Method for producing collagen-containing exosome and collagen-containing exosome produced therefrom

Abstract

The present invention relates to a method for producing a collagen-containing exosome, a collagen-containing exosome produced therefrom, and a use thereof. The method according to the present invention can use ultrasound to maximize the loading efficiency of collagen into a plant-derived exosome while maintaining the functionality of collagen, and thus can be used in technology for effectively delivering collagen to target cells.

Description

Method for producing collagen-containing exosomes and collagen-containing exosomes produced therefrom

[0001] The present invention relates to a method for producing collagen-containing exosomes, collagen-containing exosomes produced therefrom, and uses thereof.

[0002]

[0003] Many attempts have been made to load proteins into exosomes, including 'incubation', 'electroporation', 'extrusion', 'freeze-thaw cycle', 'chimeric exosome method', 'endogenous loading', and 'sonication'. Each loading method has various advantages and disadvantages. Incubation has a low impact on the loading material and exosomes, but it also has the disadvantage of extremely low loading efficiency. In addition, the remaining methods, including 'electroporation', are known to have a negative effect on the sample to which they are applied, even if they are relatively efficient. In this regard, a method applying ultrasound can be an appropriate loading method that can minimize the gap between advantages and disadvantages depending on the application technology.

[0004] However, our study showed that when ultrasound was applied to macrophage-derived exosomes under specific conditions, 'Catalase' and 'TPP1' were induced at 26.1+ / -1.2% and 70㎍ TPP1 / 10, respectively. 11 exosomes showed an efficiency similar to that of human exosomes. Furthermore, Cas9 RNP, used in gene editing, showed an efficiency of approximately 15% under specific conditions targeting HEK-293T exosomes. As such, protein loading technology into exosomes reflects the structure and various characteristics of the protein, and it is very difficult to optimize the conditions for the exosomes that receive the protein to be loaded, depending on the donor cell, resulting in low efficiency.

[0005] Therefore, increasing loading efficiency not only offers significant benefits for future processes and costs, but also offers the significant advantage of maximizing the effect on recipient cells even with small quantities. Therefore, we sought to load collagen, a useful protein used in various cosmetics, health functional foods, and medical devices, onto plant-derived exosomes. While conducting research on methods to maximize the loading effect, we completed the present invention.

[0006]

[0007] The technical problem to be achieved by the present invention is to provide a method for producing collagen-containing exosomes and collagen-containing exosomes produced therefrom.

[0008] In addition, the technical task to be achieved by the present invention is to provide a cosmetic composition and a health functional food containing the exosome.

[0009]

[0010] The technical problems to be solved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0011]

[0012] In order to achieve the above technical task, one embodiment of the present invention provides a method for producing collagen-containing exosomes, including the steps of providing ultrasonic stimulation to plant-derived exosomes and collagen, respectively; and the step of providing ultrasonic stimulation to a mixture of the ultrasonic-stimulated exosomes and collagen.

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

[0014] In an embodiment of the present invention, the step of providing ultrasound stimulation to the plant-derived exosomes may be performed at an intensity of 20% to 40% of AMP for 30 to 120 seconds.

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

[0016] In an embodiment of the present invention, the step of providing ultrasonic stimulation to the mixture may be performed at an intensity of 15% to 40% of AMP for 30 to 120 seconds.

[0017] In an embodiment of the present invention, after providing ultrasonic stimulation to the mixture, a step of culturing the mixture on ice may be further included.

[0018] In order to achieve the above technical task, another embodiment of the present invention provides a collagen-containing exosome manufactured by the above manufacturing method.

[0019] In order to achieve the above technical task, another embodiment of the present invention provides a cosmetic composition or health functional food comprising the collagen-containing exosome.

[0020]

[0021] The present invention relates to a method for producing collagen-containing exosomes, collagen-containing exosomes produced therefrom, and their uses. The method of the present invention utilizes ultrasound to maximize the loading efficiency of collagen into plant-derived exosomes while maintaining the functionality of collagen, and can be utilized in a technology for effectively delivering collagen to target cells.

[0022]

[0023] The effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the composition of the invention described in the description or claims of the present invention.

[0024]

[0025] Figures 1 to 3 show the distribution and concentration of collagen-loaded plant-derived exosomes.

[0026] Figures 4 and 5 show the intracellular uptake of plant-derived exosomes loaded with collagen through immunofluorescence analysis.

