Composition containing exosome and application of composition in product with effect of promoting tissue repair

Abstract

The invention provides a composition containing exosome, which is prepared from the following components in parts by weight: 20 to 30 parts of exosome, 10 to 15 parts of fermentation liquor, 5 to 10 parts of modified gelatin, 2 to 5 parts of beta-1, 3-glucan and 1 to 3 parts of methoxy polyethylene glycol. The preparation method comprises the following steps: mixing the exosome-containing composition, sorbic acid, propyl gallate and licorice essential oil, and stirring and mixing for 2-4 hours at the temperature of 50-70 DEG C at the speed of 400-500 r / min to prepare the product with the effect of promoting tissue repair, so that cell proliferation and repair can be promoted, rapid tissue repair can be promoted, and the biocompatibility is good.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of exosome-containing composition preparation, and particularly relates to an exosome-containing composition and application thereof in a product with a function of promoting tissue repair. BACKGROUND

[0002] Exosomes are derived from intracellular multivesicular bodies (MVBs), which are released into the extracellular matrix after fusion with the cell membrane. The generation of exosomes involves a series of complex biological processes, including the formation of endocytic vesicles by cell membrane invagination, followed by the formation of multivesicular bodies (MVBs) in the cell, and finally the fusion of MVBs with the cell membrane and the release of exosomes into the extracellular environment.

[0003] Extraction methods of exosomes include but are not limited to: ultracentrifugation: this is the most common method for extracting exosomes, but the purity may not be sufficient; density gradient centrifugation: can obtain exosomes with higher purity, but the operation is complex; immunomagnetic bead method: can ensure the integrity of the exosome morphology, and has high specificity; PS affinity method: uses phosphatidylserine (PS) to combine with magnetic beads to obtain exosomes with high purity. Due to its unique biological characteristics, exosomes have shown wide application prospects in the biomedical field, including disease diagnosis, treatment, regenerative medicine, etc.

[0004] Tissue repair refers to the process by which the body restores damaged tissue structure and function under the state of injury or disease through its own mechanism or external intervention; this process involves the coordinated action of various biomolecules, cells and signaling pathways. The goal of tissue repair is not only to restore the integrity of the tissue, but more importantly, to restore its normal function. Tissue repair products have a wide range of applications in medicine, surgery, wound treatment, skin care and other fields; these products aim to accelerate tissue repair and wound healing by promoting cell proliferation, reducing inflammation, improving blood circulation, etc. The existing products with the function of promoting tissue repair still have room for improvement in terms of repair effect. SUMMARY

[0005] The purpose of the present application is to provide an exosome-containing composition and apply it to a product with the function of promoting tissue repair, so that the exosomes, fermentation liquid and modified gelatin work together to promote cell proliferation and repair, promote rapid tissue repair, and have good biocompatibility.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: An exosome-containing composition, comprising the following components in parts by weight: 20-30 parts of exosomes, 10-15 parts of fermentation liquid, 5-10 parts of modified gelatin, 2-5 parts of beta-1, 3-glucan and 1-3 parts of methoxy polyethylene glycol.

[0007] Further, the exosome is extracted from the fat stem cell.

[0008] Further, the fermentation liquor is a purple coneflower fermentation liquor; and the modified gelatin is a diisocyanate and epichlorohydrin modified gelatin.

[0009] The preparation method of the exosome-containing composition comprises the following steps: S1: extracting exosomes; and preparing a fermentation liquor; S2: preparing a modified gelatin; S3: taking 20-30 parts of exosomes, 10-15 parts of the fermentation liquor, 5-10 parts of the modified gelatin, and 2-5 parts of beta-1,3-glucan, and stirring and mixing at a temperature of 30-40℃; then adding 1-3 parts of methoxy polyethylene glycol, and ultrasonic treatment to obtain the exosome-containing composition.

[0010] Further, the preparation process of the exosome in step S1 is as follows: using a tangential flow filtration method to concentrate cell supernatant to obtain a concentrated solution, then adding nucleic acid enzyme and water-soluble metal salt to the concentrated solution for nucleic acid removal treatment; and then performing secondary centrifugal filtration on the obtained precipitate to obtain the exosome.

