the International Society for Extracellular Vesicles in 2018 and the MISEV2014 guidelines, nanoparticles extracted from plants are usually called exosome-like nanoparticles. Exosome-like vesicles are a type of nanovesicle with a lipid bilayer membrane structure secreted by cells, rich in contents such as proteins, lipids, and nucleic acids. Due to their wide sources and stable structures, they have received extensive attention in disease diagnosis, prognosis assessment, and immunotherapy in recent years. The existing plant exosome-like vesicle extraction methods are not uniform and have low efficiency, which limits their effectiveness in practical applications. It is urgent to find a drug with significant efficacy and low price. The exosome-like vesicles from Hedyotis diffusa are based on active substances of traditional Chinese medicine, have rich contents and green advantages, and show good biocompatibility and low toxicity. However, whether Hedyotis diffusa exosome-like vesicles can treat colitis has not been reported in the prior art. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing exosome-like nanoparticles of Hedyotis diffusa and its application to solve the problems existing in the above-mentioned prior art. The present invention extracts and applies exosomes of Hedyotis diffusa according to its plant characteristics, and proves that it has the effect of improving colitis.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: A method for preparing exosome-like nanoparticles of Hedyotis diffusa, comprising the steps of extracting exosomes by ultracentrifugation and purifying the exosomes by ultracentrifugation using sucrose gradients of different concentrations.

[0007] Furthermore, the steps specifically include:

[0008] The Hedyotis diffusa juice is filtered and then subjected to low-speed centrifugation, medium-speed centrifugation and high-speed centrifugation in sequence. The supernatant is collected and filtered through a 0.22 μm filter to obtain a crude extract. Exosomes are extracted from the crude extract by ultracentrifugation, and purified exosomes are separated by ultracentrifugation using sucrose gradients of different concentrations. The purified exosomes are naturally precipitated and dried to obtain the Hedyotis diffusa exosome-like nanoparticles.

[0009] Furthermore, the Hedyotis diffusa juice is obtained by squeezing fresh and clean whole Hedyotis diffusa with ice PBS.

[0010] Furthermore, the filtration of the Hedyotis diffusa juice is performed using a 100-mesh filter.

[0011] Furthermore, the low-speed centrifugation treatment is centrifugation at 1000g and 4°C for 15 minutes; the medium-speed centrifugation treatment is centrifugation at 2000g and 4°C for 20 minutes, and at 3000g and 4°C for 30 minutes; the high-speed centrifugation treatment is centrifugation at 10000g and 4°C for 30 minutes.

[0012] Furthermore, the ultracentrifugation in extracting exosomes by ultracentrifugation is centrifugation at 150000g and 4°C for 90 min.

[0013] Furthermore, the sucrose gradients of different concentrations include sucrose solutions containing 8%, 30%, 45% and 60% sucrose concentrations respectively prepared with PBS.

[0014] Furthermore, the purified exosomes were separated by ultracentrifugation using sucrose gradients of different concentrations, and the ultracentrifugation was performed at 150,000 g and 4° C. for 2 h.

[0015] Furthermore, the natural precipitation is resuspended in PBS.

[0016] Technical solution 2: Application of Hedyotis diffusa exosome-like nanoparticles prepared by the preparation method in the preparation of drugs for treating intestinal inflammation.

[0017] The present invention discloses the following technical effects:

[0018] The present invention extracts the key active ingredients of Hedyotis diffusa, thereby enhancing the biological activity of exosomes in anti-inflammatory aspects. Furthermore, the present invention uses the extracted exosomes to develop treatment plans for intestinal inflammation, giving full play to its potential in improving intestinal health and regulating the immune system. The Hedyotis diffusa-derived exosome-like nanoparticles prepared by the present invention have high concentrations, simple and feasible operation methods, and can be used for the extraction of plant exosomes from large-volume, low-concentration samples. It has been identified that the Hedyotis diffusa-derived exosome-like nanoparticles prepared by the present invention have typical "tray-like" or "hemispherical" results, have a lipid bilayer, and have a uniform particle size distribution, an average particle size of 140.1nm, and a concentration of 7.4×10 10 Particles / mL.

