Application and preparation method of exosome-like nanoparticles derived from Chinese yam

Abstract

The invention discloses an application of Chinese yam derived exosome-like nanoparticles in preparation of skin injury repairing products, the exosome-like nanoparticles are derived from natural medicinal and edible plants, and have no cytotoxicity to skin keratinocytes HaCaT; scratch cell migration experiments are carried out, and it is found that healing can be rapidly promoted, and the anti-inflammatory effect is achieved. The invention further provides a preparation method of the Chinese yam-derived exosome-like nano-particles, Chinese yam tissue raw stock is separated through centrifugal treatment and sucrose gradient centrifugation, the high-purity exosome-like nano-particles are prepared, and the method is simple in technological process and easy to implement.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to the application of yam-derived exosome-like nanoparticles and a method for preparing the same. Background Art

[0002] Yam (Dioscorea opposita) is the tuber of Dioscorea opposita Thunb. in the Dioscoreaceae family, and is a traditional medicinal and edible plant, mainly containing polysaccharides, flavonoids, polyphenols, allantoin and other active ingredients. According to the Pharmacopoeia, yam has the remarkable effects of moistening the intestines and promoting defecation, improving chronic diarrhea, and in addition, has functions such as immune regulation, antioxidant and anti-tumor effects.

[0003] Plant-derived exosome-like nanovesicles (PENs) contain miRNAs, bioactive lipids, mRNAs and proteins, can enter the extracellular space, act as intercellular messengers, promote intercellular communication, and provide biological defense against pathological diseases. Recent studies have shown that plant-derived exosome-like nanoparticles (PENs) can be derived from roots, tubers, nuts, seeds, as well as fresh and dried plant bodies, and have great potential as drug delivery systems. PENs have good biocompatibility and biodegradability, do not cause negative effects on intestinal barrier function or other organs, have extremely high safety, and can be prepared on a large scale. Summary of the Invention

[0004] The present invention aims to at least solve one of the above technical problems existing in the prior art. For this reason, the purpose of the present invention is to provide a kind of.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides the application of yam-derived exosome-like nanovesicles (YENs) in the preparation of products for repairing skin damage.

[0007] In the present invention, "yam-derived exosome-like nanovesicles" refers to exosome-like nanovesicles prepared from yam as a raw material. In the present invention, "products for repairing skin damage" refers to products that help the body repair and restore the formed defect when the skin tissue is severed or damaged.

[0008] The present invention uses yam-derived exosome-like nanovesicles to prepare products for repairing skin damage, which has no cytotoxicity to skin keratinocytes, can quickly promote healing, and has an anti-inflammatory effect.

[0009] In some embodiments, the yam-derived exosome-like nanoparticles have a saucer-like structure.

[0010] In some embodiments, the particle size of the yam-derived exosome-like nanoparticles is 50 - 300 nm.

[0011] In some embodiments, the particle size of the yam-derived exosome-like nanoparticles is 100 - 200 nm.

[0012] In some embodiments, the average particle size of the yam-derived exosome-like nanoparticles is 145 - 155 nm.

[0013] In some embodiments, the yam-derived exosome-like nanoparticles are negatively charged.

[0014] In some embodiments, the Zeta potential of the yam-derived exosome-like nanoparticles is -10 to -1 mV.

[0015] In some embodiments, the Zeta potential of the yam-derived exosome-like nanoparticles is -5.43 mV.

[0016] In some embodiments, the skin damage repair product is used to promote skin wound healing and / or inhibit skin inflammation.

[0017] In some embodiments, the skin damage repair product is a cosmetic and a drug.

[0018] In some embodiments, the cosmetic includes any one of a nourishing cream and a nourishing facial mask.

[0019] In some embodiments, the dosage form of the drug is a skin administration dosage form, including any one of an external solution, an ointment, a plaster, a paste, and a patch.

[0020] In some embodiments, the skin damage repair product is a wound dressing.

