Stem cell exosome repairing agent and preparation method thereof

Abstract

The invention provides a stem cell exosome repairing agent and a preparation method thereof, and the repairing agent comprises stem cell exosome, hyaluronic acid, collagen, vitamin E, glutathione, an anti-inflammatory agent, liposome nanoparticles and sterile water according to specific parts by weight. Wherein the anti-inflammatory agent is prepared from a selfheal extract, an aloe extract and a liquorice extract in a specific proportion. The preparation method comprises the following steps: separation and purification of stem cell exosomes, preparation of liposome nanoparticles, formation of a compound, preparation of a mixed solution and preparation of a final repairing agent. High-purity stem cell exosomes are obtained through specific centrifugation, incubation, separation, elution and purification steps and fused with liposome nano-particles, then the fused product is mixed with an anti-inflammatory agent and other components, and the repairing agent is obtained through ultrasonic treatment. The repairing agent disclosed by the invention has a good clinical application prospect and provides a new choice for skin injury treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a stem cell exosome repair agent and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body and has multiple functions, including protecting the body from external damage, regulating body temperature and sensory input. Skin damage is a common and complex pathological process that may be caused by disease, trauma, surgery or aging. Traditional treatments for skin damage include medication, physical therapy and surgery, but these methods have certain limitations and shortcomings.

[0003] With the rapid development of biomedical technology, stem cells and their derivatives have shown great potential in the fields of regenerative medicine and beauty skin care. In the field of regenerative medicine, stem cell therapy has attracted widespread attention due to its potential in tissue repair and regeneration. In particular, exosomes derived from adult stem cells, such as umbilical cord mesenchymal stem cells (UC-MSCs), human adipose-derived mesenchymal stem cells (ADSCs), bone marrow mesenchymal stem cells (BM-MSCs) and placental mesenchymal stem cells, have been shown to play an important role in skin damage repair. As a kind of tiny vesicles secreted by stem cells, stem cell exosomes contain rich bioactive molecules such as mRNA, miRNA, proteins and lipids, which can participate in intercellular communication, regulate cell function and promote tissue repair, promote wound healing and tissue regeneration. In recent years, stem cell exosomes have attracted widespread attention in skin repair, anti-aging and immune regulation due to their unique biological properties and safety.

[0004] However, the existing technology still has some shortcomings in the separation and purification of stem cell exosomes, activity maintenance, and drug delivery. First, although traditional exosome separation methods such as ultracentrifugation can obtain exosomes, the operation is complicated, time-consuming, and costly, and may damage the biological activity of exosomes. Secondly, the purification technology of exosomes is still imperfect, and it is difficult to obtain high-purity and high-quality exosome products, which affects its clinical application effect. Furthermore, the drug delivery system lacks the ability to target specific cells or tissues, resulting in uneven drug distribution and limited efficacy; it may also cause non-specific cellular uptake, thereby affecting the safety and effectiveness of the drug; in addition, the drug release kinetics in the existing technology are often not ideal, and the targeted release efficiency is low.

[0005] In terms of drug delivery, how to effectively combine exosomes with other active ingredients to improve the comprehensive efficacy of skin damage repair agents is also a technical challenge. Existing skin damage repair agents often have a single ingredient and lack the ability to comprehensively regulate inflammation, promote cell proliferation, and rebuild the extracellular matrix. Summary of the invention

[0006] In view of this, the present invention proposes a stem cell exosome repair agent and a preparation method thereof to solve the above problems.

[0007] The technical solution of the present invention is achieved as follows: a stem cell exosome repair agent, comprising the following components in parts by weight: 5-15 parts of stem cell exosomes, 1-5 parts of hyaluronic acid, 2-8 parts of collagen, 0.5-2 parts of vitamin E, 0.2-1 parts of glutathione, 3-6 parts of anti-inflammatory agent, 20-28 parts of liposome nanoparticles, and 50-80 parts of sterile water; the concentration of the stem cell exosomes is greater than 5.0×10 10 particles / mL, and the protein ratio of stem cell exosomes was higher than 4.17×10 8 particles / μg.