[0011] Further, the exosome in step S1 is prepared as follows: using a hollow fiber column with a molecular weight cut-off of 450-650kDa to concentrate cell supernatant by tangential flow, and controlling the concentration multiple to be 10-20 to obtain a concentrated solution; then adding nucleic acid enzyme accounting for 2-4% of the mass of the concentrated solution and water-soluble metal salt accounting for 5-8% of the mass of the concentrated solution to the concentrated solution, controlling the temperature to be 30-40℃, and reacting for 10-20min; first centrifuging at a centrifugal force of 80000-100000g for 3-6min; then centrifuging at a centrifugal force of 100000-200000g for 5-10min, and filtering the obtained precipitate to obtain the exosome.

[0012] Further, the preparation process of the fermentation liquor in step S1 is as follows: mixing purple coneflower powder and deionized water according to a solid-liquid ratio of 1:20-30 and stirring; introducing carbon dioxide from the bottom; filtering to obtain supernatant; then adding DTM medium to the supernatant; culturing in a fermentation tank at 35-45℃ and at a speed of 100-200rpm for 10-20h; inactivating by stirring at a temperature of 85-95℃ for 30-50min, and filtering and pressing to obtain sterile fermentation liquor.

[0013] Further, the preparation process of the modified gelatin in step S2 is as follows: taking 10-20 parts of dihydroxy polyethylene glycol, 3-8 parts of polyhydroxybutyrate, and 30-50 parts of gelatin by weight, and stirring and reacting at a temperature of 80-100℃ and a rate of 350-450r / min for 1-2h.

[0014] Further, the power of the ultrasonic treatment in step S3 is 800-1000 W, and the time is 15-25 min.

[0015] The exosome-containing composition is applied to a product with a tissue repair-promoting effect, including the following steps: mixing 20-30 parts of the exosome-containing composition, 5-15 parts of sorbic acid, 3-6 parts of propyl gallate, and 12-18 parts of licorice essential oil, stirring and mixing at a temperature of 50-70 DEG C at a rate of 400-500 r / min for 2-4 h, to obtain the product with the tissue repair-promoting effect.

[0016] Echinacea is a herbaceous plant native to North America, which has been widely studied and applied due to its various health benefits. Echinacea is mainly composed of three varieties: Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida. Among them, Echinacea purpurea is the most commonly used one. Echinacea purpurea contains various active ingredients, including polysaccharides, caffeic acid derivatives, alkyl amides, and phenolic compounds.

[0017] Nucleases are a class of enzymes that can catalyze the hydrolytic cleavage of nucleic acids (DNA or RNA). They are divided into endonucleases and exonucleases. Endonucleases include restriction endonucleases and non-specific endonucleases; exonucleases include 5'→3' exonucleases and 3'→5' exonucleases.

[0018] Gelatin is a protein extracted from animal tissues, usually skin, bones, and connective tissue. It has a wide range of applications in the food industry, pharmaceutical industry, and cosmetics industry. Gelatin is widely used due to its unique physical and chemical properties, especially in food processing and drug formulation.

[0019] Compared with the prior art, the advantages and beneficial effects of the present application are: The present application extracts exosomes with high purity from cell supernatant by tangential flow filtration, nuclease and water-soluble metal salt treatment, centrifugation, etc.; at the same time, the fermentation broth is prepared by culturing the Echinacea purpurea powder medium, so that the active ingredients in the fermentation broth are higher; the gelatin is modified by using dihydroxy polyethylene glycol and polyhydroxybutyrate, so that the gelatin is more compact and the thickening stability is more stable; the exosome-containing composition composed of the above ingredients is applied to a tissue repair product, and under the synergistic action of them, the product can promote cell proliferation and repair, and the repair effect is further improved. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0021] The raw materials in the present application are as follows: The nuclease is from Shanghai Yuanye Biotechnology Co., Ltd. with the product number S25821; the Echinacea purpurea powder is from Wuhan Fengzhulin Chemical Technology Co., Ltd. with the product number 1275; the DTM culture medium is from DTM culture medium with the product number HB0289; the polyhydroxybutyric acid is from Shanghai Yuanye Biotechnology Co., Ltd. with the product number 3215; the gelatin is from Shanghai Yuanye Biotechnology Co., Ltd. with the product number S30952; and the beta-1, 3-glucan is from Shanghai Yuanye Biotechnology Co., Ltd. with the product number S11046.

