Method for preparing collagen-containing exosomes and collagen-containing exosomes prepared thereby

Abstract

The present invention relates to a method for preparing collagen-containing exosomes, collagen-containing exosomes prepared thereby, and uses thereof. The method of the present invention utilizes ultrasound to maximize the loading efficiency of collagen into plant-derived exosomes while maintaining the functionality of the collagen, and thus can be applied in technologies for efficient delivery of collagen to target cells.

Description

Technical Field

[0001] This invention relates to a method for preparing collagen-containing exosomes, the collagen-containing exosomes prepared thereby, and their uses. Background Technology

[0002] Numerous attempts have been made to load proteins into exosomes, with representative methods including incubation, electroporation, extrusion, freeze-thaw cycles, chimeric exosome methods, endogenous loading, and sonication. Each method has its own advantages and disadvantages. Incubation has minimal impact on the loaded material and exosomes, but suffers from extremely low loading efficiency. Other methods, including electroporation, can also adversely affect the sample when their efficiency is relatively high. Therefore, depending on the specific ultrasonic technology used, ultrasound can be considered a suitable loading method that minimizes the gap between its advantages and disadvantages.

[0003] However, according to the investigation, when macrophage-derived exosomes were subjected to ultrasound under specific conditions to load Catalase and TPP1, the efficiencies were approximately 26.1+ / -1.2% and 70 μg TPP1 / 10, respectively. 11 Exosomes. Furthermore, for Cas9 RNPs used as gene-editing agents, the efficiency is approximately 15% when using exosomes derived from HEK-293T under specific conditions. Thus, protein loading techniques into exosomes are affected by protein structure and various characteristics, and optimizing the conditions for the exosomes receiving the protein to be loaded based on the donor cells is very difficult and inefficient.

[0004] Therefore, improving loading efficiency not only offers significant advantages in subsequent processes and costs, but also has the remarkable benefit of maximizing its effect on recipient cells even with only small amounts. Thus, this invention was developed in the process of researching methods to maximize the loading efficiency of collagen, a useful protein used in various cosmetics, health foods, or medical devices, loaded into plant-derived exosomes. Summary of the Invention

[0005] Technical issues

[0006] The technical problem to be solved by the present invention is to provide a method for preparing collagen-containing exosomes and the collagen-containing exosomes prepared therefrom.

[0007] Furthermore, the technical problem to be solved by the present invention is to provide cosmetic compositions and health functional foods containing the exosomes.

[0008] The technical problems to be solved by the present invention are not limited to those described above. Those skilled in the art can clearly understand other technical problems not mentioned based on the following description.

[0009] Problem-solving methods

[0010] To address the aforementioned technical problems, one embodiment of the present invention provides a method for preparing collagen-containing exosomes, comprising:

[0011] The steps of applying ultrasound stimulation to plant-derived exosomes and collagen, respectively; and

[0012] The step of applying ultrasonic stimulation to a mixture obtained by mixing exosomes and collagen that have been stimulated by the ultrasound.

[0013] In embodiments of the present invention, the plant may be dandelion, broccoli, or carrot.

[0014] In embodiments of the present invention, the step of applying ultrasonic stimulation to the plant-derived exosomes can be performed for 30 to 120 seconds at an intensity of 20% to 40% AMP.

[0015] In embodiments of the present invention, the step of applying ultrasound stimulation to the collagen can be performed for 1 to 5 hours at an intensity of 50% to 90% of AMP.

[0016] In embodiments of the invention, the step of applying ultrasonic stimulation to the mixture can be performed for 30 to 120 seconds at an intensity of 15% to 40% of the AMP.

[0017] In an embodiment of the present invention, the preparation method may further include the step of incubating the mixture on ice after applying ultrasonic stimulation to the mixture.

[0018] To address the aforementioned technical problems, another embodiment of the present invention provides collagen-containing exosomes prepared by the preparation method described above.

[0019] To address the aforementioned technical problems, another embodiment of the present invention provides a cosmetic composition or health food containing the collagen-containing exosomes.

[0020] The effects of the invention

[0021] This invention relates to a method for preparing collagen-containing exosomes, the collagen-containing exosomes prepared thereby, and their uses. The method of this invention utilizes ultrasound to maximize the loading efficiency of collagen in plant-derived exosomes while maintaining the functionality of collagen, and therefore can be applied to techniques for effectively delivering collagen to target cells.

