Exosome carrying amino acid and collagen peptide as well as preparation method and application of exosome

Abstract

The invention provides a preparation method of exosomes carrying amino acid and collagen peptide (AmiSYN). The preparation method comprises the following steps: (1) extracting exosomes from various sources; (2) packaging amino acids and collagen peptide, wherein the amino acids comprise glycine, proline, arginine, lysine, hydroxyproline, aspartic acid and cystine; (3) purifying to remove uncombined components; (4) detecting the particle size and form of the exosome; (5) calculating the packaging rate; (6) verifying an anti-aging effect by using a cell model; and (7) evaluating the anti-wrinkle effect in practical application. The invention further provides the exosome carrying the amino acid and the collagen peptide prepared according to the preparation method and application of the exosome in skin aging resistance and skin care. The prepared exosome carrying the AmiSYN is high in packaging efficiency, the skin absorption rate of the AmiSYN is effectively improved, and the anti-aging effect of the AmiSYN can be remarkably enhanced.

Description

Technical Field

[0001] The present application generally relates to the field of biomedicine technology, and more specifically, to exosomes carrying amino acids and collagen peptides, and preparation methods and applications thereof. Background Art

[0002] Ageing, free radical accumulation, ultraviolet radiation, environmental pollution and bad living habits are the main factors leading to skin aging. Skin aging is not only a hot issue in the field of beauty, but also directly affects the health of the public. From the appearance, aging skin is characterized by looseness, sagging, deepening of wrinkles and melanin deposition; from a histological perspective, aging skin is usually characterized by atrophy of the epidermis and dermis, flattening of the epidermal reticular ridges, and a decrease in the number of fibroblasts and mast cells. In addition, aging is also accompanied by a decrease in the number of collagen fibers and an imbalance in the ratio of type III collagen to type I collagen, which further weakens the support and elasticity of the skin. External factors such as ultraviolet rays, blue light and pollution can also trigger inflammatory reactions in skin tissue. In this process, immune cells such as mast cells and macrophages release a large number of inflammatory factors, stimulate the production of proteolytic enzymes and matrix metalloproteinases (MMPs), further destroy collagen and elastic fibers in the skin, lead to the disintegration of the extracellular matrix and accelerate skin aging. The various mechanisms of skin aging ultimately point to a core factor - the loss of collagen.

[0003] Collagen is one of the most important structural proteins in the skin. It not only provides support and strength to the tissue, but also gives the skin elasticity and radiance by maintaining tissue integrity. Collagen can re-establish connections in damaged tissues and promote skin repair and regeneration. In addition, collagen also plays a vital role in wound healing and reducing inflammation. However, as we age, the synthesis of collagen gradually decreases, while the degradation rate accelerates, which gradually degrades the structural integrity and function of the skin.

[0004] However, the practical application of collagen faces many technical bottlenecks. First, due to the large molecular weight of collagen, its transdermal absorption efficiency is low, and it is difficult to effectively penetrate the skin barrier and reach deep tissues, which in turn affects its actual effect. Secondly, the chemical properties of collagen are relatively unstable and are easily affected by environmental factors such as light, heat and oxidation, resulting in loss of its activity. In addition, the systemic application of collagen may cause side effects such as local irritation or allergic reactions, limiting its widespread application in skin care products.

[0005] Exosomes are nano-scale membrane vesicles secreted by cells. In recent years, they have shown broad application prospects in the biomedical field, especially in drug delivery. Exosomes have excellent biocompatibility and can be efficiently recognized and taken up by body cells. Their natural membrane structure enables them to cross the skin barrier and avoid triggering immune responses. This unique property makes exosomes an ideal drug delivery carrier, especially in the precise targeted delivery of active molecules to the dermis, showing significant advantages.

[0006] Therefore, the present invention is proposed to overcome the technical difficulties in the practical application of collagen, improve the synthesis efficiency of collagen, enhance its transdermal absorption efficiency and reduce potential side effects. Summary of the invention

[0007] In view of this, the present application provides exosomes carrying amino acids and collagen peptides, and preparation methods and applications thereof. Amino acids and collagen peptides are the basic raw materials for collagen synthesis, directly participate in the formation of collagen fibers, and further promote collagen synthesis and tissue repair; the nanoscale size and unique membrane structure of exosomes enable them to smoothly pass through the skin barrier, ensuring that amino acids and collagen peptides penetrate deep into the skin, which can effectively promote the regeneration of skin collagen and thus significantly enhance the anti-aging effect.