[0008] Furthermore, a stem cell exosome repair agent is prepared by including the following components in parts by weight: 10 parts of stem cell exosomes, 3 parts of hyaluronic acid, 5 parts of collagen, 1 part of vitamin E, 0.6 parts of glutathione, 5 parts of anti-inflammatory agent, 24 parts of liposome nanoparticles, and 60 parts of sterile water.

[0009] Furthermore, the anti-inflammatory agent is a Prunella Vulgaris extract, an aloe vera extract and a licorice extract in a mass ratio of (2-5):(3-8):(1-3).

[0010] The above-mentioned Prunella vulgaris extract uses 95 vol% ethanol aqueous solution as the extraction solvent, adds 8 times the mass of the raw material for extraction, and the extraction temperature is 95° C., and the temperature is kept for 2 hours. After elution and filtration, the Prunella vulgaris extract is obtained.

[0011] The aloe extract is prepared by mixing a complex enzyme made of cellulase and pectinase in a mass ratio of 1:1 with aloe pulp, wherein the added amount of the complex enzyme is 0.3% of the mass of the aloe pulp, performing enzymolysis at a temperature of 50°C for 2 hours, and using ultrasonic assisted extraction for 3 hours with an ultrasonic power of 300 W and an ultrasonic frequency of 40 kHz to obtain the aloe extract.

[0012] The licorice extract is prepared by crushing licorice, passing through a 40-60 mesh sieve, adding 70% ethanol solution, with a solid-liquid ratio of 1:12, reflux extraction at 55°C for 2h, filtering, and concentrating the filtrate under reduced pressure at 55°C to obtain the licorice extract.

[0013] Furthermore, a method for preparing a stem cell exosome repair agent comprises the following steps:

[0014] S1. Isolation and purification of stem cell exosomes: The collected stem cell supernatant is centrifuged to remove cells, cell debris and other large particles, and then magnetic beads are added for incubation. The incubated mixture is separated under a magnetic field, the supernatant is discarded, the exosomes on the magnetic beads are eluted, and the stem cell exosomes are purified by ultrafiltration;

[0015] S2. Preparation of liposome nanoparticles: phospholipids and cholesterol are mixed in a mass ratio of 2-5:1, dissolved in chloroform to form a uniform solution, and chloroform is removed by rotary evaporation in a round-bottom flask at 30-40°C to form a uniform lipid film, and then PBS buffer is added to hydrate the formed liposome suspension, and liposome nanoparticles are prepared by extrusion; vesicles composed of phospholipid bilayers can simulate the structure of cell membranes, encapsulate and protect exosomes, so as to improve the stability and targeting of exosomes;

[0016] S3, preparing a complex: mixing the stem cell exosomes obtained in S1 and the liposome nanoparticles in S2, adding 0.3-1.5% of the mass of the complex enzyme to the mixture, stirring and fusing at 30-40°C for 3-6 hours, and using intermittent electron beam irradiation to assist fusion during the stirring process, each irradiation is 10-15 minutes, the interval time is 15-25 minutes, the electron beam irradiation dose is 2-8 kGy, the electron beam energy is 2.5-5.5 MeV, and the beam current intensity is 3-10 mA, to obtain a complex;

[0017] S4. preparing a mixed solution: mixing hyaluronic acid, collagen, vitamin E, glutathione and sterile water in proportion, and stirring evenly to obtain a mixed solution;

[0018] S5. Preparation of repair agent: adding the complex prepared in S3 and the anti-inflammatory agent to the mixed solution, ultrasonically treating for 10-30 minutes to fully disperse the mixture, and obtaining the stem cell exosome repair agent.