[0022] The effects of this invention are not limited to those described above, but should be understood to include all effects that can be inferred from the invention as described in the specification or claims. Attached Figure Description

[0023] Figures 1 to 3 The particle size distribution and concentration of plant-derived exosomes loaded with collagen are shown.

[0024] Figure 4 and Figure 5 The extent of cellular uptake is shown by immunofluorescence analysis of plant-derived exosomes loaded with collagen.

[0025] Figures 6 to 8 The results of flow cytometry analysis of plant-derived exosomes loaded with collagen are shown.

[0026] Figure 9 and Figure 10 The figures show cell viability and the amount of collagen-related proliferation genes measured to confirm the cell proliferation effect after treatment with plant-derived exosomes loaded with collagen.

[0027] Figures 11 to 13 The study demonstrates the aging and regeneration effects confirmed after treatment with plant-derived exosomes loaded with collagen to validate the functionality of collagen.

[0028] Figures 14 to 18 The loading efficiency of collagen in exosomes is shown under conditions other than the optimal conditions of the ultrasonic treatment of the present invention. Detailed Implementation

[0029] The present invention will now be described in detail.

[0030] This invention relates to a method for preparing collagen-containing exosomes.

[0031] The method of the present invention includes:

[0032] The steps of applying ultrasound stimulation to plant-derived exosomes and collagen, respectively; and

[0033] The step of applying ultrasonic stimulation to a mixture obtained by mixing exosomes and collagen that have been stimulated by the ultrasound.

[0034] The plant is not limited to this; for example, it could be dandelion, broccoli, or carrot.

[0035] One or more parts selected from the group consisting of the stem, leaves, roots and above-ground parts of the plant may be used. For example, leaves may be used for dandelions, and the whole plant may be used for broccoli or carrots.

[0036] The exosomes are isolated from plants and can be separated using known methods and conditions. For example, the plant sample can be sonicated, the sonicated sample can be centrifuged to obtain a supernatant, and the exosomes can be separated from the filtrate obtained by filtering the supernatant using a filter.

[0037] The step of applying the ultrasound stimulation can be performed using known methods and conditions, and by known means.

[0038] As a specific example, the step of applying ultrasound stimulation to the plant-derived exosomes can be performed for 30 to 120 seconds or 50 to 70 seconds at an intensity of 20% to 40% or 22% to 30%, preferably 25% AMP.

[0039] Furthermore, the step of applying ultrasound stimulation to the collagen can be performed for 1 to 5 hours or 2 to 4 hours at an intensity of AMP 50% to 90% or AMP 60% to 80%, preferably AMP 70%.

[0040] Furthermore, the step of applying ultrasonic stimulation to the mixture can be performed for 30 to 120 seconds or 50 to 70 seconds at an intensity of AMP 15% to 40% or AMP 20% to 35%, preferably AMP 30%.

[0041] Ultrasound stimulation conditions are not limited to the above range, but when performed within the above range, the degree of damage to collagen can be minimized and the loading efficiency can be maximized.

[0042] If necessary, the method of the present invention may further include the step of incubating the mixture on ice after applying ultrasonic stimulation to the mixture.

[0043] The incubation can, for example, last from 5 to 20 minutes.

[0044] In addition, after incubation on ice, it can be further incubated at room temperature for 1 to 3 hours.

[0045] This invention relates to collagen-containing exosomes prepared by the aforementioned preparation method.

[0046] The preparation method is as described above.

[0047] The collagen-containing exosomes effectively maintain the functionality of collagen while containing collagen within the exosomes, thus facilitating delivery to target cells and maximizing the effect of collagen in target cells.

[0048] This invention relates to cosmetic compositions or health foods containing said collagen-containing exosomes.

[0049] Regarding the collagen-containing exosomes, as previously described.

[0050] The cosmetic compositions of the present invention can be formulated into dosage forms such as lotion, emulsion, toner, beauty soap, shower gel, serum, facial cleanser, essence, nourishing cream, mask, and massage cream. In addition, they can also be formulated into dosage forms such as softening lotion, astringent lotion, nourishing lotion, eye cream, eye serum, facial foam, makeup remover, powder, body lotion, body cream, body oil, body serum, makeup primer, foundation, shampoo, or conditioner, but are not limited thereto.