[0019] The results of the present invention show that due to the nano-scale size of the extracellular vesicles, they are easily absorbed. The exosome-like nanoparticles derived from Hedyotis diffusa prepared by the present invention can overcome the gastrointestinal barrier, and can still maintain good biological activity in the variable gastrointestinal environment, have a good improvement effect on inflammatory bowel inflammation, have a relatively high internalization rate, and are non-toxic. It is expected to provide new ideas for the preparation of drugs to improve intestinal inflammation in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is an electron micrograph of exosome-like nanoparticles derived from Hedyotis diffusa, with a resolution of 100 nm;

[0022] Figure 2 The particle size distribution and concentration of exosome-like nanoparticles derived from Hedyotis diffusa;

[0023] Figure 3 This is a graph showing the effect of administering different numbers of Hedyotis diffusa-derived exosome-like nanoparticles on the viability of IPEC-J2 cells;

[0024] Figure 4 The weight loss of mice in each treatment group, * refers to p < 0.05, ** refers to p < 0.01, *** refers to p < 0.001, **** refers to p < 0.0001;

[0025] Figure 5 The health index diagram of each treatment group for colitis disease in mice;

[0026] Figure 6The photos show the colon length of mice in each treatment group;

[0027] Figure 7 Statistical diagram of the effect of each treatment group on the colon length of mice, * means p < 0.05;

[0028] Figure 8 H&E sections of the colon tissues of mice in each treatment group at a magnification of 4X, where A is the Control group, B is the DSS group, C is the L-SELNs group, D is the M-SELNs group, and E is the H-SELNs group;

[0029] Fig. 9 H&E sections of colon tissues of mice in each treatment group at a magnification of 10X, where A is the Control group, B is the DSS group, C is the L-SELNs group, D is the M-SELNs group, and E is the H-SELNs group. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1. A method for preparing an exosome sample derived from Hedyotis diffusa, comprising the following steps:

[0037] (1) Preparation of Hedyotis diffusa juice: Take 250 g of fresh Hedyotis diffusa whole plant, wash it carefully 5 times with distilled water to remove surface stains, and then wash it twice with PBS. Pour 150 mL of ice PBS into the juice, mix and crush, and obtain Hedyotis diffusa whole plant juice;

[0038] (2) filtering the juice obtained in step (1) through a 100-mesh filter to obtain a coarsely filtered juice, and dispensing the coarsely filtered juice into 50 mL centrifuge tubes and placing them in an ice box;

[0039] (3) Precool the centrifuge in advance and perform low, medium and high speed centrifugation. First, perform low speed centrifugation (1000g, 4℃, 15min) to remove plant fibers and large particles, and collect the supernatant into a new centrifuge tube; secondly, perform medium speed centrifugation (2000g, 4℃, 20min) to remove larger debris and organelles, collect the supernatant into a new centrifuge tube, and then perform 3000g, 4℃, 30min treatment, collect the supernatant into a new centrifuge tube; filter the supernatant in sequence: 1μm filter → 0.45μm filter, and continue to collect the supernatant into a centrifuge tube; perform high speed centrifugation (10000g, 4℃, 30min), collect the supernatant into a new centrifuge tube, and repeat this step 3 times; uniformly filter the supernatant after high speed centrifugation through a 0.22μm filter, and collect the crude extract for later use;

[0040] (4) The supernatant of the crude extract was transferred to an ultracentrifuge tube and ultracentrifuged at 150,000 g, 4°C, 90 min. The exosomes were at the bottom of the ultracentrifuge tube. The supernatant was discarded and 20 mL of pre-cooled PBS was added to each tube to resuspend the precipitate. The supernatant was centrifuged again at 150,000 g, 4°C, 90 min. The supernatant was discarded and the precipitate was resuspended with PBS again. After drying, the exosome sample derived from Hedyotis diffusa was obtained for use.

[0041] 2. Purification of exosome-like nanoparticles from Hedyotis diffusa

[0042] Use PBS to prepare sucrose solutions containing 8%, 30%, 45%, and 60% sucrose, respectively. Slowly push the sucrose solutions from low concentration to high concentration from the bottom of the horizontal ultracentrifuge tube. Be careful and do not shake violently. After preparation, obvious stratification can be observed. Slowly add 1 mL of the crude exosome extract extracted in step (4) to the prepared gradient sucrose solution. After 150,000 g, 4°C, and 2 h, the solution is obviously stratified. Use a 1 mL syringe to carefully collect the exosomes between the 30%-45% sucrose solution, transfer them to a new ultracentrifuge tube, add 20 mL of PBS, and perform 150,000 g, 4°C, 2 h again to wash away the sucrose. Finally, discard the supernatant, add 1 mL of ice PBS to resuspend, if there are insoluble particles, perform 3000 g, 4°C, 5 min centrifugation, collect the supernatant, and divide it into aliquots in a -80°C refrigerator for later use.

[0043] 3. Characterization and identification of exosome-like nanoparticles derived from Hedyotis diffusa

[0044] (1) Observation of the morphology of Hedyotis diffusa-derived exosome-like nanoparticles by transmission electron microscopy: 10 μL of Hedyotis diffusa-derived exosome sample was added dropwise on a copper mesh to precipitate for 1 min, and the floating liquid was removed by filter paper; 10 μL of uranyl acetate was added dropwise on a copper mesh to precipitate for 1 min, and the floating liquid was removed by filter paper; and the mixture was dried at room temperature for 3 min to obtain Hedyotis diffusa-derived exosome-like nanoparticles, and the morphology was observed by transmission electron microscopy. The specific results are shown in the table. Figure 1 . Figure 1 It shows that the Hedyotis diffusa-derived exosome-like nanoparticles prepared in the present invention have a typical "tray-like" or "hemispherical" result.