[0027] Figures 6 to 8 show the results of flow cytometry analysis of plant-derived exosomes loaded with collagen.

[0028] Figures 9 and 10 show cell viability and the amount of collagen-related proliferation genes to confirm the cell proliferation effect after treatment with plant-derived exosomes loaded with collagen.

[0029] Figures 11 to 13 show the aging and regenerative effects after treatment with plant-derived exosomes loaded with collagen to confirm the functionality of collagen.

[0030] Figures 14 to 18 show the results of confirming the collagen loading efficiency in exosomes under conditions other than the optimal conditions of the ultrasonic treatment of the present invention.

[0031]

[0032] Hereinafter, the present invention will be described in detail.

[0033]

[0034] The present invention relates to a method for producing collagen-containing exosomes.

[0035] The method of the present invention comprises the steps of providing ultrasonic stimulation to plant-derived exosomes and collagen, respectively; and the step of providing ultrasonic stimulation to a mixture of the ultrasonic-stimulated exosomes and collagen.

[0036] The above plants may include, but are not limited to, dandelions, broccoli or carrots, for example.

[0037] The above plant may be used in one or more parts selected from the group consisting of stems, leaves, roots and aerial parts, and for example, dandelions may be used in leaves, and for example, broccoli or carrots may be used in whole plants.

[0038] The above exosomes are isolated from plants and can be isolated using known methods and conditions using known means. For example, exosomes can be isolated by treating a plant sample with ultrasound, centrifuging the sonicated sample to obtain a supernatant, and then filtering the obtained supernatant through a filter to isolate the exosomes from the filtrate.

[0039] The step of providing the above ultrasonic stimulation can be separated using known means and under known methods and conditions.

[0040] For example, the step of providing ultrasound stimulation to the plant-derived exosomes may be performed at an intensity of AMP 20% to 40% or AMP 22% to 30%, preferably AMP 25%, for 30 to 120 seconds or 50 to 70 seconds.

[0041] Additionally, the step of providing ultrasound stimulation to the collagen may be performed at an intensity of AMP 50% to 90% or AMP 60% to 80%, preferably AMP 70%, for 1 to 5 hours or 2 to 4 hours.

[0042] Additionally, the step of providing ultrasonic stimulation to the mixture may be performed at an intensity of 15% to 40% AMP or 20% to 35% AMP, preferably 30% AMP, for 30 to 120 seconds or 50 to 70 seconds.

[0043] Although the ultrasonic stimulation conditions are not limited to the above range, when performed within the above range, the degree of damage to collagen can be minimized and the loading efficiency can be maximized.

[0044] The method of the present invention may further include, if necessary, a step of providing ultrasonic stimulation to the mixture and then culturing the mixture on ice.

[0045] The above incubation can be performed for, for example, 5 to 20 minutes.

[0046] Additionally, after incubation on ice, it can be further incubated at room temperature for 1 to 3 hours.

[0047] The present invention relates to collagen-containing exosomes manufactured by the above manufacturing method.

[0048] The manufacturing method is as described above.

[0049] The above collagen-containing exosomes effectively maintain the functionality of collagen, and since collagen is contained within the exosomes, they are easily delivered to target cells, and the effect of collagen in the target cells can be maximized.

[0050] The present invention relates to a cosmetic composition or health functional food comprising the collagen-containing exosome.

[0051] Regarding collagen-containing exosomes, the same is as described above.

[0052] The cosmetic composition of the present invention can be formulated as a skin, lotion, toner, cosmetic soap, body wash, serum, cleansing lotion, essence, nourishing cream, pack, massage cream, etc., and can also be formulated as an emollient toner, an astringent toner, a nourishing toner, eye cream, eye essence, cleansing foam, cleansing water, powder, body lotion, body cream, body oil, body essence, makeup base, foundation, shampoo, or rinse, but is not limited thereto.

[0053] In addition, when used as a cosmetic composition, additional substances may be added according to the formulation of the external skin agent or cosmetic. For example, but not limited to, when the formulation is a paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component, and when the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included. In addition, when the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and preferably, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used, but is not limited thereto.When the formulation is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as carrier components, and when the formulation is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester may be used as carrier components, but are not limited thereto.

[0054] The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc.

[0055] The health functional food of the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body, and is meant to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.