[0051] Furthermore, when used in cosmetic compositions, substances may be added depending on the dosage form of the topical skin agent or cosmetic. For example, but not limited to, when the dosage form is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin wax, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as carrier components; when the dosage form is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as carrier components. In particular, when the dosage form is a spray, propellants such as chlorofluorocarbons, propane / butane, or dimethyl ether may also be included.

[0052] In addition, when the dosage form is a solution or emulsion, a solvent, solubilizer or emulsifier can be used as the carrier component, preferably water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol fatty acid ester, polyethylene glycol or sorbitan fatty acid ester, but not limited to these.

[0053] When the dosage form is a suspension, liquid diluents such as water, ethanol, or propylene glycol can be used as carrier components; suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan anhydride ester; microcrystalline cellulose; aluminum hydroxide; bentonite; agar; tragali gum, etc.

[0054] When the dosage form is a cleaning preparation containing surfactants, the carrier component may include fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethanesulfonates, imidazoline derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters, but is not limited to these.

[0055] The health functional food of the present invention can be prepared and processed into tablets, capsules, powders, granules, liquids, pills and other forms.

[0056] In this invention, "health functional food" refers to food prepared and processed using raw materials or ingredients that have beneficial functions to the human body, and refers to food consumed for health purposes, such as regulating nutrients or exerting physiological effects on the structure and function of the human body.

[0057] The health functional food of the present invention may contain common food additives. Unless otherwise specified, suitability as a food additive shall be determined in accordance with the general principles and general test methods of the Codex Alimentarius of Food Additives approved by the Korean Ministry of Food and Drug Safety, and in accordance with the specifications and standards of the relevant category.

[0058] The items included in the aforementioned "Compendium of Food Additives" include, but are not limited to: chemically synthesized compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, crystalline cellulose, sorghum pigment, and guar gum; and compound preparations such as L-glutamate preparations, alkali additives for flour products, preservative preparations, and tar pigment preparations.

[0059] For example, health functional foods in tablet form can be prepared by: granulating a mixture obtained by mixing the exosomes with excipients, binders, disintegrants, and other additives using conventional methods, and then adding lubricants and compressing the mixture; or, the mixture can be directly compressed. Furthermore, the health functional foods in tablet form may also contain flavoring agents, etc., as needed.

[0060] In health functional foods in capsule form, hard capsules can be prepared by filling conventional hard capsules with a mixture obtained by mixing the exosomes with excipients and other additives; soft capsules can be prepared by filling a capsule matrix such as gelatin with a mixture obtained by mixing the exosomes with excipients and other additives. Depending on the requirements, the soft capsules may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc.

[0061] Health functional foods in pill form can be prepared by molding a mixture obtained by mixing the exosomes with excipients, binders, disintegrants, etc., according to existing known methods; if necessary, it can be coated with sucrose or other coating agents, or the surface can be coated with substances such as starch or talc.

[0062] Health functional foods in granule form can be made into granules by mixing the exosomes with excipients, binders, disintegrants, etc., according to existing known methods, and may contain flavoring agents, taste enhancers, etc. as needed.

[0063] The health functional foods mentioned can be beverages, meats, chocolates, food products, pastries, pizzas, instant noodles, other noodle products, chewing gum, candies, ice cream, alcoholic beverages, multivitamins, and health supplements, etc.

[0064] The aforementioned health functional foods can be taken orally as nutritional supplements, and there are no particular restrictions on their form of administration.

[0065] The present invention will be described in detail below through embodiments.

[0066]

Example

[0067] Example 1. Loading collagen into dandelion-derived exosomes

[0068] To isolate dandelion-derived exosomes, dandelion leaves were used, and the characteristics of the exosomes isolated from dandelion leaves were analyzed and collagen loaded.

[0069] First, the weight of dandelion leaf samples was measured and aliquoted into 50 mL tubes at 1 g per tube, with 10 mL of DPBS added to each tube. The dandelion leaf samples were homogenized using a homogenizer and then sonicated for 15 seconds. The sonicated samples were then filtered through a 40 μm filter. The filtered samples were then sonicated for another 15 minutes.