[0008] In order to achieve the above objectives, this application provides the following technical solutions:

[0009] The present application provides a method for preparing exosomes carrying amino acids and collagen peptides, comprising the following steps: (1) extracting exosomes from multiple sources; (2) encapsulating amino acids and collagen peptides, wherein the amino acids include glycine, proline, arginine, lysine, hydroxyproline, aspartic acid and cysteine; (3) purifying and removing unbound components; (4) detecting the particle size and morphology of exosomes; (5) calculating the encapsulation rate; (6) verifying the anti-aging effect with a cell model; and (7) evaluating the anti-wrinkle effect in practical applications.

[0010] Preferably, the extraction sources of exosomes include a variety of cell, tissue and body fluid sources, such as mesenchymal stem cells (such as bone marrow-derived (BM-MSCs), adipose-derived (AD-MSCs), umbilical cord-derived (UC-MSCs), placenta-derived (PL-MSCs)), milk-derived (such as human milk, cow milk, goat milk, horse milk, camel milk, deer milk), blood-derived (such as human blood, animal blood) and exosomes derived from induced pluripotent stem cells (iPSCs).

[0011] More preferably, different treatment methods are adopted for different sources: For cell sources: including mesenchymal stem cells and induced pluripotent stem cells (iPSCs) from bone marrow, fat, umbilical cord, and placenta, the corresponding cells are first cultured and allowed to reach the logarithmic growth phase, and then the cell culture supernatant is collected, followed by conventional exosome extraction. For tissue sources: including umbilical cord, placenta and other tissue sources, the supernatant is first extracted by tissue homogenization, and then conventional exosome extraction is performed. For body fluid sources: including body fluid sources such as milk and blood, after collecting fresh samples, use a 40μm filter for preliminary filtration to remove fat particles and larger particulate matter, and then perform conventional exosome extraction.

[0012] Preferably, the step (1) comprises: preliminary centrifugation: centrifuging a variety of exosome-derived samples at room temperature at 3,000×g twice for 10 minutes each time; fine filtration: continuously filtering the supernatant using a 0.22 μm filter to remove residual impurities; ultrafiltration concentration: ultrafiltration using a 100k Dalton ultrafiltration tube, and performing short-term centrifugation at 6,000×g for 4 minutes, 7 minutes, 14 minutes and 30 minutes in sequence, and repeating the process until all the supernatant has been processed to obtain a concentrated exosome sample; PBS washing: adding PBS solution to the concentrated sample and washing 2-3 times to remove residual culture medium; sterile filtration and storage: filtering the exosome sample using a sterile filter, aliquoting it into cryopreservation tubes, and storing it at -80°C.

[0013] Preferably, in step (2), the encapsulation process of amino acids and collagen peptides adopts electroporation, and microcurrent poration is performed using an electroporator, and the conditions of microcurrent poration are set to CA-137 mode.

[0014] Preferably, in the step (2), the amounts of amino acids and collagen peptides are as follows: 0.2 g to 0.4 g collagen peptide, 0.1 g to 0.2 g glycine, 0.1 g to 0.2 g proline, 0.1 g to 0.2 g arginine, 0.1 g to 0.2 g lysine, 0.1 g to 0.2 g hydroxyproline, 0.1 g to 0.2 g aspartic acid, 0.1 g to 0.2 g cystine, and all the ingredients are dissolved in 1 ml of exosome solution.

[0015] Preferably, the step (3) comprises: selecting an ultrafiltration tube with a molecular cutoff limit of 100k Daltons, diluting the mixed solution after microcurrent perforation to 15 ml and adding it to an ultrafiltration device, ultrafiltration is performed at a centrifugal force of 6,000×g, the initial centrifugation time is 10-15 minutes, repeating the ultrafiltration step 2-3 times, adding PBS or other buffer after each ultrafiltration, adjusting the solution volume, and collecting the concentrated exosome solution in the ultrafiltration tube after the ultrafiltration is completed.

[0016] Preferably, in step (4), the exosome solution collected in the ultrafiltration step is analyzed for particle size using a nanoparticle tracking analyzer (NTA), and the particle size of the exosomes is about 115 nm (see Figure 2 In addition, a portion of the exosome samples were taken and imaged using a transmission electron microscope (TEM) to observe the morphology and structure of the exosomes to confirm their integrity and morphological characteristics (see Figure 1 ).