[0056] The health functional food of the present invention may contain conventional food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0057] The items listed in the above food additive code include, but are not limited to, chemical 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 mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0058] For example, a health functional food in tablet form can be made by mixing the exosomes with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, adding a lubricant, etc., and then compression molding the mixture, or by directly compression molding the mixture. In addition, the health functional food in tablet form can also contain a maturing agent, etc., as needed.

[0059] Among health functional foods in capsule form, hard capsules can be manufactured by filling a mixture of the exosomes and additives such as excipients into a regular hard capsule, and soft capsules can be manufactured by filling a mixture of the exosomes and additives such as excipients into a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

[0060] The health functional food in the form of a ring can be prepared by mixing the above exosomes with excipients, binders, disintegrants, etc., and molding the mixture using a method known in the art. If necessary, the mixture can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc.

[0061] Granular health functional foods can be manufactured into granules by mixing the exosomes and excipients, binders, disintegrants, etc. using a conventionally known method, and may contain flavoring agents, flavor enhancers, etc., as needed.

[0062] The above health functional foods may include beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0063] The above health functional food can be taken orally as a nutritional supplement, and the form of application is not particularly limited.

[0064]

[0065] Hereinafter, the present invention will be described in detail by way of examples to specifically explain the present invention.

[0066]

[0067] Example

[0068] Example 1. Collagen loading into dandelion-derived exosomes

[0069] Dandelion leaves were used to isolate exosomes derived from dandelion, and the characteristics of exosomes isolated from dandelion leaves were analyzed and collagen loading was performed.

[0070] First, the dandelion leaf samples were weighed and distributed 1 g each into 50 ml tubes, and 10 ml of DPBS was added to each tube. Each sample was homogenized and crushed, and then ultrasonicated for 15 seconds. The ultrasonicated samples were filtered through a 40 μm filter. The filtered samples were then ultrasonicated for an additional 15 minutes.

[0071] A centrifuge was applied to the previously prepared sample to separate the sediment. The conditions were as follows. First, the rotation speed was set to 1,000xg, the time and temperature were set to 10 min, and 4℃, and the supernatant was separated from the plant tissue or residue. The separated supernatant was transferred to a new 50ml tube and centrifuged again, and the conditions were as follows (rotation speed: 3,000xg, the time and temperature were set to 20 min, and 4℃). After two rounds of centrifugation, the supernatant was filtered through a 0.2 μm filter and transferred to a new 50 ml tube. The filtered sample was finally placed in an Amicon®Ultra-15 Centrifugal Filter Unit (UFC9100, Millipore) and subjected to 10,000 g at 4 °C for 30 minutes. The concentrated dandelion exosome sample was dispensed into 1.5 ml tubes and stored in a -80 °C freezer.

[0072] Hydrolyzed collagen powder was diluted in DPBS to a concentration of 1 mg / ml and then sonicated. The sonication conditions for collagen were 70% intensity, 10 seconds of treatment, 10 seconds of pause, and 1,440 cycles for a total of 4 hours. 10 10 Dandelion exosomes that had been sonicated (AMP 25% (pulse 10 s on / 10 s off), 1 min) were added and adjusted to a total volume of 500 μl. Then, sonication was performed at 25% intensity for 10 s, followed by 10 s pause for a total of 6 cycles. After sonication, the mixture was allowed to react on ice for 10 min, followed by an additional 2 h at room temperature.

[0073]

[0074] Example 2. Collagen loading into broccoli-derived exosomes

[0075] Same as Example 1 except that the whole broccoli plant was used as a sample.

[0076]

[0077] Example 3. Collagen loading into carrot-derived exosomes

[0078] Same as Example 1 except that the whole carrot was used as a sample.

[0079]

[0080] Experimental methods and results

[0081] 1. NTA measurement

[0082] To measure the size distribution of exosomes, NTA was performed using a Nanosight instrument (Nanosight NS300, Malvern Panalytical). After separating exosomes and loading collagen into exosomes, an appropriate concentration range of exosomes (10) was determined for accurate measurement. 7 ~ 10 9 ) was diluted to match the sample. In addition, filtration was performed using a 0.22 μm filter to remove impurities and particles that may interfere with particle size and concentration analysis, and the size and size distribution of exosomes were measured.

[0083] The size distribution and concentration of collagen-containing exosomes derived from dandelion, broccoli, and carrot are shown in Figures 1 to 3, respectively.

[0084]

[0085] 2. Intracellular absorption analysis (immunofluorescence analysis)

[0086] To confirm the intracellular uptake of collagen-loaded exosomes, fluorescently stained collagen and fluorescently stained exosomes with DID were examined using immunofluorescence.