[0045] (2) Particle size analysis of Hedyotis diffusa-derived exosome-like nanoparticles by NTA: frozen samples were taken, thawed on ice, and diluted 1000 times with 1× PBS for detection. The specific results are shown in Figure 2 . Figure 2 The results showed that the exosomes derived from Hedyotis diffusa prepared by the present invention were consistent with the size of exosomes, had a lipid bilayer, had a uniform particle size distribution, an average particle size of 140.1 nm, and a concentration of 7.4×10 10 Particles / mL.

[0046] 4. Detection of cytotoxicity of exosome-like nanoparticles derived from Hedyotis diffusa on IPEC-J2 cells

[0047] (1) Take IPEC-J2 cells in the logarithmic growth phase, wash them twice with PBS, digest them with trypsin for 6 minutes, centrifuge them at 1000 rpm for 5 minutes, discard the supernatant, add 3 mL of complete culture medium, and prepare a single cell suspension. After counting the cells, spread them on a 96-well plate and add 100 μL of culture medium to each well to make the cell density 1×10 5 / hole;

[0048] (2) Cultivate cells in a 5% CO2, 37°C cell culture incubator for 12 h;

[0049] (3) When the cell confluence reaches 60%-70%, discard the culture medium in the wells, wash once with PBS, add serum-free culture medium, and starve for 12 h;

[0050] (4) The culture medium in the well was discarded and washed once with PBS. According to the exosome-like nanoparticles derived from Hedyotis diffusa obtained in Example 1 of the present invention, a blank group, an experimental group, and a control group were set up: the Hedyotis diffusa exosomes and culture medium with different particle numbers were added as the experimental group (0-2.22×10 9 ), the culture medium without exosomes of Hedyotis diffusa was the control group, and the blank group without cells. 6 replicate wells, 100 μL per well. Cultured in a cell culture incubator at 5% CO2 and 37°C;

[0051] (5) After incubation for 48 h, discard the culture medium in the wells, add 100 μL of culture medium and 10 μL of CCK-8 solution to each well, and continue incubating in the cell culture incubator for 1 h;

[0052] (6) The absorbance of each well was measured at 450 nm using an ELISA reader. Figure 3 . Figure 3 The results showed that exosomes from low to high concentrations had no toxic effects on cells, indicating that exosomes were biocompatible within the tested concentration range and did not cause toxicity or damage to cells. This means that the nanoparticles prepared by the present invention are safe at the cellular level and are suitable for further application research or as a component of a drug delivery system.

[0053] 5. Animal testing

[0054] Eight-week-old C57BL / 6 mice were randomly divided into five groups, including PBS group, DSS group, low-concentration exosome group (L-SELNs), medium-concentration exosome group (M-SELNs) and high-concentration exosome group (H-SELNs). In the drinking water of the experimental groups, the PBS group only contained PBS, while the DSS group, low-concentration exosome group, medium-concentration exosome group and high-concentration exosome group were all added with 2.5% DSS to establish an inflammatory bowel disease (IBD) model. During the experiment, mice in the DSS group received 200 μL / mouse PBS gavage every day for 7 consecutive days; while mice in the low, medium and high exosome groups received 200 μL / mouse of Hedyotis diffusa exosome-like nanoparticles every day for 7 consecutive days, with particle concentrations of low concentration (1.48×10 8 ), medium concentration (1.48×10 9 ) and high concentration (1.48×10 10 During the experiment, the blood in the stool, body weight changes and stool characteristics of the mice were recorded.

[0055] After the experiment, the mice were weighed on the 7th day and then anesthetized and killed for subsequent analysis. These data are key indicators to verify whether the inflammatory bowel disease model is successfully established, and are used to evaluate the health status of the mice and the efficacy of exosomes. Specifically, after killing on the 7th day, the mouse colon tissue specimens were obtained. The results were analyzed by fixation (fixed in 4% paraformaldehyde for 48 hours), dehydration (after washing the excess fixative with running water, refer to Table 1 for dehydration, transparency and wax immersion, where benzyl alcohol is xylene: anhydrous ethanol = 1:1), embedding, sectioning, and H&E staining.

[0056] Table 1

[0057]

[0058]

[0059] Preparation of paraffin sections: Slice the embedded tissue blocks with an automatic rotary slicer, set the slice thickness to 5 μm, and then place the slices in a 37°C incubator overnight to prevent tissue detachment during subsequent experiments.

