Exosome vehicle, method of preparation and use thereof

Abstract

The application belongs to the technical field of cosmetics or high-end biomedical field, and discloses an exosome solvent, a preparation method and application thereof. The exosome solvent comprises a sodium hyaluronate compound, an active type I collagen alpha 1 precursor chain compound, a complex polypeptide and a solvent. The exosome solvent fully utilizes the sodium hyaluronate compound, the active type I collagen alpha 1 precursor chain compound and the complex polypeptide in the exosome solvent to produce a synergistic effect with the exosome, and additionally endows the exosome with synergistic functions such as moisturizing, repairing and anti-aging on the basis of stabilizing the exosome, so that the exosome solvent is a compound functional solvent integrating stable storage, active maintenance and function enhancement.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics or high-end biomedical field, and relates to an exosome solvent, a preparation method and application thereof. BACKGROUND

[0002] In the field of injection medical aesthetics, cosmetic skin care, an exosome solvent is a solution specially used for preserving and resolubilizing exosomes. The main function of the common exosome solvent is to dissolve exosome lyophilized powder. The main components of the exosome solvent are one or a combination of physiological saline, deionized water, glycerol and butanediol. In order to increase the function or improve the service life of the traditional solvent, humectants (such as sodium hyaluronate, dextran, collagen, etc.), osmotic pressure regulators (such as mannitol, Tween 80, trehalose, etc.), preservatives (such as 1,2-hexanediol, pentanediol, sorbic acid, hydroxybenzoic acid, phenoxyethanol, etc.), pH regulators (such as triethanolamine, etc.), viscosity regulators (such as carbomer, hydroxyethyl cellulose, etc.) and other additives are added. However, these additives only increase the other effects of the solvent from the surface and do not participate in active reactions. Therefore, the existing solvent products are mostly limited to physical dissolution functions, lack of systematic protection of the stability of active ingredients, and are prone to introduce particulate pollution during the resolubilization process, resulting in an increase in the risk of local irritation. In addition, some preservatives such as hydroxybenzoic acid have the risk of sensitization, and long-term use may damage the skin microecological balance. After the lyophilized powder is resolubilized, the bioavailability of the active ingredients cannot be effectively maintained, especially the protection of sensitive ingredients such as polypeptides and growth factors, resulting in a decrease in efficacy. SUMMARY

[0003] In view of the above problems, the main purpose of the present application is to provide an exosome solvent, a preparation method and application thereof. The exosome solvent is not only a solvent with only dissolution and preservation functions in the prior art, but also a complex functional solution with stable preservation, active maintenance and function enhancement. The exosome solvent not only protects the exosomes, but also has multiple functions by combining with the exosomes.

[0004] To achieve the above-mentioned purpose of the application, the first aspect of the present application provides an exosome solvent, which comprises a sodium hyaluronate complex, an active type I collagen alpha 1 precursor chain complex, a complex polypeptide and a solvent.

[0005] The second aspect of the present application provides a preparation method of the exosome solvent according to the first aspect, which comprises: weighing the sodium hyaluronate complex, the active type I collagen alpha 1 precursor chain complex and the complex polypeptide according to the weight percentage, dissolving them in a solvent and mixing them uniformly to obtain the exosome solvent.

[0006] The third aspect of the present application provides a use of the exosome solvent of the first aspect or the exosome solvent prepared by the preparation method of the second aspect in preserving exosomes.

[0007] Compared with the prior art, the present application has at least the following advantages:

[0008] (1) The exosome solvent provided by the present application contains sodium hyaluronate complex, active collagen type I alpha 1 precursor chain complex, and composite polypeptide. The sodium hyaluronate complex, active collagen type I alpha 1 precursor chain complex, and composite polypeptide in the exosome solvent are fully utilized to produce a synergistic effect with the exosomes, thereby endowing the exosomes with additional functions such as moisturizing, repairing, and anti-aging on the basis of stabilizing the exosomes. The exosome solvent is a complex functional solvent that integrates stable preservation, active maintenance, and function enhancement.

[0009] (2) In the preparation method of the exosome solvent provided by the present application, only the sodium hyaluronate complex, active collagen type I alpha 1 precursor chain complex, and composite polypeptide need to be uniformly mixed and dissolved in a solvent, which is simple and easy to operate.