[0087] After culturing HDF cells for 3 days in DMEM high glucose culture medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C, 5x10 cells were seeded in 24-well plates. 4 Cells were inoculated at 1x10 9PBS was added to the exosomes loaded with dog collagen to make 50 μL, and 0.25 μL of DiD staining reagent was added, mixed well, and incubated at 37°C for 30 minutes. 50 μL of DiD-stained exosomes were treated with cells seeded in a 24-well plate and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. The next day, the culture medium was aspirated and removed, and the cells were harvested by treating them with a mixture of trypsin and EDTA. Centrifugation was performed at 1,000 rpm for 5 minutes, the supernatant was removed, and the pellet was washed with 1 mL of PBS and centrifuged at 1,000 rpm for 5 minutes. The obtained pellet was resuspended in 1 mL of PBS, 200 μL of PBS was sprayed, and only 170 μL was removed without allowing it to completely dry. 4% paraformaldehyde / PBS was applied to 150 μL of cover glass and fixed for 5 minutes at room temperature. After removing 4% paraformaldehyde / PBS, 150 μL of 0.3% TritonX-100 / PBS was added and permeabilized. After removing 0.3% TritonX-100 / PBS, 3% BSA / PBS was added and blocked for 1 hour at room temperature. The primary antibody (Collagen type I-col1a1) was diluted 1:1000 in 100 μL of 3% BSA / PBS and treated for 2 hours at room temperature. After removing the antibody, the sections were washed three times with 200 μL of 0.03% Tween20 / PBS. The secondary antibody was diluted 1:500 in 100 μL of 3% BSA / PBS and treated and reacted for 1 hour at room temperature. After antibody removal, the slides were washed three times with 200 μL of 0.03% Tween 20 / PBS and twice with clean PBS. 5 μL of mounting solution was added to the slide glass, the cover glass was inverted, and mounted. After 10 minutes, air was blocked with nail polish, and then photographs were taken under a microscope.

[0088] The results are shown in Figures 4 and 5.

[0089]

[0090] 3. Flow cytometry analysis

[0091] To confirm the loading rate of collagen in exosomes after loading collagen in exosomes, flow cytometry (FACSMelodyTM, BD Biosciences) analysis was performed using Qdot655 and Col1a1 antibodies.

[0092] 0.5 μL of aldehyde / sulfate-latex beads (A37304, Thermofisher) were added to 50 μL of collagen-loaded exosomes and incubated at room temperature for 15 minutes. An additional 1 mL of 0.1% BSA solution was added, and the mixture was mixed well and incubated overnight at 4°C. The following day, the mixture was centrifuged at 2,000 g for 10 minutes, the supernatant was removed, and the mixture was washed with 1 mL of DPBS. The mixture was centrifuged again at 2,000 g for 10 minutes and the supernatant was removed. The resulting pellet was resuspended in 50 μL of DPBS.

[0093] After transferring 20 μL of the resuspended pellet to an amber tube, 80 μL of DPBS was added to make the total volume 100 μL. For exosome staining, Qdot655 and for collagen staining, 5 μL of col1a1 antibody were added, mixed well, and incubated at 4°C for 1 hour. After adding 1 mL of DPBS and washing, centrifugation was performed at 2,000 g for 10 minutes, the supernatant was removed, resuspended in 1 mL of DPBS, and measured by flow cytometry.

[0094] The results for collagen-containing exosomes derived from dandelion, broccoli, and carrot are shown in Figures 6 to 8, respectively.

[0095]

[0096] 4. Evaluation of cell viability in exosomes

[0097] To evaluate the toxicity of collagen-loaded exosomes, a cell viability experiment was conducted. Human skin keratinocytes (HaCaT) were cultured in DMEM high glucose culture medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and then seeded in 96-well plates at a density of 1x10 per well. 4 Cells were seeded. The next day, the cells were treated with control, collagen, plant exosomes, and collagen-loaded plant exosomes. After 24 hours, 10 μL of Ez-cytox (DoGenBio, South Korea) was added per 100 μL of culture medium, and the reaction was incubated at 37°C for 1 hour and 30 minutes in a darkened environment. The absorbance was measured at 450 nm.

[0098] The results are shown in Fig. 9.