[0022] Embodiment 1 The present embodiment provides a preparation method of an exosome-containing composition, comprising the following steps: S1: a hollow fiber column with a molecular weight cut-off of 450 kDa is used to tangential flow concentrate cell supernatant, and the concentration multiple is controlled to be 10 times to obtain a concentrated solution; then 2% nuclease and 5% magnesium chloride aqueous solution with a mass fraction of 15% are added to the concentrated solution, the temperature is controlled to be 30°C, and the stirring rate is controlled to be 400 r / min for 10 min; first centrifugal treatment is performed at a centrifugal force of 80000 g for 3 min; then centrifugal treatment is performed at a centrifugal force of 100000 g for 5 min, and the obtained precipitate is filtered to obtain exosomes; The Echinacea purpurea powder and deionized water are mixed and stirred according to a solid-liquid ratio of 1:20; carbon dioxide is introduced from the bottom, and the supernatant is filtered; then the DTM culture medium is added to the supernatant; the fermentation tank is cultured at 35°C and 100 rpm for 10 h; inactivation is performed at a temperature of 85°C and a stirring rate of 600 r / min for 30 min, and the sterile fermentation liquid is obtained by filtering and pressing the membrane; S2: 10 g of dihydroxy polyethylene glycol, 3 g of polyhydroxybutyric acid and 30 g of gelatin are taken, and stirring reaction is performed at a temperature of 80°C and a stirring rate of 350 r / min for 1 h; S3: 20 g of exosomes, 10 g of fermentation liquid, 5 g of modified gelatin and 2 g of beta-1, 3-glucan are taken, and stirring and mixing are performed at a temperature of 30°C for 2 h; 1 g of methoxy polyethylene glycol is further added, and ultrasonic treatment is performed at a power of 800 W for 15 min to obtain an exosome-containing composition.

[0023] Embodiment 2 The present embodiment provides a preparation method of an exosome-containing composition, comprising the following steps: S1: adopt hollow fiber column tangential flow to concentrate cell supernatant with molecular weight cut-off of 650 kDa, control the concentration multiple to be 20 times, obtain concentrated liquid; then add 4% nucleic acid enzyme and 8% mass fraction 25% copper sulfate aqueous solution to the concentrated liquid, control the temperature to be 40 DEG C, stir at the rate of 600 r / min for 20 min; first centrifugal treatment under centrifugal force 100000 g for 6 min; then centrifugal under centrifugal force 200000 g for 10 min, filter the obtained precipitate to obtain exosome; According to the solid-liquid ratio 1:30, mix and stir the Echinacea purpurea powder and deionized water; introduce carbon dioxide from the bottom, filter to obtain supernatant; then add DTM medium to the supernatant; culture in a fermenter at 45 DEG C, 200 rpm for 20 h; inactivate at a temperature of 95 DEG C with a stirring rate of 500 r / min for 50 min, filter and press to obtain sterile fermentation liquid; S2: take 20g dihydroxy polyethylene glycol, 8g polyhydroxybutyrate and 50g gelatin, stir at a rate of 450 r / min at a temperature of 100 DEG C for 2h to obtain modified gelatin; S3: take 30g exosome, 15g fermentation liquid, 10g modified gelatin and 5g beta-1, 3-glucan, stir at a rate at a temperature of 40 DEG C for 1h; then add 3g methoxy polyethylene glycol, ultrasonic treatment at a power of 1000 W for 25 min to obtain an exosome-containing composition.