[0021] The second aspect of the present invention provides a method for preparing yam-derived exosome-like nanoparticles, comprising the following steps:

[0022] (a) Add a buffer solution to yam, stir and break it into yam juice to obtain tissue homogenate;

[0023] (b) Centrifuge the tissue homogenate to obtain tissue supernatant;

[0024] (c) Centrifuge the tissue supernatant to obtain a precipitate;

[0025] (d) Resuspend the precipitate with a buffer solution to obtain a resuspension;

[0026] (e) Add sucrose solutions with mass concentrations of 8%, 30% and 45% to the resuspension solution in sequence, and perform centrifugation.

[0027] (f) Collect the liquid layer between the sucrose solution layers corresponding to 30% and 45% in mass concentration.

[0028] Yam has a lot of mucus and fibers, and it is difficult to remove impurities completely during centrifugation. The present invention provides a method for preparing yam-derived exosome-like nanoparticles. After centrifugation, the yam-derived exosome-like nanoparticles are separated by sucrose density gradient centrifugation. When sucrose solution is centrifuged, a discontinuous density gradient will be formed in the centrifuge tube, and substances with different molecular weights can be separated by the action of centrifugal force. In the present invention, sucrose solutions with mass concentrations of 8%, 30% and 45% are used, and it is found that the boundaries of the three different concentrations of sucrose solutions at 8%, 30% and 45% are very obvious and easy to distinguish during centrifugation; and it is found that collecting the liquid layer between the sucrose solution layers corresponding to 30% and 45% in mass concentration can obtain high-purity yam-derived exosome-like nanoparticles.

[0029] In some embodiments, the buffer solution in step (a) is a buffer solution pre-cooled at 2-6°C.

[0030] In some embodiments, the buffer solution in step (a) is a buffer solution pre-cooled at 4°C.

[0031] Preparing tissue homogenate with pre-cooled buffer solution and yam is beneficial to maintaining the activity of substances.

[0032] In some embodiments, the mass ratio of yam to buffer solution in step (a) is 1:(1-5).

[0033] In some embodiments, the mass ratio of yam to buffer solution in step (a) is 1:1.

[0034] In some embodiments, the buffer solution in step (a) is a phosphate buffer solution.

[0035] In some embodiments, the molar concentration of the phosphate buffer solution in step (a) is 0.01 M and the pH is 7.4.

[0036] In some embodiments, the centrifugation treatment in step (b) means centrifuging at a centrifugation rate of 6000-15000 rpm for 1-5 hours.

[0037] In some embodiments, the centrifugation treatment in step (b) means centrifuging at a centrifugation rate of 10000 rpm for 3 hours.

[0038] In some embodiments, the centrifugation treatment in step (c) refers to centrifuging at a rate of 50,000 - 80,000 rpm for 1 - 5 hours.

[0039] In some embodiments, the centrifugation treatment in step (c) refers to centrifuging at a rate of 65,000 rpm for 3 hours.

[0040] In some embodiments, the buffer solution in step (d) is a phosphate buffer solution.

[0041] In some embodiments, the phosphate buffer solution in step (d) has a molar concentration of 0.01 M and a pH of 7.4.

[0042] In some embodiments, the sucrose solution in step (e) is obtained by dissolving sucrose in Tris - HCl buffer solution.

[0043] In some embodiments, the Tris - HCl buffer solution has a pH of 7.2 and a volume concentration of 20 mM / L.

[0044] In some embodiments, the centrifugation treatment in step (e) refers to centrifuging at a rate of 50,000 - 80,000 rpm for 1 - 5 hours.

[0045] In some embodiments, the centrifugation treatment in step (e) refers to centrifuging at a rate of 65,000 rpm for 3 hours.

[0046] In some embodiments, the centrifugation treatments in steps (b), (c), and (e) are carried out at 4°C.

[0047] During ultra - centrifugation, the high - speed rotation of the rotor generates heat, leading to a temperature increase. Using a refrigeration system to maintain the temperature at 4°C helps to preserve the activity of substances.