[0019] Furthermore, the method for separating and purifying S1 stem cell exosomes comprises the following steps:

[0020] S1-1, centrifugation: collect stem cell supernatant into a sterile centrifuge tube and centrifuge at 3000-10000g for 10-25min to remove cells, cell debris and other large particles;

[0021] S1-2, incubation: add the magnetic beads to the above centrifuge tube and incubate in segments using a pulsed magnetic field. Initial incubation: adjust the temperature of the mixture to 3-6°C, stir at 30-50rpm, and incubate for 1-3h; intermediate incubation: temperature is 8-12°C, stirring at 60-100rpm, and incubate for 3-6h; late incubation: temperature is 3-6°C, 40-60rpm, and incubate for 2-4h. The pulsed magnetic field strength of the above segmented incubation is 100-500mT; the pulse frequency is 30-80Hz, and the number of pulses is 10-50;

[0022] S1-3, separation: the incubated mixture was placed in a magnetic field with a strength of 500-1000 Gauss for 3-5 hours, and the supernatant was aspirated and discarded;

[0023] S1-4, elution: Use a phosphate buffer with a pH of 5.0 and a concentration of 0.1 mol / L to elute the exosomes bound to the magnetic beads, and transfer the eluted exosome solution to a new centrifuge tube;

[0024] S1-5. Purification: The exosome solution is ultrafiltered 2-4 times using an ultrafiltration membrane with a pore size of 10-100 kDa, and the liquid in the lower chamber is collected to obtain stem cell exosomes.

[0025] Furthermore, the stem cells are selected from any one of umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, and placental mesenchymal stem cells.

[0026] Furthermore, the magnetic beads are superparamagnetic ferroferric oxide magnetic beads or ferric oxide nanoparticles.

[0027] Furthermore, the S2 phospholipid includes but is not limited to phosphatidylcholine or phosphatidylserine.

[0028] Furthermore, the complex enzyme of S3 is α-glucosidase and phosphatase in a mass ratio of 1-3:5, and the enzyme activity of the α-glucosidase is greater than 0.8×10 4 U / mL; the phosphatase activity is greater than 1.35×10 4 U / mL.

[0029] Furthermore, the ultrasonic power of S5 is 200-500W and the frequency is 20-40kHz.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The repair agent of the present invention utilizes the natural targeting ability and biocompatibility of stem cell exosomes, combined with the synergistic effect of multiple bioactive ingredients, not only promoting cell regeneration and repair, but also effectively improving skin texture, enhancing skin elasticity and luster. At the same time, the introduced anti-inflammatory agent is formulated in a scientific and reasonable mass ratio, has a significant anti-inflammatory effect, can alleviate the inflammatory response caused by damage, accelerate the recovery process, and comprehensively promote skin repair and regeneration.

[0032] 2. A specific separation and purification method is used to obtain stem cell exosomes. Through centrifugation, pulsed magnetic field segmented incubation, and ultrafiltration, the purity and activity of the exosomes are effectively improved, ensuring the quality and effect of the repair agent.

[0033] 3. The use of compound enzyme and electron beam irradiation assisted fusion technology improves the fusion efficiency of exosomes and liposome nanoparticles, ensures the uniform dispersion and stability of active ingredients, and achieves efficient drug loading and controlled release; by encapsulating stem cell exosomes in liposome nanoparticles, not only the biological activity of exosomes is protected, but also their stability and targeting in the body are significantly improved. Liposomal nanoparticles can serve as carriers to guide exosomes to accurately reach damaged tissues or cells, thereby achieving a more precise repair effect, reducing nonspecific distribution in non-target areas, and reducing the risk of side effects;

[0034] 4. The stem cell exosome repair agent of the present invention has good targeting, stability and repair effect, and has broad application prospects. It is suitable for various types of skin damage and repair, including but not limited to burns, trauma, surgical incisions and chronic wounds, and has broad clinical application potential. DETAILED DESCRIPTION

[0035] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0036] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0037] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0038] Example 1

[0039] The stem cell exosome repair agent provided in this embodiment includes the following components in weight: 5 parts of stem cell exosomes, 1 part of hyaluronic acid, 2 parts of collagen, 0.5 parts of vitamin E mixture, 0.2 parts of glutathione, 3 parts of anti-inflammatory agent, 20 parts of liposome nanoparticles, and 50 parts of sterile water; the anti-inflammatory agent is a selfheal extract, an aloe extract, and a licorice extract in a mass ratio of 2:3:1.