[0010] (3) When the exosome solvent provided by the present application is applied in preserving exosomes, the exosome stock solution can stabilize the exosomes after being mixed with the exosome solvent. In addition, the exosome solvent can endow the exosomes with certain whitening, anti-inflammatory, antioxidant, and anti-aging effects, thereby promoting the clinical transformation of the exosomes to a certain extent. DETAILED DESCRIPTION

[0011] The technical solutions of the present application will be explained and described in more detail below. However, it should be understood that the above-mentioned technical features of the present application and the technical features described in detail below (such as the examples) can be combined with each other to form new or preferred technical solutions within the scope of the present application. Due to the limited space, they will not be listed one by one here.

[0012] As mentioned above, the technical solutions of the present application are obtained through long-term and in-depth research and a large number of practices, and will be described in detail below.

[0013] One aspect of the present application provides an exosome solvent, which includes sodium hyaluronate complex, active collagen type I alpha 1 precursor chain complex, composite polypeptide, and solvent.

[0014] The exosome solvent of the present application discards the traditional solvent for the purpose of dissolving the exosome freeze-dried powder and the solvent composition in the components mainly for dissolving the exosome freeze-dried powder, and creatively proposes a complex functional solvent integrating stable storage, activity maintenance and function enhancement, which comprises a sodium hyaluronate complex, an active type I collagen alpha 1 precursor chain complex and a complex polypeptide. The present application fully utilizes the sodium hyaluronate complex, the active type I collagen alpha 1 precursor chain complex and the complex polypeptide in the exosome solvent to produce a synergistic effect with the exosome, and additionally endows the exosome with synergistic functions such as moisturizing, repairing and anti-aging on the basis of stabilizing the exosome.

[0015] The sodium hyaluronate complex in the exosome solvent of the present application provides a three-dimensional moisturizing network from the surface layer to the deep layer, and creates a hydrophilic protective environment for the exosome; the active type I collagen alpha 1 precursor chain complex in the exosome solvent can supplement collagen for the exosome, which helps to maintain the activity of the exosome, and at the same time, the signals (such as miRNA) carried by the exosome can activate skin fibroblasts, while the collagen precursor in the solvent provides "ready-made bricks and tiles" for the synthesis of new collagen by cells, and the combination of the two can greatly improve the efficiency of skin repair and regeneration; the complex polypeptide in the exosome solvent provides precise and target-specific biological signals, which synergistically act with the complex and cell-level regulation network provided by the exosome to achieve full coverage from macroscopic regulation to microscopic target, amplify the biological activity signal, so that the exosome solvent of the present application surpasses the traditional solvent and creates a "full-function" delivery system that not only protects the exosome but also combines with it to additionally endow the exosome with synergistic functions such as moisturizing, repairing and anti-aging.

[0016] In some embodiments, the sodium hyaluronate complex comprises a combination of low molecular weight / oligomer / ultra-low molecular weight, medium molecular weight, ultra-high / high molecular weight sodium hyaluronate. In this embodiment, the sodium hyaluronate complex uses a combination of low molecular weight / oligomer / ultra-low molecular weight, medium molecular weight, ultra-high / high molecular weight sodium hyaluronate, which can fully utilize the synergy of sodium hyaluronate with different molecular weights. The ultra-high / high molecular weight sodium hyaluronate forms a water-locking film on the outermost layer of the skin to prevent water evaporation, the medium molecular weight sodium hyaluronate moisturizes the stratum corneum, and the low molecular weight / oligomer / ultra-low molecular weight sodium hyaluronate penetrates into the dermis to fill the skin from the inside, while providing hydration protection for the exosome, achieving all-around moisturizing from the surface layer to the deep layer.

[0017] In some preferred embodiments, the low molecular weight / oligomer / ultra-low molecular weight is 0.2-1.4KDa. In this preferred embodiment, the sodium hyaluronate with a molecular weight of 0.2-1.4KDa can penetrate deep into the stratum corneum and even into the dermis due to its extremely small molecules, promote damage repair from the cellular level, and improve the skin's own moisturizing ability, while having certain anti-inflammatory and soothing effects.