[0024] Example 3 The present embodiment provides a preparation method of an exosome-containing composition, comprising the following steps: S1: adopt hollow fiber column tangential flow to concentrate cell supernatant with molecular weight cut-off of 550 kDa, control the concentration multiple to be 16 times, obtain concentrated liquid; then add 3% nucleic acid enzyme and 7% mass fraction 22% sodium nitrate aqueous solution to the concentrated liquid, control the temperature to be 35 DEG C, stir at the rate of 400 r / min for 15 min; first centrifugal treatment under centrifugal force 95000 g for 5 min; then centrifugal under centrifugal force 150000 g for 8 min, filter the obtained precipitate to obtain exosome; According to the solid-liquid ratio 1:25, mix and stir the Echinacea purpurea powder and deionized water; introduce carbon dioxide from the bottom, filter to obtain supernatant; then add DTM medium to the supernatant; culture in a fermenter at 42 DEG C, 160 rpm for 15 h; inactivate at a temperature of 90 DEG C with a stirring rate of 450 r / min for 40 min, filter and press to obtain sterile fermentation liquid; S2: take 16g dihydroxy polyethylene glycol, 5g polyhydroxybutyrate and 45g gelatin, stir at a rate of 400 r / min at a temperature of 90 DEG C for 1.5h to obtain modified gelatin; S3: 23 g of exosomes, 12 g of fermentation broth, 7 g of modified gelatin, and 4 g of β-1,3-glucan were mixed at a temperature of 35°C and a stirring rate of 600 r / min for 1.5 h; 2 g of methoxypolyethylene glycol was then added, and ultrasonic treatment was performed at a power of 9500 W for 22 min to obtain an exosome-containing composition.

[0025] Comparative Example 1 The difference between this comparative example and Example 1 is that no nuclease treatment is added in step S1, and the other steps are the same.

[0026] Comparative Example 2 The difference between this comparative example and Example 1 is that no magnesium chloride aqueous solution treatment is added in step S1, and the other steps are the same.

[0027] Comparative Example 3 The difference between this comparative example and Example 1 is that there is no sterile fermentation broth in step S1, and the other steps are the same.

[0028] Comparative Example 4 The difference between this comparative example and Example 1 is that step S2 is omitted, i.e., unmodified gelatin is directly added in step S3, and the other steps are the same.

[0029] Comparative Example 5 The difference between this comparative example and Example 1 is that no methoxypolyethylene glycol is added in step S3, and the other steps are the same.

[0030] Comparative Example 6 The difference between this comparative example and Example 1 is that the product is physiological saline.

[0031] The exosome-containing compositions obtained in the above examples and comparative examples are applied to products having a tissue repair-promoting effect. 25 g of the exosome-containing composition, 10 g of sorbic acid, 5 g of propyl gallate, and 15 g of licorice essential oil are mixed, and stirring and mixing are performed at a temperature of 60°C and a stirring rate of 450 r / min for 3 h to obtain a product having a tissue repair-promoting effect.

[0032] Twenty-seven 8- to 10-week-old male Kunming mice (purchased from the Guangdong Provincial Animal Center) were selected and housed in cages. The mice were anesthetized, the back was shaved and disinfected, a punch with a diameter of 6 mm was used to punch the exposed skin on the back of the mouse, a circular skin including the epidermis and dermis layer was cut off, and the wound site was pasted with a transparent oxygen-permeable dressing to play a shielding and protective role. The product was given twice a day, 2 mg / mL each time, and the obtained repair product was given continuously for 10 days, and the wound area size change was measured every day. The wound healing rate was calculated according to the formula wound healing rate % = (S1-Sn) / S1 (S1 is the wound area on the first day, and Sn is the wound area on the nth day). The results are shown in Table 1.

[0033] Table 1 Performance test results

[0034] P<0.05 has significant difference. From the performance test results, it can be seen that the products prepared by the exosome-containing compositions of Examples 1-3 can obviously promote the healing of mouse skin wounds, and there is a significant difference compared with the comparative examples; in particular, the comprehensive performance of Example 3 is the most outstanding. While Comparative Examples 1-6 did not use the necessary technical solutions, resulting in a significant difference in the corresponding performance test compared with the examples. The above experimental results further prove the importance of the technical solutions defined in the application to its technical effects.