[0017] Preferably, in step (5), the amino acid content is detected by liquid chromatograph (Model 1260, Agilent Technologies, Inc.). The seven amino acids (glycine, proline, arginine, lysine, hydroxyproline, aspartic acid, cysteine) in the exosome solution are quantitatively analyzed. First, a standard solution of each amino acid is prepared and a standard curve is established. Then, the exosome sample to be tested is subjected to liquid chromatography analysis after pretreatment, and the concentration of each amino acid in the sample is obtained by comparison with the standard curve.

[0018] Preferably, the collagen peptide content is also detected using a liquid chromatograph (Model 1260, Agilent Technologies Inc.) Purified collagen peptide is used as a standard to establish a standard curve and determine the collagen peptide content in the exosome sample.

[0019] Preferably, the encapsulation efficiency (EE) is calculated by comparing the amino acid and collagen peptide contents actually detected before exosome loading (initial addition amount) and after loading (content after ultrafiltration and washing steps to remove unencapsulated components).

[0020] Preferably, in step (6), a cell model for anti-aging efficacy is established. In order to evaluate the anti-aging effect of exosomes carrying AmiSYN, human skin fibroblasts (HSF) are selected as an experimental model. A senescent cell model is established by continuous passage to the 10th generation. The 10th generation senescent cells are randomly divided into three groups: a control group (PBS buffer), an Exo group (addition of exosome solution without active ingredient encapsulation) and a Pro-Exo group (addition of exosomes carrying AmiSYN). After each group of cells is cultured under standard culture conditions for 24 hours, cell samples are collected for gene expression analysis.

[0021] Preferably, in step (7), an anti-wrinkle effect evaluation in practical application is performed. In order to further evaluate the anti-wrinkle effect of exosomes carrying AmiSYN in practical application, a clinical effect evaluation experiment was designed. Chinese healthy men or women aged between 40 and 60 years old who met the test requirements were selected and randomly divided into three groups, 10 people in each group, a total of 30 people, for anti-wrinkle effect evaluation.

[0022] The present invention also provides exosomes carrying amino acids and collagen peptides prepared by any of the preparation methods described above, and use thereof in anti-skin aging and skin care.

[0023] The preparation method of exosomes carrying amino acids and collagen peptides provided in the present application can promote collagen synthesis and tissue repair by carrying AmiSYN, the basic raw material for collagen synthesis, through exosomes, and using exosomes as carriers. The double-layer phospholipids can isolate oxygen, ultraviolet rays and moisture in the external environment, reduce the risks of oxidation, photodegradation and hydrolysis, thereby extending the shelf life of its active ingredients. Compared with traditional methods, the extraction process of the exosomes of the present invention is simple and low in cost. Since exosomes have excellent biocompatibility and usually do not induce significant immune responses or toxicity, they are safer. In addition, exosomes are similar to the components of human cell membranes and can effectively fuse with cell membranes, thereby ensuring that active substances can enter cells in a targeted manner and be fully absorbed. The natural biocompatibility of exosomes makes them an ideal drug delivery carrier, which helps to reduce the occurrence of adverse reactions. The nanoscale size and unique membrane structure of exosomes enable them to penetrate biological barriers, such as skin barriers and blood-brain barriers, thereby achieving more efficient drug delivery. Exosomes not only prolong the release time of drugs, but also enhance the therapeutic effect and ensure the stable delivery of active ingredients to target tissues. In addition, exosomes contain a variety of bioactive molecules, such as proteins, lipids, and RNA, which may have immunomodulatory effects and help provide additional therapeutic benefits during drug delivery.

[0024] In summary, the exosomes carrying AmiSYN of the present application provide a safer, gentler and more effective anti-aging solution. Compared with traditional anti-aging therapies, this solution effectively avoids possible side effects and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the present application are specifically set forth in the appended claims. The features and advantages of the present application will be better understood by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the present application are utilized. The accompanying drawings are only used for the purpose of illustrating the embodiments and should not be considered as limiting the present application. Also, throughout the drawings, the same reference numerals are used to represent the same elements.