[0048] In some embodiments, the preparation method further includes a step of washing the liquid collected in step (f).

[0049] In some embodiments, the washing refers to centrifuging the collected liquid to remove the supernatant, resuspending the precipitate to obtain a resuspension, and repeating the centrifugation and resuspension steps until the resuspension is clear. The centrifugation is carried out at 4°C, with a centrifugation rate of 65,000 rpm and a centrifugation time of 3 hours. Resuspending the precipitate means resuspending it using a phosphate buffer solution. The phosphate buffer solution has a molar concentration of 0.01 M and a pH of 7.4.

[0050] The third aspect of the present invention provides a yam - derived exosome - like nanoparticle prepared by the preparation method of the second aspect.

[0051] The beneficial effects of the present invention are:

[0052] The present invention provides the application of yam-derived exosome-like nanoparticles in the preparation of products for repairing skin damage. These exosome-like nanoparticles are derived from natural medicinal and edible plants and have no cytotoxicity to the skin keratinocyte HaCaT. Through a scratch cell migration experiment, it was found that they can rapidly promote healing and have an anti-inflammatory effect.

[0053] The present invention also provides a method for preparing yam-derived exosome-like nanoparticles. The original pulp of yam tissue is separated by centrifugation and sucrose density gradient centrifugation. The liquid between the 30% sucrose concentration layer and the 45% sucrose concentration layer is collected to obtain a liquid layer with a high purity of exosome-like nanoparticles. This method has a simple process flow and is easy to implement. Description of the Drawings

[0054] Figure 1 It is a transmission electron microscope image of yam-derived exosome-like nanoparticles.

[0055] Figure 2 It is a graph of the particle size distribution results of yam-derived exosome-like nanoparticles.

[0056] Figure 3 It is a Zeta potential graph of yam-derived exosome-like nanoparticles.

[0057] Figure 4 It is an analysis graph of the lipid and protein components of yam-derived exosome-like nanoparticles.

[0058] Figure 5 It is a cytotoxicity graph of yam-derived exosome-like nanoparticles.

[0059] Figure 6 It is a photo of yam-derived exosome-like nanoparticles promoting scratch migration and its quantification graph.

[0060] Figure 7 It is an anti-inflammatory effect graph of yam-derived exosome-like nanoparticles. Detailed Description of the Invention

[0061] The following further elaborates on the content of the present invention through specific examples. The raw materials, reagents, or devices used in the examples can be obtained from conventional commercial channels or by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0062] Example 1 Preparation of Yam-derived Exosome-like Nanoparticles (YENs)

[0063] A method for preparing yam-derived exosome-like nanoparticles provided by the present invention includes the following steps:

[0064] (1) Place fresh Chinese yams that can be used both as medicine and food in a juicer, add phosphate buffer solution pre-cooled at 4°C in a mass ratio of 1:1, stir and crush to obtain Chinese yam juice, and get the tissue raw pulp. The molar concentration of the phosphate buffer solution is 0.01M, and the pH is 7.4. The phosphate buffer solution is made by dissolving 3.473 g of Na2HPO4·12H2O, 0.226 g of NaH2PO4·12H2O, and 0.9 g of NaCl in triple-distilled water and making up the volume to 1000 mL.

[0065] (2) Centrifuge the tissue raw pulp obtained in step (1) at 4°C, with a centrifugation rate of 10000 rpm and a centrifugation time of 3 hours to obtain the tissue supernatant.

[0066] During ultracentrifugation, the high-speed rotation of the rotor generates heat, which causes the temperature to rise. Therefore, a refrigeration system must be used to keep the temperature at 4°C to maintain the activity of the substances.

[0067] (3) Centrifuge the tissue supernatant collected in step (2) at 4°C, with a centrifugation rate of 65000 rpm and a centrifugation time of 3 hours to obtain the precipitate.