[0040] Example 2

[0041] A stem cell exosome repair agent comprises the following components in parts by weight: 15 parts of stem cell exosomes, 5 parts of hyaluronic acid, 8 parts of collagen, 2 parts of vitamin E mixture, 1 part of glutathione, 6 parts of anti-inflammatory agent, 28 parts of liposome nanoparticles, and 80 parts of sterile water; the anti-inflammatory agent is a selfheal extract, an aloe extract, and a licorice extract in a mass ratio of 5:8:3.

[0042] Example 3

[0043] A stem cell exosome repair agent, comprising the following components in parts by weight: 10 parts of stem cell exosomes, 3 parts of hyaluronic acid, 5 parts of collagen, 1 part of vitamin E, 0.6 parts of glutathione, 5 parts of anti-inflammatory agent, 24 parts of liposome nanoparticles, and 60 parts of sterile water; the anti-inflammatory agent is a selfheal extract, an aloe extract, and a licorice extract in a mass ratio of 3:5:2;

[0044] The above examples 1-3 adopt the following preparation method:

[0045] S1. Isolation and purification of stem cell exosomes:

[0046] S1-1, centrifugation: collect the supernatant of umbilical cord mesenchymal stem cells into a sterile centrifuge tube, and centrifuge at 6000g for 16 minutes to remove cells, cell debris and other large particles;

[0047] S1-2, incubation: add superparamagnetic ferroferric oxide magnetic beads to the above centrifuge tube and use pulsed magnetic field for segmented incubation, initial incubation: adjust the temperature to 5°C, stirring rate to 40rpm, incubate for 2h; intermediate incubation: temperature to 10°C, stirring rate to 80rpm, incubate for 5h; late incubation: temperature to 5°C, 50rpm, incubate for 3h, the pulsed magnetic field intensity of the above segmented incubation is 1300mT; the pulse frequency is 50Hz, and the number of pulses is 30;

[0048] S1-3, separation: the incubated mixture was placed in a magnetic field with a strength of 800 Gauss for 4 h, and the supernatant was aspirated and discarded;

[0049] S1-4, elution: Use a phosphate buffer with a pH of 5.0 and a concentration of 0.1 mol / L to elute the exosomes bound to the magnetic beads, and transfer the eluted exosome solution to a new centrifuge tube;

[0050] S1-5. Purification: The exosome solution was ultrafiltered three times using an ultrafiltration membrane with a pore size of 80 kDa, and the lower chamber liquid was collected to obtain stem cell exosomes. The concentration of stem cell exosomes was greater than 5.0×10 10 particles / mL, and the protein ratio of stem cell exosomes was higher than 4.17×10 8particles / μg;

[0051] S2. Preparation of liposome nanoparticles: phosphatidylcholine and cholesterol were mixed in a mass ratio of 3:1, dissolved in chloroform to form a uniform solution, and the chloroform was removed by rotary evaporation in a round-bottom flask at 35° C. to form a uniform lipid film, and then PBS buffer was added to hydrate the formed liposome suspension, and liposome nanoparticles were prepared by extrusion method;

[0052] S3, preparing a complex: mixing the stem cell exosomes obtained in S1 and the liposome nanoparticles in S2, adding 0.9% of the mass of the mixture of complex enzymes, wherein the complex enzymes are α-glucosidase and phosphatase in a mass ratio of 2:5, stirring and fusing at 35°C for 5 hours, and using intermittent electron beam irradiation to assist fusion during the stirring process, each irradiation for 12 minutes, with an interval of 20 minutes, the electron beam irradiation dose of 6 kGy, the electron beam energy of 4.0 MeV, and the beam current intensity of 6 mA, to obtain a complex;

[0053] S4. preparing a mixed solution: mixing hyaluronic acid, collagen, vitamin E, glutathione and sterile water in proportion, and stirring evenly to obtain a mixed solution;

[0054] S5. Preparation of repair agent: adding the complex and anti-inflammatory agent prepared in S3 to the mixed solution, and ultrasonically treating the mixture for 20 min at an ultrasonic power of 350 W and a frequency of 30 kHz to fully disperse the mixture, thereby obtaining a stem cell exosome repair agent.