[0070] The prepared sample was centrifuged to separate the precipitate. The conditions were as follows: First, the rotation speed, time, and temperature were set to 1,000 × g, 10 min, and 4 °C, respectively, to initially separate the supernatant from the plant tissue or residue. The separated supernatant was transferred to a new 50 mL tube and centrifuged a second time under the following conditions: rotation speed of 3,000 × g, time of 20 min, and temperature of 4 °C.

[0071] After two centrifugations, the supernatant was filtered through a 0.2 μm filter and transferred to a new 50 mL tube. The filtered sample was then added to an Amicon® Ultra-15 Centrifugal Filter Unit (UFC9100, Millipore) and treated at 10,000 × g and 4 °C for 30 minutes. The concentrated dandelion exosome sample was aliquoted into 1.5 mL tubes and stored at −80 °C.

[0072] Hydrolyzed collagen powder was diluted to a concentration of 1 mg / mL with DPBS and then sonicated. The collagen was sonicated at 70% intensity, with 10 seconds of treatment followed by 10 seconds of rest constituting one cycle, for a total of 4 hours and 1,440 cycles.

[0073] Add 10 to the ultrasonically treated collagen 10 Dandelion exosomes were sonicated [AMP 25% (pulse on 10 s / off for 1 minute)], and the total volume was adjusted to 500 μL. Subsequently, sonication was performed at 25% intensity, with a cycle of 10 seconds on, 10 seconds off, for a total of 6 cycles. After sonication, the mixture was allowed to react on ice for 10 minutes, followed by a further reaction at room temperature for 2 hours.

[0074] Example 2. Loading collagen into broccoli-derived exosomes

[0075] Except that the whole broccoli plant was used as the sample, the procedure was the same as in Example 1.

[0076] Example 3. Loading collagen into carrot-derived exosomes

[0077] Except that the whole carrot plant was used as the sample, the procedure was the same as in Example 1.

[0078] Experimental methods and results

[0079] 1. NTA determination

[0080] To determine the particle size distribution of exosomes, NTA measurements were performed using a NanoSight instrument (NanoSight NS300, Malvern Panalytical). After isolating exosomes and loading collagen into them, the samples were diluted to an appropriate exosome concentration range (10⁻⁶) for accurate measurement. 7 Up to 10 9 ).

[0081] In addition, to remove impurities and particles that may interfere with particle size and concentration analysis, a 0.22 μm filter was used for filtration, and the particle size and particle size distribution of exosomes were determined.

[0082] The particle size distribution and concentration of collagen-containing exosomes from dandelion, broccoli, and carrot are shown in the figures. Figures 1 to 3 middle.

[0083] 2. Cellular uptake analysis (immunofluorescence analysis)

[0084] To confirm the extent of cellular uptake of collagen-loaded exosomes, immunofluorescence was used to observe fluorescently stained collagen and exosomes stained with DiD.

[0085] HDF cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C for 3 days, followed by inoculation with 5 × 10⁻⁶ cells per cell line. 4 A sample of cells was seeded into 24-well plates.

[0086] To 1×10 9 Add PBS to each exosome loaded with collagen to make a total volume of 50 μL, add 0.25 μL of DiD staining reagent, mix thoroughly, and incubate at 37°C for 30 minutes.

[0087] 50 μL of DiD-stained exosomes were added to cells seeded in 24-well plates and then cultured for 24 hours in an incubator at 37°C and 5% CO2.

[0088] The next day, the culture medium was removed by aspiration, and a mixture of trypsin and EDTA was added to obtain cells. The cells were then centrifuged at 1,000 rpm for 5 minutes, and the supernatant was removed. The pellet was washed with 1 mL of PBS and centrifuged at 1,000 rpm for 5 minutes.

[0089] The obtained precipitate was resuspended in 1 mL PBS, and 200 μL of the PBS resuspension was used for layering. Then only 170 μL was removed to prevent the sample from drying completely.

[0090] Add 150 μL of 4% paraformaldehyde / PBS to the coverslip and fix for 5 minutes at room temperature. After removing the 4% paraformaldehyde / PBS, add 150 μL of 0.3% Triton X-100 / PBS for permeation treatment.