[0035] The above is the preferred embodiment of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. An exosome-containing composition, characterized in that, The composition comprises the following components in parts by weight: 20-30 parts of exosomes, 10-15 parts of fermentation liquor, 5-10 parts of modified gelatin, 2-5 parts of beta-1, 3-glucan and 1-3 parts of methoxy polyethylene glycol.

2. The exosome-containing composition of claim 1, wherein, The exosomes are extracted from fat stem cells.

3. The exosome-containing composition of claim 1, wherein, The fermentation liquor is Echinacea purpurea fermentation liquor; and the modified gelatin is diisocyanate and epichlorohydrin modified gelatin.

4. A method of preparing an exosome-containing composition, characterized by, The method comprises the following steps: S1: extracting exosomes; and preparing fermentation liquor; S2: preparing modified gelatin; S3: taking 20-30 parts of exosomes, 10-15 parts of fermentation liquor, 5-10 parts of modified gelatin and 2-5 parts of beta-1, 3-glucan, stirring and mixing at a temperature of 30-40 DEG C; then adding 1-3 parts of methoxy polyethylene glycol, and ultrasonic treatment to obtain a composition containing exosomes.

5. The method of claim 4, wherein the exosome-containing composition is prepared by, In step S1, the preparation process of the extracted exosomes is as follows: the cell supernatant is concentrated by tangential flow filtration to obtain a concentrated solution, then nucleic acid removal treatment is performed on the concentrated solution by adding nucleic acid enzyme and water-soluble metal salt, and the obtained precipitate after secondary centrifugal filtration is the exosomes.

6. The method of claim 5, wherein the exosome-containing composition is prepared by, In step S1, the extracted exosomes are prepared as follows: the cell supernatant is concentrated by a hollow fiber column with a molecular weight cut-off of 450-650 kDa, and the concentration multiple is controlled to be 10-20 times to obtain a concentrated solution; then, 2-4% nucleic acid enzyme and 5-8% water-soluble metal salt based on the mass of the concentrated solution are added to the concentrated solution, the temperature is controlled to be 30-40 DEG C, and the reaction is performed for 10-20 min; first, centrifugal treatment is performed at a centrifugal force of 80000-100000 g for 3-6 min; then, centrifugal treatment is performed at a centrifugal force of 100000-200000 g for 5-10 min, and the obtained precipitate after filtration is the exosomes.

7. The method of claim 4, wherein the exosome-containing composition is prepared by, In step S1, the preparation process of the fermentation liquor is as follows: Echinacea purpurea powder and deionized water are mixed and stirred according to a solid-liquid ratio of 1:20-30; carbon dioxide is introduced from the bottom; the supernatant is obtained by filtration; then, DTM medium is added to the supernatant; the fermentation tank is cultured at 35-45 DEG C and at a speed of 100-200 rpm for 10-20 h; inactivation is performed by stirring at a temperature of 85-95 DEG C for 30-50 min, and sterile fermentation liquor is obtained by filtration and membrane pressing.

8. The method of claim 4, wherein the exosome-containing composition is prepared by, In step S2, the preparation process of the modified gelatin is as follows: 10-20 parts of dihydroxy polyethylene glycol, 3-8 parts of polyhydroxybutyrate and 30-50 parts of gelatin are taken in parts by weight, and stirring reaction is performed at a temperature of 80-100 DEG C and at a speed of 350-450 r / min for 1-2 h.

9. The method of claim 4, wherein the exosome-containing composition is prepared by, In step S3, the ultrasonic treatment is performed at a power of 800-1000 W for 15-25 min.

10. The exosome-containing composition prepared according to any one of claims 1 to 9 for use in the preparation of a product having an effect of promoting tissue repair, characterized in that, The method comprises the following steps: 20-30 parts of the composition containing exosomes, 5-15 parts of sorbic acid, 3-6 parts of propyl gallate and 12-18 parts of licorice essential oil are mixed, stirred and mixed at a temperature of 50-70 DEG C and at a speed of 400-500 r / min for 2-4 h to obtain a product with the function of promoting tissue repair.

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