[0026] Figure 1 This is an image of the exosomes carrying AmiSYN provided in the present application under a transmission electron microscope (TEM);

[0027] Figure 2 Tracking analysis of exosome nanoparticles carrying AmiSYN provided in this application; and

[0028] Figure 3 In order to establish an aging model by passaged human skin fibroblasts to the 10th generation, PBS, Exo, and Pro-Exo were added for intervention, and the expression of aging-related genes p21 and p53 was detected. DETAILED DESCRIPTION

[0029] The present application provides milk exosomes carrying AmiSYN, and a preparation method and application thereof, which achieve full adsorption and encapsulation of AmiSYN by exosomes, effectively enhance the skin's absorption of AmiSYN, promote collagen synthesis and tissue repair, and thus exert a significant anti-aging effect, improve skin elasticity, and reduce wrinkles.

[0030] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present application, the technical solution of the present application is now described in detail below, but it should not be understood as limiting the scope of implementation of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods for which specific conditions are not specified in the embodiments are operated according to the conventional conditions of the field. The starting reagent materials used in the embodiments are all commercially available unless otherwise specified, or prepared with reference to the prior art in the field.

[0031] Example 1 Extraction of exosomes from various sources

[0032] This example describes the process of extracting exosomes from various sources. Exosomes can come from the following different sources: mesenchymal stem cells (such as bone marrow-derived (BM-MSCs), adipose-derived (AD-MSCs), umbilical cord-derived (UC-MSCs), placenta-derived (PL-MSCs)), milk-derived (such as human milk, cow milk, goat milk, horse milk, camel milk, deer milk), blood-derived (such as human blood, animal blood) and exosomes derived from induced pluripotent stem cells (iPSCs).

[0033] Different sources are handled differently:

[0034] For cell sources: including mesenchymal stem cells and induced pluripotent stem cells (iPSCs) from bone marrow, fat, umbilical cord, and placenta, the corresponding cells are first cultured and allowed to reach the logarithmic growth phase, and then the cell culture supernatant is collected, followed by conventional exosome extraction.

[0035] For tissue sources: including umbilical cord, placenta and other tissue sources, first extract the supernatant through tissue homogenization, and then perform conventional exosome extraction.

[0036] For body fluid sources: including breast milk, blood and other body fluid sources, after collecting fresh samples, use a 40μm filter to perform preliminary filtration to remove fat particles and larger particulate matter, and then perform conventional exosome extraction.

[0037] The conventional exosome extraction process is as follows: Initial centrifugation: Centrifuge the body fluid sample twice at 3,000 × g at room temperature for 10 minutes each time. Fine filtration: Continuously filter the supernatant using a 0.22 μm filter to remove residual impurities. Ultrafiltration concentration: Ultrafiltration was performed using a 100k Dalton ultrafiltration tube. First, short-term centrifugation was performed at 6,000 × g for 4, 7, 14, and 30 minutes, and the process was repeated until all the supernatant was processed to obtain a concentrated exosome sample. PBS wash: Add PBS solution to the concentrated sample and wash 2 to 3 times to remove residual culture medium. Sterile filtration and storage: Filter the exosome samples using a sterile filter, dispense them into cryovials, and store them at -80°C.

[0038] Example 2 AmiSYN packaging into exosomes

[0039] Collagen peptides (0.2g-0.4g), glycine (0.1g-0.2g), proline (0.1g-0.2g), arginine (0.1g-0.2g), lysine (0.1g-0.2g), hydroxyproline (0.1g-0.2g), aspartic acid (0.1g-0.2g) and cystine (0.1g-0.2g) were dissolved in 1ml of exosome solution. Subsequently, microcurrent perforation was performed using an electroporator produced by Lonza, Germany, in CA-137 mode to ensure that AmiSYN can be fully adsorbed and encapsulated in exosomes.

[0040] Example 3 Removal of unbound AmiSYN

[0041] Select an ultrafiltration tube with a molecular cutoff limit of 100k Dalton to ensure that only unbound small molecule AmiSYN is removed, while exosomes carrying AmiSYN are retained. Dilute the mixed solution after microcurrent perforation to 15 ml and add it to the ultrafiltration device. Ultrafiltration is performed at a centrifugal force of 6,000×g, and the initial centrifugation time is 10 to 15 minutes. Through the centrifugation process, unbound free collagen peptides, seven amino acids and other small molecule impurities will be filtered out. To ensure that unbound AmiSYN is completely removed, repeat the ultrafiltration step 2 to 3 times. After each ultrafiltration, add PBS or other buffer to adjust the solution volume and maintain the stability of the exosomes. After the ultrafiltration is completed, collect the concentrated exosome solution in the ultrafiltration tube to ensure that AmiSYN is completely encapsulated in the exosomes and no free AmiSYN remains.