[0091] After removing 0.3% Triton X-100 / PBS, add 3% BSA / PBS and block at room temperature for 1 hour. Dilute the primary antibody (type I collagen-Col1a1) 1:1000 in 100 μL of 3% BSA / PBS and treat at room temperature for 2 hours.

[0092] After removing the antibody, wash three times with 200 μL of 0.03% Tween 20 / PBS. Dilute the secondary antibody 1:500 in 100 μL of 3% BSA / PBS and treat with the solution, then react at room temperature for 1 hour.

[0093] After removing the antibodies, wash three times with 200 μL of 0.03% Tween 20 / PBS, and then twice with clean PBS. Add 5 μL of mounting solution to the slide, invert the coverslip and mount it. After 10 minutes, use nail polish to isolate it from air, and then take pictures using a microscope.

[0094] The results are shown in Figure 4 and Figure 5 middle.

[0095] 3. Flow cytometry analysis

[0096] After loading collagen into exosomes, flow cytometry (FACSMelody™, BD Biosciences) was performed using Qdot655 and Col1a1 antibodies to confirm the collagen loading rate in the exosomes.

[0097] Add 0.5 μL of aldehyde / sulfate-latex beads (A37304, Thermo Fisher) to 50 μL of exosomes loaded with collagen and react at room temperature for 15 minutes. Then add 1 mL of 0.1% BSA solution, mix thoroughly, and react overnight at 4°C.

[0098] The next day, the sample was centrifuged at 2,000×g for 10 minutes, and the supernatant was removed. After washing with 1 mL of DPBS, the sample was centrifuged again at 2,000×g for 10 minutes, and the supernatant was removed. The resulting precipitate was resuspended in 50 μL of DPBS.

[0099] Transfer 20 μL of the resuspended precipitate to a brown tube, and add 80 μL of DPBS to bring the total volume to 100 μL. Add Qdot655 for exosome staining and 5 μL of Col1a1 antibody for collagen staining, mix thoroughly, and incubate at 4°C for 1 hour.

[0100] After washing with 1 mL of DPBS, centrifuge at 2,000×g for 10 minutes, remove the supernatant and resuspend in 1 mL of DPBS, and then perform the assay by flow cytometry.

[0101] Results of collagen-containing exosomes from dandelion, broccoli, and carrot are shown in the figures below. Figures 6 to 8 middle.

[0102] 4. Evaluation of cell viability after exosome treatment

[0103] To evaluate the toxicity of collagen-loaded exosomes, cell viability assays were performed.

[0104] Human keratinocytes (HaCaT) were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, followed by inoculation with 1 × 10⁶ cells per well. 4 A sample of cells was seeded into 96-well plates.

[0105] On the second day, the control group, collagen, plant exosomes, and plant exosomes loaded with collagen were used for treatment, respectively.

[0106] After 24 hours, 10 μL of Ez-Cytox (DoGenBio, South Korea) was added per 100 μL of culture medium, and the mixture was reacted in the dark at 37°C for 1 hour and 30 minutes. The absorbance was then measured at 450 nm.

[0107] The results are shown in Figure 9 middle.

[0108] 5. RNA isolation

[0109] 5×10 5 Personal fibroblasts were cultured in 60 mm culture dishes. When the cells reached approximately 50% to 60% confluence, collagen at a concentration of 5 mg / mL and collagen at a concentration of 1×10⁻⁶ were respectively administered. 7 Plant exosomes particles / mL and a concentration of 1×10⁻⁶ 7 Exosomes loaded with collagen were processed at a particle / mL and cultured for 24 hours.

[0110] After 24 hours of treatment, the culture medium was removed, 1 mL of TRIzol was added to harvest the cells, and the cells were transferred to a 1.5 mL tube. The sample was then incubated at room temperature for 5 minutes.

[0111] Add 200 μL of chloroform for every 1 mL of TRIzol, vortex for 15 seconds, and let stand at room temperature for 10 minutes.

[0112] Centrifuge the mixture at 14,000 rpm and 4°C for 20 minutes, and transfer the supernatant to a new 1.5 mL tube. Then add 600 μL of isopropanol, gently invert the tube to mix, and let stand at room temperature for 10 minutes.