[0068] (4) Resuspend the precipitate obtained in step (3) with phosphate buffer solution to obtain the resuspended solution; the precipitate will adhere to the wall after centrifugation and needs to be resuspended and dispersed; among them, the molar concentration of the phosphate buffer solution is 0.01M, and the pH is 7.4. The phosphate buffer solution is made by dissolving 3.473 g of Na2HPO4·12H2O, 0.226 g of NaH2PO4·12H2O, and 0.9 g of NaCl in triple-distilled water and making up the volume to 1000 mL.

[0069] (5) Perform sucrose density gradient centrifugation on the resuspended solution obtained in step (4), where the concentrations of the sucrose solutions are 8%, 30%, and 45%. The solvent for preparing the sucrose is Tris-Hcl, the pH of Tris-Hcl is 7.2, and the volume concentration is 20 mM / L; the centrifugation rate is 65000 rpm, and the centrifugation time is 3 hours.

[0070] The 8% concentration layer cannot be removed because different molecular weight substances need to be separated. During centrifugation, the boundaries of the three different concentration sucrose solutions of 8%, 30%, and 45% are very obvious and easy to distinguish; when the sucrose solution is centrifuged, a discontinuous density gradient will be formed in the centrifuge tube, and different molecular weight substances can be separated by the action of centrifugal force.

[0071] (6) Collect the liquid between the 30% and 45% sucrose solutions and centrifuge at 4°C at a rate of 65,000 rpm for 3 hours to obtain a precipitate; resuspend the obtained precipitate in phosphate buffer to obtain a resuspension, centrifuge at 4°C at a rate of 65,000 rpm for 3 hours to obtain a precipitate; resuspend the obtained precipitate in phosphate buffer to obtain a resuspension, and centrifuge at 4°C at a rate of 65,000 rpm for 3 hours to obtain a precipitate; resuspend the obtained precipitate in phosphate buffer to obtain a resuspension, at which time the resuspension is clear, centrifuge at 4°C at a rate of 65,000 rpm for 3 hours to obtain a precipitate, which is the exosome-like nanoparticles derived from yam. Resuspend the dispersed precipitate in PBS and store at -80°C for long-term storage.

[0072] YENs Characterization

[0073] The desalted YENs working solution was dropped onto the ultra-thin carbon film copper grid. After natural air drying, 20 μL of 1% uranyl acetate solution was added for negative staining. After natural air drying again, the YENs appearance was observed under a high-resolution transmission electron microscope.

[0074] Transmission electron microscopy results Figure 1 As shown, yam-derived exosome-like nanoparticles (YENs) exhibited a saucer-like structure similar to exosomes without obvious impurities.

[0075] After the YENs stock solution was diluted to an appropriate multiple, the average particle size, particle size distribution and charge state were analyzed using a nanoparticle tracking analyzer.

[0076] like Figure 2 As shown, the results of the nanoparticle tracking analyzer showed that the nanoparticles were evenly distributed, with a particle size of about 50 to 300 nm and an average particle size of 150.3 nm.

[0077] like Figure 3 As shown, the nanoparticle tracking analyzer results showed that YENs were negatively charged with a Zeta potential of -5.43 mV.

[0078] To explore the composition of the contents of YENs, we performed lipidomics and proteomics analyses.

[0079] Lipidomics results Figure 4As shown in A, YENs are rich in hexosylceramide (Hex1Cer, 27.9%), digalactosyldiacylglycerol (DGDG, 18.3%), and ceramide (Cer, 16.1%). In addition, the protein composition of YENs was also analyzed. A total of 4,866 proteins were identified by proteomics. Further, bioinformatics analysis was performed on all proteins. Using the GO database, the proteins were classified and annotated in terms of biological process, cellular component, and molecular function. The results showed ( Figure 4 in B–D), approximately 3,061 proteins were related to biological processes, 2,642 proteins were related to cellular components, and 3,022 proteins were related to molecular functions.

[0080] The above characterization results indicate that exosome-like nanoparticles can be successfully extracted and isolated from fresh yam by the preparation method of this example, and the isolated nanoparticles have a high purity.