[0018] In some preferred embodiments, the medium molecular weight is 20-80 KDa. In this preferred embodiment, sodium hyaluronate with a molecular weight of 20-80 KDa can further penetrate the dermis layer of the skin while further forming a breathable film on the surface layer of the skin, locking in moisture and also having certain penetration ability.

[0019] In some preferred embodiments, the ultra-high / high molecular weight is 120-200 KDa. In this preferred embodiment, sodium hyaluronate with a molecular weight of 120-200 KDa can further lock in moisture on the surface of the skin, prevent water evaporation, and long-lasting moisturizing.

[0020] In some specific embodiments, the sodium hyaluronate complex is a combination of sodium hyaluronate with molecular weights of 0.2-0.5 KDa, 1.0-1.4 KDa, 20-40 KDa, 60-80 KDa, and 120-200 KDa, respectively. The sodium hyaluronate complex composed of sodium hyaluronate with molecular weights of 0.2-0.5 KDa, 1.0-1.4 KDa, 20-40 KDa, 60-80 KDa, and 120-200 KDa can form a three-dimensional moisturizing network, further providing all-around moisturizing from the surface layer to the deep layer.

[0021] In some embodiments, the active collagen type I alpha 1 precursor chain complex includes at least two active collagen type I alpha 1 precursor chains with molecular weights of 90 KDa-110 KDa, 130 KDa-155 KDa, and 200 KDa-350 KDa. In this embodiment, the use of an active collagen type I alpha 1 precursor chain complex composed of at least two active collagen type I alpha 1 precursor chains with molecular weights of 90 KDa-110 KDa, 130 KDa-155 KDa, and 200 KDa-350 KDa can further significantly promote the migration, proliferation of fibroblasts, and self-synthesis of collagen, while maintaining high collagen activity and further effectively capturing moisture, playing a moisturizing role.

[0022] In some embodiments, the complex polypeptide includes a combination of neurotransmitter inhibitory peptides, signal peptides, and repair peptides. In this embodiment, the use of a complex polypeptide composed of neurotransmitter inhibitory peptides, signal peptides, and repair peptides can further promote the proliferation and differentiation of true epidermal cells, accelerate the synthesis rate of the sodium hyaluronate complex and the active collagen type I alpha 1 precursor chain complex, repair aging and broken collagen elastic fibers, and effectively eliminate free radicals, improve the cell growth microenvironment, thereby further enhancing the exosome solvent to have repair, anti-aging, and other functions.

[0023] In some preferred embodiments, the neurotransmitter inhibitory peptide includes at least one of a snake venom-like peptide and an acetyl oligopeptide.

[0024] In some preferred embodiments, the signal peptide-like substance comprises at least one of palmitoyl tripeptide-5, palmitoyl pentapeptide-4, palmitoyl dipeptide-7, and decapeptide-4.

[0025] In some preferred embodiments, the repair peptide-like substance comprises at least one of oligopeptide-1 and oligopeptide-3.

[0026] In some specific embodiments, the complex polypeptide is a combination of oligopeptide-1, oligopeptide-3, snake venom peptide-like substance, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, palmitoyl dipeptide-7, acetyl tetrapeptide-11, and decapeptide-4. The complex polypeptide composed of oligopeptide-1, oligopeptide-3, snake venom peptide-like substance, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, palmitoyl dipeptide-7, acetyl tetrapeptide-11, and decapeptide-4 can further promote collagen regeneration, improve static wrinkles and sagging, reduce inflammatory aging, and achieve comprehensive and multi-pathway anti-aging effects.

[0027] In some embodiments, the complex polypeptide is 0.4wt%-2.2wt%, the sodium hyaluronate complex is 0.3wt%-0.8wt%, the active collagen type I alpha 1 precursor chain complex is 0.2wt-0.6wt%, and the rest is solvent. Dissolving 0.4wt%-2.2wt% complex polypeptide, 0.2wt%-0.8wt% sodium hyaluronate complex, and 0.2wt-0.6wt% active collagen type I alpha 1 precursor chain complex in a solvent can balance the cost of exosome solvent and the compatibility stability, transdermal absorption, and synergy with exosomes of multiple active ingredients.

[0028] It should be noted that in the present application, the complex polypeptide, the sodium hyaluronate complex, and the active collagen type I alpha 1 precursor chain complex are dissolved in a solvent, which only needs to maintain the uniformity and stability of the active molecules and exosomes, and the type of solvent is not limited, which can be selected from at least one of physiological saline, mannitol, and butanediol, but is not limited thereto.