[0055] Example 4

[0056] A stem cell exosome repair agent, comprising the following components in parts by weight: 10 parts of stem cell exosomes, 3 parts of hyaluronic acid, 5 parts of collagen, 1 part of vitamin E, 0.6 parts of glutathione, 5 parts of anti-inflammatory agent, 24 parts of liposome nanoparticles, and 60 parts of sterile water; the anti-inflammatory agent is a herbaceous herb extract, an aloe extract, and a licorice extract in a mass ratio of 3:5:2;

[0057] The above-mentioned Example 4 adopts the following preparation method:

[0058] S1. Isolation and purification of stem cell exosomes:

[0059] S1-1, centrifugation: collect the supernatant of umbilical cord mesenchymal stem cells into a sterile centrifuge tube, and centrifuge at 3000g for 10 minutes to remove cells, cell debris and other large particles;

[0060] S1-2, incubation: add superparamagnetic ferroferric oxide magnetic beads to the above centrifuge tube and incubate in segments using a pulsed magnetic field. Initial incubation: adjust the temperature of the mixture to 3°C, the stirring rate to 30 rpm, and incubate for 1 hour; intermediate incubation: the temperature is 8°C, the stirring rate is 60 rpm, and the incubation is 3 hours; late incubation: the temperature is 3°C, the stirring rate is 60 rpm, and the incubation is 2 hours. The pulsed magnetic field intensity of the above segmented incubation is 100 mT; the pulse frequency is 30 Hz, and the number of pulses is 10;

[0061] S1-3, separation: the incubated mixture was placed in a magnetic field with a strength of 500 Gauss for 3 h, and the supernatant was aspirated and discarded;

[0062] S1-4, elution: Use a phosphate buffer with a pH of 5.0 and a concentration of 0.1 mol / L to elute the exosomes bound to the magnetic beads, and transfer the eluted exosome solution to a new centrifuge tube;

[0063] S1-5. Purification: The exosome solution was ultrafiltered twice using an ultrafiltration membrane with a pore size of 10 kDa, and the lower chamber liquid was collected to obtain stem cell exosomes. The concentration of stem cell exosomes was greater than 5.0×10 10 particles / mL, and the protein ratio of stem cell exosomes was higher than 4.17×10 8 particles / μg;

[0064] S2. Preparation of liposome nanoparticles: phosphatidylcholine and cholesterol were mixed in a mass ratio of 2:1, dissolved in chloroform to form a uniform solution, and the chloroform was removed by rotary evaporation in a round-bottom flask at 30° C. to form a uniform lipid film, and then PBS buffer was added to hydrate the formed liposome suspension, and liposome nanoparticles were prepared by extrusion method;

[0065] S3, preparing a complex: mixing the stem cell exosomes obtained in S1 and the liposome nanoparticles in S2, adding 0.3% of the mass of the mixture of complex enzymes, wherein the complex enzymes are α-glucosidase and phosphatase in a mass ratio of 1:5, stirring and fusing at 30°C for 3 hours, and using intermittent electron beam irradiation to assist fusion during the stirring process, each irradiation is 10 minutes, the interval time is 15 minutes, the electron beam irradiation dose is 2 kGy, the electron beam energy is 2.5 MeV, and the beam current intensity is 3 mA, to obtain a complex;

[0066] S4. preparing a mixed solution: mixing hyaluronic acid, collagen, vitamin E, glutathione and sterile water in proportion, and stirring evenly to obtain a mixed solution;

[0067] S5. Preparation of repair agent: adding the complex prepared in S3 and the anti-inflammatory agent to the mixed solution, ultrasonically treating the mixture for 10 min at an ultrasonic power of 200 W and a frequency of 20 kHz to fully disperse the mixture, thereby obtaining a stem cell exosome repair agent.