[0060] H&E staining steps: ① bake the slices at 60℃ for 2 minutes; ② place the slices in a combined staining jar and operate according to the following process: xylene I (15 minutes) → xylene II (15 minutes) → xylene: anhydrous ethanol = 1:1 (10 minutes) → 100% ethanol (5 minutes) → 95% ethanol (5 minutes) → 80% ethanol (5 minutes) → 70% ethanol (2 minutes) → 50% ethanol (5 minutes) → hematoxylin staining of cell nuclei (1 minute) → self-staining Rinse with fresh water for 10 min → wash with distilled water → 70% ethanol (5 min) → 80% ethanol (5 min) → 90% ethanol (3 min) → eosin staining (30 s) → 95% ethanol I (3 s) → 95% ethanol II (3 s) → 95% ethanol III (3 s) → 100% ethanol I (3 min) → 100% ethanol II (3 min) → xylene: anhydrous ethanol = 1:1 (8 min) → xylene I (10 min) → xylene II (10 min).

[0061] The results showed that Hedyotis diffusa-derived exosome-like nanoparticles (SELNs) could effectively improve the weight loss and bloody stool of mice, and significantly prevent the shortening of colon length. This suggests that the SELNs used in inflammatory bowel disease have a certain anti-inflammatory effect. Figure 4 As shown in the figure, under the intervention of SELNs, compared with the DSS group, low, medium and high concentrations can significantly inhibit the weight loss caused by DSS, that is, SELNs have a certain improvement effect; the weight loss rate, stool characteristics, and stool color (dark blue, occult blood, occult blood, etc.) were tested for comprehensive scores. Figure 5 As shown, after the fifth day, under the intervention of low, medium and high concentrations of SELNs groups, compared with the DSS group, all played a certain improvement role, that is, SELNs had a certain improvement effect; Figure 6 and Figure 7 As shown in the figure, the colon length of mice in each treatment group showed significant differences. The results showed that SELNs have a good therapeutic effect on improving inflammatory bowel inflammation, can effectively improve intestinal damage induced by DSS, and effectively prevent the occurrence and development of inflammation. Figure 8 and Fig. 9 As shown, the results showed that the DSS group had a missing crypt structure, destroyed mucosal epithelial structure and a large number of inflammatory cells infiltrated. However, under the intervention of SELNs, the low, medium and high concentration groups all showed significant improvements, indicating that SELNs have a certain protective effect on the mouse colon.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing exosome-like nanoparticles of Hedyotis diffusa, characterized in that: The method comprises the steps of extracting exosomes by ultracentrifugation and separating and purifying the exosomes by ultracentrifugation using sucrose gradients of different concentrations.

2. The preparation method according to claim 1, characterized in that: The steps specifically include: The Hedyotis diffusa juice is filtered and then subjected to low-speed centrifugation, medium-speed centrifugation and high-speed centrifugation in sequence. The supernatant is collected and filtered through a 0.22 μm filter to obtain a crude extract. Exosomes are extracted from the crude extract by ultracentrifugation, and purified exosomes are separated by ultracentrifugation using sucrose gradients of different concentrations. The purified exosomes are naturally precipitated and dried to obtain the Hedyotis diffusa exosome-like nanoparticles.

3. The preparation method according to claim 2, characterized in that: The Hedyotis diffusa juice is obtained by squeezing fresh and clean whole Hedyotis diffusa with ice PBS.

4. The preparation method according to claim 2, characterized in that: The filtration of the Hedyotis diffusa juice is performed using a 100-mesh filter.

5. The preparation method according to claim 2, characterized in that: The low-speed centrifugation treatment is centrifugation at 1000g and 4°C for 15 minutes; the medium-speed centrifugation treatment is centrifugation at 2000g and 4°C for 20 minutes, and at 3000g and 4°C for 30 minutes; the high-speed centrifugation treatment is centrifugation at 10000g and 4°C for 30 minutes.

6. The preparation method according to claim 2, characterized in that: The ultracentrifugation for extracting exosomes by ultracentrifugation is performed at 150,000 g and 4° C. for 90 min.

7. The preparation method according to claim 2, characterized in that: The sucrose gradients of different concentrations include sucrose solutions prepared with PBS and containing 8%, 30%, 45% and 60% sucrose concentrations, respectively.

8. The preparation method according to claim 2, characterized in that: The purified exosomes were separated by ultracentrifugation using sucrose gradients of different concentrations, and the ultracentrifugation was performed at 150,000 g and 4° C. for 2 h.

9. The preparation method according to claim 2, characterized in that: The natural precipitate was resuspended in PBS.

10. Use of the Hedyotis diffusa exosome-like nanoparticles prepared by the preparation method according to any one of claims 1 to 9 in the preparation of a drug for improving intestinal inflammation.

Images