[0029] To increase other effects of the exosome solvent, at least one of a stabilizing agent, a preservative, a penetration enhancer, an antioxidant, an anti-inflammatory soothing agent, and a skin repair agent can be selectively added to the exosome solvent.

[0030] In some specific embodiments, the stabilizing agent is at least one of trehalose, glycerol, and taurine.

[0031] In some specific embodiments, the preservative is a mixture of phenoxyethanol and ethylhexylglycerin.

[0032] In some specific embodiments, the penetration enhancer is at least one of azone, a liposome, and poloxamer 188.

[0033] In some embodiments, the antioxidant is at least one of vitamin E, ergothioneine, superoxide dismutase.

[0034] In some embodiments, the anti-inflammatory soothing agent is at least one of dipotassium glycyrrhizinate, bisabolol, Centella asiatica extract.

[0035] In some embodiments, the skin repair agent is at least one of ceramide, squalane, cholesterol.

[0036] The second aspect of the present application provides a preparation method of the exosome vehicle, comprising: weighing the sodium hyaluronate compound, the active collagen type I alpha 1 precursor chain compound and the complex polypeptide according to the weight percentage, dissolving them in a solvent and mixing them uniformly to obtain the exosome vehicle. The preparation method of the exosome vehicle provided by the present application only needs to mix the sodium hyaluronate compound, the active collagen type I alpha 1 precursor chain compound and the complex polypeptide uniformly in a solvent to obtain the exosome vehicle, which is simple and easy to operate.

[0037] In some embodiments, the preparation method of the exosome vehicle comprises: dissolving the complex polypeptide into normal saline to form a colorless transparent liquid A; dissolving the sodium hyaluronate compound into normal saline to form a transparent colorless gel B; dissolving the active collagen type I alpha 1 precursor chain compound into mannitol to form a stable colloidal dispersion C; and then mixing ABC three phases uniformly to form a single colorless transparent solution, i.e. the exosome vehicle.

[0038] The third aspect of the present application provides an application of the exosome vehicle of the first aspect or the exosome vehicle prepared by the method of the second aspect in preserving exosomes.

[0039] In some embodiments, the exosome stock solution is mixed with the exosome vehicle at a ratio of 1:5-1:20 (v / v) to obtain an exosome solution. Generally, the exosome stock solution is wrapped by a lipid bilayer membrane and contains rich bioactive molecules inside, including proteins, lipids, mRNA, miRNA and DNA, etc.

[0040] The exosome solution is generally programmed to cool down to-80℃ at a speed of 1-2℃ / min for cryopreservation. When needed, the cryopreserved suspension is recovered and then subjected to relevant bioactivity tests.

[0041] To make the present invention easier to understand, the technical solution of the present invention will be further described below in conjunction with several embodiments. It should be noted that these embodiments are only illustrative of the present invention. The various reaction participants and process conditions used are all typical examples. However, after a large number of experiments, it has been verified that other types of reaction participants and other process conditions listed above are also applicable and can achieve the technical effects claimed by the present invention.

[0042] Example 1

[0043] Oligopeptide-1, oligopeptide-3, snake venom-like peptide, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, palmitoyl dipeptide-7, acetyl tetrapeptide-11, and decapeptide-4 were dissolved in physiological saline for injection in a certain proportion and stirred evenly to form a colorless and transparent liquid A.

[0044] Sodium hyaluronate with molecular weights of 0.2-0.5 kDa, 1.0-1.4 kDa, 20-40 kDa, 60-80 kDa, and 120-200 kDa were dissolved in injectable saline in a certain proportion and stirred evenly at 30-40°C to form a transparent and colorless gel B.

[0045] Active type I collagen α1 precursor chains with molecular weights of 90KDa-110KDa, 130KDa-155KDa, and 200KDa-350KDa were dissolved in mannitol and stirred evenly to form a stable colloidal dispersion C.

[0046] Then mix the three phases ABC thoroughly and stir to form a single colorless and transparent solution, which is the exosome solvent.

[0047] The proportions of each component in the exosome solvent are shown in Table 1.