[0081] Example 2 Cytotoxicity test of yam-derived exosome-like nanoparticles (YENs)

[0082] In this example, the skin keratinocyte HaCaT was used to perform a cytotoxicity experiment on YENs prepared according to the method of Example 1.

[0083] The wound healing experiment includes the following steps:

[0084] (1) YENs were prepared with reference to the preparation method of Example 1.

[0085] (2) HaCaT cells were seeded in a 96-well plate and cultured overnight in a cell culture incubator.

[0086] (3) Different concentrations of YENs samples were added to the culture media of the experimental groups, and no YENs sample was added to the culture media of the blank control. Culturing was continued for 24 hours.

[0087] (4) The culture media in the 96-well plate were aspirated, 0.5 mg / mL MTT solution was added, and the plate was cultured in the incubator for 4 hours.

[0088] (5) DMSO solution was added and shaken on a shaker for 15 minutes, and the absorbance at 570 nm was measured to analyze the cell viability. Each group was cultured in 3 replicate wells, and the experiment was repeated 3 times.

[0089] By measuring the OD value and calculating the cell viability of each group, as Figure 5 shown, there was no cytotoxicity when the addition amount of YENs was within 50 μg / mL, and there was a certain effect of promoting cell proliferation at low concentrations (1–5 μg / mL). When the concentration reached 100 μg / mL, there was a significant inhibitory effect on cell activity. The above results show the low cytotoxicity of YENs.

[0090] Example 3: Study on the function of yam-derived exosome-like nanoparticles (YENs) in promoting wound healing

[0091] In this example, the skin keratinocyte HaCaT was used to conduct a wound healing experiment on the YENs prepared according to the method of Example 1.

[0092] The wound healing experiment included the following steps:

[0093] (1) YENs were prepared with reference to the preparation method of Example 1.

[0094] (2) HaCaT cells were seeded in a 6-well plate and placed in a cell culture incubator until the cell density reached 80%.

[0095] (3) Use a 200 μL pipette tip to draw a cross at the center of the bottom of each well in the 6-well plate to simulate wound formation, and take a picture of the cell layer coverage rate under a 10-fold magnification microscope for recording.

[0096] (4) Add 20 ng / mL vascular endothelial growth factor (VEGF) to the positive control 6-well plate, add different concentrations of YENs to the experimental group 6-well plate, and the blank control was not treated additionally.

[0097] (5) After culturing with the samples for 24 hours, take pictures of the cell layer coverage rate again.

[0098] (6) Use Image J software to analyze and compare the cell layer coverage rate.

[0099] The wound healing rate was calculated by measuring the cell wound area at 0 hour and 24 hours, as Figure 6 shown. The results showed that when the concentration of YENs was 10 μg / mL, it had a significant effect on promoting wound healing, and the wound healing rate reached 53.4% at this time. When the concentration of YENs was increased to 50 μg / mL, the wound healing rate reached 73.2%, which was significantly higher than 61.3% of the positive control VEGF.

[0100] The experimental results showed that YENs had a significant effect on promoting wound healing and showed a dose-dependent relationship.

[0101] Example 4: Anti-inflammatory function study of yam-derived exosome-like nanoparticles (YENs)

[0102] In this example, lipopolysaccharide (LPS)-induced RAW264.7 macrophages and TNF-α-induced HaCaT cells were used to conduct an anti-inflammatory experiment on the YENs prepared according to the method of Example 1.

[0103] The wound healing experiment included the following steps:

[0104] (1) YENs were prepared according to the preparation method of Example 1.

[0105] (2) The cells were seeded in a 12-well plate and cultured overnight in a cell incubator.

[0106] (3) The culture medium was aspirated, and culture medium (blank control), culture medium containing 10 nM dexamethasone (Dex) (positive control), and culture medium containing different concentrations of YENs (experimental group) were added to the 12-well plate respectively.

[0107] (4) After culturing in the incubator for 20 hours, the culture medium was aspirated and the cells were washed 3 times with phosphate buffer.