[0113] The mixed sample was centrifuged at 14,000 rpm and 4°C for 10 minutes to precipitate RNA.

[0114] To wash away the precipitate, add 1 mL of ethanol (EtOH), gently tap the tube, and centrifuge at 14,000 rpm and 4°C for 5 minutes. Use a pipette to remove as much residual ethanol as possible, and open the tube cap to allow the precipitate to dry at room temperature for about 10 minutes.

[0115] Finally, add an appropriate amount of sterile water according to the size of the RNA precipitate to resuspend the RNA, and determine the amount of RNA by absorbance.

[0116] The results are shown in Figure 10 middle.

[0117] 6. cDNA synthesis and real-time PCR

[0118] First, cDNA synthesis was performed using SuperiorScript II Reverse Transcriptase (RT005M). The experiment was conducted according to the kit's operating procedures. 4 μL of 5× First-Strand Buffer, 1 μL of SuperiorScript II Reverse Transcriptase (200 units / μL), 1 μL of dNTP mixture, 2 μL of 0.1 M DTT, 2 μg of RNA, and 1 μL of oligo dT were mixed, and sterile water was added to bring the final volume to 20 μL. The mixture was incubated at 42°C for 1 hour, and then heat-treated at 95°C for 5 minutes to terminate the reaction.

[0119] Subsequently, real-time PCR was performed using TOPreal™ SYBR Green qPCR PreMIX (RT500M). Following the kit instructions, 10 μL of TOPreal qPCR 2× PreMIX, 1 μL of forward primer (10 pmol), 1 μL of reverse primer (10 pmol), 5 μL of sterile water, and 30 ng of cDNA template were mixed to prepare the PCR reaction solution.

[0120] The PCR cycle settings were as follows. For initial denaturation, the reaction was carried out at 95°C for 10 minutes. Subsequently, 55 cycles were performed, consisting of denaturation at 95°C for 10 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 30 seconds.

[0121] Treatment with plant-derived collagen-containing exosomes confirmed aging and regeneration effects, the results of which are shown in... Figures 11 to 13 middle.

[0122] 7. Determine the optimal conditions for loading collagen into plant exosomes.

[0123] Under conditions in which more than one of the following substances—collagen, plant-derived exosomes, and mixtures thereof—was not subjected to sonication, the loading degree was confirmed, and the results are shown in… Figures 14 to 16 middle.

[0124] 8. Determine the optimal conditions for ultrasonic treatment of collagen.

[0125] The optimal conditions for sonication of collagen were determined by varying the sonication process. Results showed that when collagen was sonicated at 50% AMP for 30 minutes or 1 hour, both collagen and plant exosomes were confirmed to exist in a "free" state. Figure 17 When collagen was sonicated at 60% AMP for 1 hour, 2 hours, or 4 hours, a small amount of merged parts were confirmed. Figure 18 ).

[0126] Therefore, it can be confirmed that loading efficiency decreases under conditions other than those of the present invention.

Claims

1. A method for preparing collagen-containing exosomes, comprising: The steps of applying ultrasound stimulation to plant-derived exosomes and collagen, respectively; as well as The step of applying ultrasound stimulation to a mixture obtained by mixing exosomes and collagen that have been stimulated by the ultrasound.

2. The preparation method according to claim 1, wherein, The plant in question is dandelion, broccoli, or carrot.

3. The preparation method according to claim 1, wherein, The step of applying ultrasound stimulation to the plant-derived exosomes was performed for 30 to 120 seconds at an intensity of 20% to 40% of AMP.

4. The preparation method according to claim 1, wherein, The step of applying ultrasound stimulation to the collagen was performed for 1 to 5 hours at an intensity of 50% to 90% of AMP.

5. The preparation method according to claim 1, wherein, The step of applying ultrasonic stimulation to the mixture is performed for 30 to 120 seconds at an intensity of 15% to 40% AMP.

6. The preparation method according to claim 1, further comprising: The step of incubating the mixture on ice after applying ultrasonic stimulation to the mixture.

7. An exosome containing collagen, prepared by the preparation method described in claim 1.

8. A cosmetic composition comprising the collagen-containing exosomes of claim 7.

9. A health functional food comprising the collagen-containing exosomes as described in claim 7.

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