[0048] Example 2-3

[0049] The preparation methods of the exosome solvents in Examples 2-3 are the same as those in Example 1, except that the components of the exosome solvents are different, and the specific proportions are shown in Table 1.

[0050] Table 1. Proportions of exosome solvent components in procedures 1-3

[0051]

[0052] To fully demonstrate the superior effects of the exosome solvent provided by this invention, exosome fluid will be uniformly obtained by mixing the exosome stock solution with the exosome solvent of Example 1, and then tested in experiments including B16 / F10 cell melanin synthesis inhibition, B16 / F10 cell intracellular tyrosinase activity inhibition, anti-inflammatory cell assay, anti-free radical and antioxidant cell assay, and exosome anti-aging effect on fibroblasts.

[0053] B16 / F10 cell melanin synthesis inhibition experiment

[0054] (1) Cell plating modeling: The concentration of B16 / F10 cells (mouse melanoma cells) was adjusted to 1×10⁵ cells / well, and seeded into 6-well culture plates. 200 nM α MSH (melanocyte-stimulating hormone) pretreatment was used to create the model, and the cells were cultured overnight at 37 ℃ to allow the cells to adhere to the culture vessel.

[0055] (2) Add exosomes: Add exosomes to the experimental sample group, and add 1mM kojic acid to the kojic acid positive control group as a positive control. Incubate at 37 ℃ for 48h.

[0056] (3) Collect cells: Discard the supernatant, wash with PBS, digest with 0.25% EDTA trypsin for 3 min, stop with 10% FBS complete medium, collect the cell suspension into a 1.5 ml centrifuge tube, centrifuge at 5000 rpm for 5 min, and discard the supernatant;

[0057] (4) Add NaOH: Add 1 mol / L NaOH solution containing 10% DMSO to the cell pellet to lyse the cells;

[0058] (5) Water bath: 80℃ metal bath for 2 hours, then transfer the supernatant to a 96-well plate;

[0059] (6) Microplate reader detection: A405nm absorbance value.

[0060] Relative melanin content (%) = (Experimental group absorbance value - blank solvent) ÷ (Control group average absorbance value - blank solvent) × 100%

[0061] Inhibition of intracellular tyrosinase activity in B16 / F10 cells

[0062] (1) Cell plating modeling: The concentration of B16 / F10 cells (mouse melanoma cells) was adjusted to 5×103 cells / well, and seeded into 96-well culture plates. 500 nM α MSH (melanocyte-stimulating hormone) pretreatment was used to create the model, and the cells were cultured overnight at 37 ℃ to allow the cells to adhere to the culture vessel.

[0063] (2) Add exosomes: Add exosomes to the experimental sample group, and add 1mM kojic acid to the kojic acid positive control group as a positive control. Incubate at 37 ℃ for 48 h.

[0064] (3) Triton X-100 cell lysis: Wash cells with PBS, add 100 μl of 1% Triton X-100-PBS solution to each well, freeze at -80℃ for 30 min, and incubate at 37℃ for 30 min to completely lyse the cells;

[0065] (4) After 30 min, 50 ul of 10 mM L-DOPA solution was added, and incubated at 37°C for 30 min, and the absorbance value was detected at 470 nm.

[0066] Relative tyrosinase activity (%) = (absorbance value of experimental group - blank solvent) ÷ (average absorbance value of control group - blank solvent) x 100%

[0067] Table 2 Results of melanin synthesis inhibition experiment and intracellular tyrosinase activity inhibition experiment of B16 / F10 cells

[0068]

[0069] From the results in Table 2, it can be seen that the exosome vehicle of Example 1 has a certain inhibitory effect on melanin synthesis, and the mechanism of action is related to the inhibition of key enzymes, and the exosome vehicle of Example 1 has a certain whitening effect.

[0070] Anti-inflammatory efficacy cell test

[0071] (1) Cell plating: adjust the concentration of Raw264.7 (mouse monocyte macrophage leukemia cells) cells to 5 x 104 cells / mL, and inoculate in 96-well culture plates, with 3 replicates;

[0072] (2) Add exosome liquid: add different exosome liquids according to the grouping, and culture overnight in a 37°C conventional incubator;

[0073] (3) Add LPS stimulation: add 1 ug / ml LPS (lipopolysaccharide) for 6h incubation except for the NC group;

[0074] (4) Collect the supernatant, dilute the supernatant of the NC group 25 times with 1x Calibration dilute, and detect the expression of pro-inflammatory factor IL-6 in the supernatant according to the ELISA (enzyme-linked immunosorbent assay) instructions.