[0042] Example 4 Detection of the size and morphology of exosomes

[0043] The size and morphology of exosomes were detected by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The specific steps are as follows:

[0044] Exosome solution preparation: The exosome solution collected from the ultrafiltration step was washed with PBS to remove excess impurities and buffer to ensure the sample was pure and ready for subsequent analysis.

[0045] Nanoparticle Tracking Analysis (NTA): The exosome samples were analyzed for particle size using an NTA instrument. The particle size distribution, average diameter, and concentration of the exosomes were determined by NTA. In this example, the particle size distribution of the exosomes was as follows: Figure 2 As shown in the figure, the particle size range is mainly concentrated around 115nm. Ensure that the test results are within this range to verify the quality and stability of exosomes.

[0046] Transmission electron microscopy (TEM) observation: Take a small amount of exosome sample, drop it on the carbon film, and use appropriate negative staining agent to improve the contrast of the sample. Observe the morphology and structure of exosomes by TEM to confirm their integrity and morphological characteristics. Figure 1 The specific morphology and structure of exosomes are shown.

[0047] Example 5 Calculation of AmiSYN encapsulation rate

[0048] Amino acid content was detected using a liquid chromatograph (Model 1260, Agilent Technologies). Seven amino acids (glycine, proline, arginine, lysine, hydroxyproline, aspartic acid, and cysteine) in the exosome solution were quantitatively analyzed. First, a standard solution of each amino acid was prepared and a standard curve was established. Then, the exosome sample to be tested was pre-treated and subjected to liquid chromatography analysis. By comparing with the standard curve, the concentration of each amino acid in the sample was obtained.

[0049] The collagen peptide content was also detected using a liquid chromatograph (Model 1260, Agilent Technologies, Inc.). Purified collagen peptide was used as a standard to establish a standard curve and determine the collagen peptide content in the exosome samples.

[0050] Encapsulation efficiency calculation: The encapsulation efficiency (EE) was calculated by comparing the actual detected amino acid and collagen peptide contents before exosome loading (initial addition amount) and after loading (content after ultrafiltration and washing steps to remove unencapsulated components). The encapsulation efficiency calculation formula is as follows:

[0051] The total mass of the target substance initially added: refers to the total amount of collagen peptides or amino acids added during the preparation process. The mass of the target substance encapsulated in the exosomes: refers to the initial addition amount minus the mass of the target substance not encapsulated (the mass of the target substance detected in the filtrate or supernatant).

[0052] The calculation results of the encapsulation efficiency of amino acids and collagen peptides are shown in Table 1. Table 1 Element Initial concentration (mg / ml) Encapsulation concentration (mg / ml) Encapsulation rate (%) Collagen Peptides 0.3 0.135 45％ Glycine (Gly) 0.15 0.0675 45％ Proline (Pro) 0.15 0.072 48％ Arginine (Arg) 0.15 0.06 40％ Lysine (Lys) 0.15 0.0675 45％ Hydroxyproline (Hyp) 0.15 0.0675 45％ Aspartic acid (Asp) 0.15 0.06 40％ Cystine (Cys) 0.15 0.0675 45％

[0053] Example 6 Effect on the expression of aging-related genes p21 and p53

[0054] In order to evaluate the anti-aging effect of the exosomes carrying AmiSYN provided in this application, especially its effect on the expression of aging-related genes p21 and p53, the following experiment was conducted. Human skin fibroblasts (HSF) were selected as the experimental model. A cell aging model was established by continuous passage to the 10th generation. The 10th generation aging model cells were randomly divided into three groups for treatment: a control group (PBS buffer), an Exo group (adding exosome solution without active ingredient packaging) and a Pro-Exo group (adding exosomes carrying AmiSYN). After each group of cells was cultured under standard culture conditions for 24 hours, cell samples were collected for subsequent gene expression analysis.

[0055] Real-time quantitative polymerase chain reaction (qRT-PCR) technology was used to detect the mRNA and protein expression levels of aging-related genes p21 and p53 in each group of cells. The specific steps are as follows:

[0056] RNA extraction and reverse transcription: Total RNA was extracted from cells in each group using a commercial RNA extraction kit, and then reverse transcribed to synthesize cDNA.