[0099]

[0100] 5. RNA isolation

[0101] Human fibroblasts were seeded at 5x10 in 60mm culture dishes. 5 When the cells were cultured and reached a density of about 50-60%, 5 mg / mL of collagen and 1x10 plant exosomes were added to the medium. 7 particles / mL, 1x10 collagen-loaded exosomes 7 particles / mL and cultured for 24 hours.

[0102] After 24 hours of treatment, the culture medium was removed, 1 mL of TRIzol was added, and the cells were harvested and transferred to a 1.5 mL tube. The sample was then left to stand at room temperature for 5 minutes. 200 μL of chloroform was added per 1 mL of TRIzol, mixed (vortexed) for 15 seconds, and left to stand at room temperature for 10 minutes.

[0103] The mixture was centrifuged at 14,000 rpm at 4°C for 20 minutes, and the supernatant was transferred to a new 1.5 mL tube. After adding 600 μL of isopropanol, the tube was gently inverted to mix, and left to stand at room temperature for 10 minutes. The mixed sample was centrifuged at 14,000 rpm at 4°C for 10 minutes to precipitate the RNA pellet.

[0104] To wash the precipitated pellet, 1 mL of ethanol (EtOH) was added, the tube was gently tapped, and centrifuged at 14,000 rpm at 4°C for 5 minutes. Remaining ethanol was removed as much as possible using a pipette, the tube was opened, and the pellet was dried at room temperature for approximately 10 minutes. Finally, an appropriate amount of sterile water was added depending on the size of the RNA pellet to resuspend the RNA, and the amount of RNA was measured by absorbance.

[0105] The results are shown in Fig. 10.

[0106]

[0107] 6. cDNA Synthesis and Real-Time PCR

[0108] First, cDNA synthesis was performed using SuperiorScript II Reverse Transcriptase (RT005M). The experiment was performed according to the kit protocol, and 4 μL of 5x 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 then sterilized water was added to make a final volume of 20 μL. The mixture was reacted at 42°C for 1 hour and then heat treated at 95°C for 5 minutes to terminate the reaction.

[0109] After TOPreal TMReal-time PCR was performed using SYBR Green qPCR PreMIX (RT500M). Following the kit protocol, the PCR reaction solution was prepared by mixing 10 μL of TOP real qPCR 2x 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.

[0110] The PCR cycle was set as follows: Initial denaturation was performed at 95°C for 10 minutes. Subsequently, 55 cycles were performed: denaturation at 95°C for 10 seconds, annealing at 60°C for 15 seconds, and elongation at 72°C for 30 seconds.

[0111] The anti-aging and regenerative effects were confirmed after treatment with plant-derived collagen-containing exosomes, and the results are shown in Figures 11 to 13.

[0112]

[0113] 7. Deriving optimal conditions for loading collagen into plant exosomes.

[0114] When collagen, plant-derived exosomes, and their mixtures were not treated with ultrasound, the degree of loading was confirmed and is shown in Figures 14 to 16.

[0115]

[0116] 8. Deriving optimal conditions for collagen ultrasonic treatment

[0117] The optimal conditions were derived by varying the sonication conditions for collagen. As a result, it was confirmed that collagen and plant exosomes existed in a 'free' state when treated at 50% AMP for 30 minutes and 1 hour, respectively (Fig. 17). When sonicated at 60% AMP for 1 hour, 2 hours, and 4 hours, it was confirmed that a small amount of merged parts existed (Fig. 18).

[0118] Therefore, it was confirmed that the loading efficiency was reduced outside the loading conditions of the present invention.

Claims

1. A step of providing ultrasound stimulation to plant-derived exosomes and collagen, respectively; and A method for producing collagen-containing exosomes, comprising the step of providing ultrasound stimulation to a mixture of the ultrasound-stimulated exosomes and collagen.

2. A manufacturing method according to claim 1, wherein the plant is dandelion, broccoli or carrot.

3. A manufacturing method according to claim 1, wherein the step of providing ultrasound stimulation to the plant-derived exosome is performed at an intensity of 20% to 40% of AMP for 30 to 120 seconds.

4. A manufacturing method according to claim 1, wherein the step of providing ultrasonic stimulation to the collagen is performed at an intensity of 50% to 90% of AMP for 1 to 5 hours.

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

6. A manufacturing method according to claim 1, further comprising a step of culturing the mixture on ice after providing ultrasonic stimulation to the mixture.

7. Collagen-containing exosome manufactured by the manufacturing method of claim 1.

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

9. A health functional food comprising the collagen-containing exosome of claim 7.

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