[0075] Table 3 Anti-inflammatory efficacy cell test results

[0076]

[0077] From the results in Table 3, it can be seen that the exosome vehicle of Example 1 has a certain anti-inflammatory efficacy.

[0078] Anti-free radical antioxidant cell test

[0079] (1) Preparation of positive control: prepare 100 ug / ml vitamin C solution from 10 mg / ml vitamin C solution with the extraction solution;

[0080] (2) Preparation of DPPH working solution: DPPH powder is placed in a glass bottle. Before use, 4.05 mL of anhydrous ethanol is added and shaken to dissolve, and DPPH working solution is prepared according to the ratio of DPPH: anhydrous ethanol (V:V) = 4:21, and it is prepared and used immediately;

[0081] (3) Add 100 μl of blank group, experimental group, and Xiangshen group samples to the 96-well plate;

[0082] (4) Add an equal amount of DPPH working solution and mix well to ensure complete reaction;

[0083] (5) Place at room temperature in the dark for 30 min, and measure the absorbance at 515 nm. Record Ablank, Atest, Acontrol, and Apositive control. Each test tube requires one control tube.

[0084] (6) Record the data and calculate the free radical (DPPH) clearance rate.

[0085] Positive control free radical clearance rate calculation formula:

[0086] DPPH free radical clearance rate Dvc% = [(Ablank-Apositive control) ÷ Ablank] x 100%

[0087] Sample free radical clearance rate calculation formula:

[0088] DPPH free radical clearance rate D% = [Ablank- (Atest-Acontrol)] ÷ Ablank] x 100%

[0089] Table 4 Results of anti-free radical and antioxidant cell test

[0090]

[0091] From the results in Table 4, it can be seen that the exosome solvent of Example 1 has certain anti-free radical and antioxidant efficacy.

[0092] Exosome anti-aging efficacy detection experiment on fibroblasts

[0093] (1) Cell plating: adjust the concentration of human dermal fibroblasts HDF to 5 x 104 cells / mL, and inoculate 200 μl in 48-well culture plates, with 4 replicates;

[0094] (2) Add exosome solution: add 20 μl of different sample exosome solution according to the grouping, and culture in a 37 °C conventional incubator for 48 h;

[0095] (3) Semi-automatic nucleic acid instrument to extract RNA: the cells were taken out from the -80 °C refrigerator, and the room temperature was recovered. The 96-well deep well plate of nucleic acid extraction kit was taken out, and 15 μL protease K + 200 μL sample to be tested was added to the deep well plate A1-H1, A7-H7. The 96-well plate was placed in the semi-automatic nucleic acid extraction instrument, and the 8-link stirring sleeve was inserted. Turn on the instrument and run the nucleic acid extraction program to complete the nucleic acid extraction process. After the instrument runs, the nucleic acid solution in A6-H6, A12-H12 wells is taken out in the 8-link row, 1 μL DNase I + 1 μL DNase I reaction buffer is added, 37 °C incubation for 30 min, 0.5 μl 200 mM EDTA is added as reaction termination liquid, 65C incubation for 10 min, and DNase I is inactivated.

[0096] (4) Reverse transcription to obtain cDNA:

[0097] The cDNA was synthesized by reverse transcription kit, and the specific steps were as follows: genomic DNA removal, 5 × gDNA wiper Mix 2.0 μl, total RNA 8.0 μl were added in 0.2 ml PCR tube, and mixed and incubated at 42 °C for 2 min.

[0098] Preparation of first strand cDNA synthesis reaction solution: prepare the following mixture in an RNase-free centrifuge tube: the mixture of the above step (10 μL), 10 × RT Mix (2 μL), HiScript III Enzyme Mix (2 μL), Oligo (dT) 20 VN (1 μL), Random hexamers (1 μL), and RNase-free ddH 2 O (4 μL).

[0099] The first strand cDNA synthesis reaction was performed at 37 °C for 15 min and 85 °C for 5 sec.