[0057] qRT-PCR detection: Real-time quantitative PCR was performed on p21 and p53 genes using specific primers, and fluorescence detection was performed using the SYBR Green dye method to record the relative expression level of each gene.

[0058] The experimental results are as follows Figure 3 As shown, qRT-PCR results: p21 gene expression: compared with the control group, the p21 mRNA level in the Exo group decreased significantly; the p21 mRNA level in the Pro-Exo group decreased further. p53 gene expression: the p53 mRNA level in the Exo group decreased, and the Pro-Exo group decreased significantly.

[0059] The above experimental results show that exosomes carrying AmiSYN can significantly inhibit the expression of aging-related genes p21 and p53. Compared with the Exo group with only exosomes added, the Pro-Exo group showed a stronger inhibitory effect. As key genes in cell cycle regulation and stress response, the overexpression of p21 and p53 is closely related to cell aging. The expression levels of these two genes in the Pro-Exo group were significantly reduced, indicating that the encapsulated collagen peptides and amino acids played a synergistic role in delaying cell aging.

[0060] Application example: Anti-wrinkle effect of milk exosome essence carrying AmiSYN

[0061] The preparation method of the milk exosome essence carrying AmiSYN includes the following components in mass percentage: 10% to 15% milk exosome carrying AmiSYN, 20% to 25% glycerol, 6% to 9% aloe vera water, and the balance is purified water. Other preparation steps are as described above in the present invention.

[0062] Anti-wrinkle efficacy test

[0063] Purpose of the test: To evaluate the anti-wrinkle efficacy of each sample of Example 2 by measuring the changes in facial wrinkle feature counts of healthy male and female subjects before and after using the test samples.

[0064] Subjects: Select healthy Chinese men or women who meet the test requirements, aged between 40 and 60 years old, who have not taken or applied any external medications, can cooperate well with the test, and maintain a regular life during the study. The subjects were randomly divided into three groups, 10 in each group, for a total of 30 people, and the specific groups are as follows: Group 1 (control group): 20% to 25% glycerol, 6% to 9% aloe vera water, and the remainder purified water were used. The second group (Exo group) used 10%-15% milk exosomes, 20%-25% glycerol, 6%-9% aloe vera water, and the rest was purified water. The third group (Pro-Exo group) used 10%-15% milk exosomes carrying AmiSYN, 20%-25% glycerol, 6%-9% aloe vera water, and the rest was purified water.

[0065] Test method: According to the anti-aging efficacy evaluation method in the "Evaluation Standards for Cosmetic Efficacy Claims" issued by the State Food and Drug Administration, an objective instrument evaluation method is used. The specific test parameters and steps are as follows:

[0066] Test parameter: Wrinkle feature count.

[0067] Instrument: Skin image analyzer.

[0068] Test time points: Day 0 and Day 28.

[0069] Test area: face.

[0070] Test method: Use facial cleansing products to clean the face and ensure that the skin is clean. Use dry facial tissue to dry the skin. Sit still for 30 minutes in a laboratory environment with a temperature of 21±1℃ and a relative humidity of 50±10% to stabilize the skin condition. Use a skin image analyzer to collect facial wrinkle features and perform statistical analysis.

[0071] Directions: Twice a day, morning and evening. 28 days. Apply the essence evenly to the entire face and massage gently until fully absorbed.

[0072] Anti-wrinkle efficacy test results: The following are the average values ​​of wrinkle feature counts of different treatment groups before and after use, see Table 2 for details. Table 2 Wrinkle feature counts (average value for each group) Group 0-Day Wrinkle Count 28 Day Wrinkle Count Reduction ratio (%) Control group 141.21±19.62 139.54±20.10 1.2 Exo Group 156.55±17.28 139.14±16.86 11.1 Pro-Exo Group 152.28±20.92 121.91±18.22 20.0 Reduction ratio (%) = ((0-day wrinkle count - 28-day wrinkle count) / 0-day wrinkle count) x 100%.

[0073] The experimental results showed that compared with the control group, the facial wrinkle counts in the Exo group and the Pro-Exo group were significantly reduced after 28 days of use. Among them, the number and proportion of wrinkle reduction in the Pro-Exo group were significantly higher than those in the Exo group and the control group, indicating that the milk exosome essence carrying AmiSYN has a superior anti-wrinkle effect.