[0108] (5) Total cellular RNA was extracted using RNAzol and reverse transcribed into cDNA.

[0109] (6) Using 480 SYBR Green, real-time fluorescence quantitative polymerase chain reaction was performed on equal amounts of each cDNA sample to detect the relative transcriptional levels of the genes of TNF-α, IL-1β, IL-6, iNOS in RAW264.7 cells and TNF-α, IL-1β, IL-6, COX-2 in HaCaT cells. The GAPDH gene level was used as an internal reference.

[0110] After RAW264.7 cells were treated with YENs, similar to the positive control Dex, the gene expressions of inflammatory factors such as TNF-α, IL-1β, IL-6, and iNOS were significantly down-regulated and showed a dose-dependent relationship. The inhibition rates of IL-1β and IL-6 exceeded 80% ( Figure 7 ). In the TNF-α-induced HaCaT cell inflammation model, the gene expressions of TNF-α, IL-1β, IL-6, and iNOS factors were also significantly inhibited.

[0111] The experimental results indicate that YENs have significant anti-inflammatory effects.

[0112] The results of the above examples strongly demonstrate that YENs can be successfully extracted with high purity under this preparation method. In terms of applications, YENs have low toxicity, anti-inflammatory, and wound healing-promoting effects, and have potential skin regeneration value and can be applied in the fields of cosmetics and medicine.

[0113] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of Dioscorea opposita-derived exosome-like nanoparticles in the preparation of products for repairing skin damage.

2. The application according to claim 1, wherein The Dioscorea opposita-derived exosome-like nanoparticles have a saucer-shaped structure.

3. The application according to claim 1, characterized in that, The particle size of the Dioscorea opposita-derived exosome-like nanoparticles is 50 - 300 nm. Preferably, the particle size of the Dioscorea opposita-derived exosome-like nanoparticles is 100 - 200 nm.

4. The application according to claim 1, wherein The Dioscorea opposita-derived exosome-like nanoparticles are negatively charged. Preferably, the Zeta potential of the Dioscorea opposita-derived exosome-like nanoparticles is -10 to -1 mV.

5. The application according to claim 1, characterized in that, The product for repairing skin damage is used for promoting skin wound healing and / or inhibiting skin inflammation.

6. The application according to claim 1, characterized in that, The product for repairing skin damage is cosmetics and drugs.

7. A preparation method of Dioscorea opposita-derived exosome-like nanoparticles, comprising the following steps: (a) Add a buffer solution to Dioscorea opposita, stir and break it into Dioscorea opposita juice to obtain tissue homogenate. (b) Centrifuge the tissue homogenate to obtain tissue supernatant. (c) Centrifuge the tissue supernatant to obtain a precipitate. (d) Resuspend the precipitate with a buffer solution to obtain a resuspension. (e) Sequentially add sucrose solutions with mass concentrations of 8%, 30% and 45% to the resuspension, and centrifuge. (f) Collect the liquid layer between the sucrose solution layers corresponding to 30% and 45% mass concentrations.

8. The preparation method according to claim 7, characterized in that, The centrifugation treatment in step (b) means centrifuging at a centrifugation rate of 6000 - 15000 rpm for 1 - 5 hours. Preferably, the centrifugation treatment in step (c) means centrifuging at a centrifugation rate of 50000 - 80000 rpm for 1 - 5 hours. Preferably, the centrifugation treatment in step (e) means centrifuging at a centrifugation rate of 50000 - 80000 rpm for 1 - 5 h. Preferably, the centrifugation treatments in steps (b), (c) and (e) are carried out at 4°C.

9. The preparation method according to claim 7, wherein The buffer solution in step (a) is a buffer solution pre-cooled to 2 - 6°C. Preferably, the mass ratio of Dioscorea opposita to the buffer solution in step (a) is 1:(1 - 5).

10. A Dioscorea opposita-derived exosome-like nanoparticle prepared by the preparation method according to any one of claims 7 - 9.

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