[0100] (5) Polymerase chain reaction PCR

[0101] Primer synthesis: the primers used in qPCR were synthesized by Shanghai Sangon Company, and the housekeeping gene Gapdh was used as an internal reference.

[0102]

[0103] Real time-PCR reaction system: according to the Real time-PCR reaction system to prepare the reaction solution. In the PCR reaction tube, respectively, add ddH2O, Color SYBR Green qPCR Master Mix, Forward primer, Reverse primer, cDNA template, mix well. Real time-PCR reaction system as follows:

[0104]

[0105] PCR amplification conditions:

[0106]

[0107] Real-Time PCR data processing: after PCR amplification, 2-△△CT method is used to analyze the expression difference of target gene between control group and each experimental group, and the calculation formula is as follows: △Ct = Ct target gene - Ct internal reference, recorded as △Ct control, the average value of △Ct control is obtained, and the △Ct of each group is subtracted from the average value of △Ct control, and the △△Ct value is obtained, and then the 2-△△CT value of each group is calculated, that is, the relative expression amount of gene in each group.

[0108] Table 5 Exosome liquid anti-aging efficacy detection experiment results of fibroblasts

[0109]

[0110] From the results in Table 5, it can be seen that the exosome solvent of Example 1 has certain anti-aging efficacy.

[0111] The above examples are used to explain the technical solutions of the present application in detail. It should be understood that the above description is only a specific embodiment of the present application and is not used to limit the present application. Any modification, supplement or similar replacement within the principle range of the present application should be included in the protection scope of the present application.

Claims

1. An exosome vehicle, characterized by, The exosome solvent comprises a sodium hyaluronate complex, an active type I collagen alpha 1 precursor chain complex, a complex polypeptide, and a solvent; The complex polypeptide is 0.4wt%-2.2wt%, the sodium hyaluronate complex is 0.3wt%-0.8wt%, the active type I collagen alpha 1 precursor chain complex is 0.2wt-0.6wt%, and the rest is solvent; The sodium hyaluronate complex comprises a combination of low molecular weight / oligomer / ultra-low molecular weight, medium molecular weight, and ultra-large / high molecular weight sodium hyaluronate; the low molecular weight / oligomer / ultra-low molecular weight is 0.2-1.4KDa; the medium molecular weight is 20-80KDa; and the ultra-large / high molecular weight is 120-200KDa; The active type I collagen alpha 1 precursor chain complex comprises active type I collagen alpha 1 precursor chains with molecular weights of 90KDa-110KDa and 200KDa-350KDa; The complex polypeptide is a combination of oligopeptide-1, oligopeptide-3, snake venom-like peptide, palmitoyl tripeptide-5, palmitoyl pentapeptide-4, palmitoyl dipeptide-7, acetyl tetrapeptide-11, and decapeptide-4.

2. The exosome vehicle of claim 1, wherein, The sodium hyaluronate complex is a combination of sodium hyaluronate with molecular weights of 0.2-0.5KDa, 1.0-1.4KDa, 20-40KDa, 60-80KDa, and 120-200KDa.

3. The exosome vehicle of claim 1, wherein, The exosome solvent further comprises at least one of a stabilizer, a preservative, a penetration enhancer, an anti-inflammatory soothing agent, and a skin repair agent.

4. The exosome solvent of claim 3, wherein The stabilizer is at least one of trehalose, glycerol, and taurine; The preservative is a mixture of phenoxyethanol and ethylhexylglycerin; The penetration enhancer is at least one of azone, liposomes, and poloxamer 188; The anti-inflammatory soothing agent is at least one of dipotassium glycyrrhizinate, bisabolol, and centella asiatica extract; The skin repair agent is at least one of ceramides, squalane, and cholesterol.

5. The exosome vehicle of claim 3, wherein, The exosome solvent further comprises an antioxidant, which is at least one of vitamin E, ergothioneine, and superoxide dismutase.

6. The method of claim 1-5, wherein the exosome vehicle is prepared by, The preparation method comprises: weighing the sodium hyaluronate complex, the active type I collagen alpha 1 precursor chain complex, and the complex polypeptide according to the weight percentage, dissolving them in the solvent, and mixing them uniformly to obtain the exosome solvent.

7. Use of the exosome solvent of any one of claims 1-5 or the exosome solvent prepared by the preparation method of claim 6 in preserving exosomes.

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