[0074] Based on the research results of this application example, milk exosome essence carrying AmiSYN can be widely used in anti-aging skin care products, such as essence, cream, mask and other products. This exosome carrier system not only improves the skin absorption rate and stability of active ingredients, but also significantly enhances the anti-aging effect of the product, and has broad market prospects and application value.

[0075] In the specification provided herein, a large number of specific details are described. However, it should be understood that the embodiments of the present disclosure can be practiced without these specific details. In some embodiments, well-known equipment and technology are not shown in detail so as not to obscure the understanding of this specification.

[0076] Although exemplary embodiments of the present application have been shown and described herein, it is apparent to those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will appreciate many changes, modifications and substitutions without departing from the present application. It should be understood that various alternatives to the embodiments of the present application described herein may be adopted in the process of practicing the present application. The following claims are intended to define the scope of the present application, and therefore cover the solutions and their equivalents within the scope of these claims.

Claims

1. A method for preparing exosomes carrying amino acids and collagen peptides, characterized in that: The steps include: (1) Extraction of exosomes from various sources; (2) Encapsulation of amino acids and collagen peptides, wherein the amino acids include glycine, proline, arginine, lysine, hydroxyproline, aspartic acid and cysteine; (3) purification to remove unbound components; (4) Detection of exosome particle size and morphology; (5) Calculation of encapsulation rate; (6) Cell model to verify anti-aging effects; (7) Evaluation of anti-wrinkle effects in practical applications.

2. The preparation method according to claim 1, characterized in that In step (1), the extraction sources of exosomes include mesenchymal stem cells, fat, umbilical cord, placenta, breast milk, blood and induced pluripotent stem cells.

3. The preparation method according to claim 1, characterized in that: The step (1) comprises: Initial centrifugation: Various exosome-derived samples were centrifuged twice at 3,000 × g for 10 min at room temperature; Fine filtration: Use a 0.22 μm filter to continuously filter the supernatant to remove residual impurities; Ultrafiltration concentration: ultrafiltration was performed using a 100k Dalton ultrafiltration tube, and short-term centrifugation was performed at 6,000 × g for 4 minutes, 7 minutes, 14 minutes, and 30 minutes, respectively. The process was repeated until all the supernatant was processed to obtain the concentrated exosome sample; PBS washing: Add PBS solution to the concentrated sample and wash 2-3 times to remove the residual culture medium; Sterile filtration and storage: Filter the exosome samples using a sterile filter, dispense them into cryovials, and store them at -80°C.

4. The preparation method according to claim 1, characterized in that: In the step (2), the encapsulation process of amino acids and collagen peptides adopts electroporation, and micro-current poration is performed using an electroporator, and the conditions of micro-current poration are set to CA-137 mode.

5. The preparation method according to claim 1, characterized in that: In the step (2), the amounts of amino acids and collagen peptides are as follows: 0.2 g to 0.4 g collagen peptide, 0.1 g to 0.2 g glycine, 0.1 g to 0.2 g proline, 0.1 g to 0.2 g arginine, 0.1 g to 0.2 g lysine, 0.1 g to 0.2 g hydroxyproline, 0.1 g to 0.2 g aspartic acid, and 0.1 g to 0.2 g cystine, and all the components are dissolved in 1 ml of exosome solution.

6. The preparation method according to claim 1, characterized in that: The step (3) comprises: selecting an ultrafiltration tube with a molecular cutoff limit of 100k Daltons, diluting the mixed solution after microcurrent perforation to 15 ml and adding it to an ultrafiltration device, ultrafiltration is performed at a centrifugal force of 6,000×g, the initial centrifugation time is 10-15 minutes, repeating the ultrafiltration step 2-3 times, adding PBS or other buffer after each ultrafiltration, adjusting the solution volume, and collecting the concentrated exosome solution in the ultrafiltration tube after the ultrafiltration is completed.

7. The preparation method according to claim 1, characterized in that: In the step (4), the particle size of the exosomes is 115 nm.

8. The preparation method according to claim 1, characterized in that: In step (5), the amino acid content is detected by using a liquid chromatograph to quantitatively analyze the amino acids in the exosome solution to obtain the concentration of each amino acid in the sample.

9. The preparation method according to claim 1, characterized in that: In the step (6), human skin fibroblasts are selected as the experimental model, and a senescent cell model is established by continuous passage to the 10th generation.

10. Exosomes carrying amino acids and collagen peptides prepared by the preparation method according to any one of claims 1 to 9, and use thereof in anti-skin aging and skin care.

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