[0068] Example 5

[0069] A stem cell exosome repair agent, comprising the following components in parts by weight: 10 parts of stem cell exosomes, 3 parts of hyaluronic acid, 5 parts of collagen, 1 part of vitamin E, 0.6 parts of glutathione, 5 parts of anti-inflammatory agent, 24 parts of liposome nanoparticles, and 60 parts of sterile water; the anti-inflammatory agent is a selfheal extract, an aloe extract, and a licorice extract in a mass ratio of 3:5:2;

[0070] The above-mentioned Example 5 adopts the following preparation method:

[0071] S1. Isolation and purification of stem cell exosomes:

[0072] S1-1, centrifugation: collect the supernatant of umbilical cord mesenchymal stem cells into a sterile centrifuge tube, and centrifuge at 10000g for 25 minutes to remove cells, cell debris and other large particles;

[0073] S1-2, incubation: add superparamagnetic ferroferric oxide magnetic beads to the above centrifuge tube and use pulsed magnetic field for segmented incubation, initial incubation: adjust the temperature of the mixture to 6°C, stirring rate to 50rpm, incubate for 3h; intermediate incubation: temperature to 12°C, stirring rate to 100rpm, incubate for 6h; late incubation: temperature to 6°C, 60rpm, incubate for 4h, the pulsed magnetic field intensity of the above segmented incubation is 500mT; the pulse frequency is 80Hz, and the number of pulses is 50;

[0074] S1-3, separation: the incubated mixture was placed in a magnetic field with a strength of 1000 Gauss for 5 h, and the supernatant was aspirated and discarded;

[0075] S1-4, elution: Use a phosphate buffer with a pH of 5.0 and a concentration of 0.1 mol / L to elute the exosomes bound to the magnetic beads, and transfer the eluted exosome solution to a new centrifuge tube;

[0076] S1-5. Purification: The exosome solution was ultrafiltered four times using an ultrafiltration membrane with a pore size of 100 kDa, and the lower chamber liquid was collected to obtain stem cell exosomes. The concentration of stem cell exosomes was greater than 5.0×10 10 particles / mL, and the protein ratio of stem cell exosomes was higher than 4.17×10 8 particles / μg;

[0077] S2. Preparation of liposome nanoparticles: phosphatidylcholine and cholesterol were mixed in a mass ratio of 5:1, dissolved in chloroform to form a uniform solution, and the chloroform was removed by rotary evaporation in a round-bottom flask at 40° C. to form a uniform lipid film, and then PBS buffer was added to hydrate the formed liposome suspension, and liposome nanoparticles were prepared by extrusion method;

[0078] S3, preparing a complex: mixing the stem cell exosomes obtained in S1 and the liposome nanoparticles in S2, adding 1.5% of the mass of the mixture of complex enzymes, wherein the complex enzymes are α-glucosidase and phosphatase in a mass ratio of 3:5, stirring and fusing at 40°C for 6 hours, and using intermittent electron beam irradiation to assist fusion during the stirring process, each irradiation for 15 minutes, with an interval of 25 minutes, the electron beam irradiation dose of 8 kGy, the electron beam energy of 5.5 MeV, and the beam current intensity of 10 mA, to obtain a complex;

[0079] S4. preparing a mixed solution: mixing hyaluronic acid, collagen, vitamin E, glutathione and sterile water in proportion, and stirring evenly to obtain a mixed solution;

[0080] S5. Preparation of repair agent: adding the complex prepared in S3 and the anti-inflammatory agent to the mixed solution, ultrasonically treating the mixture for 30 min at an ultrasonic power of 500 W and a frequency of 40 kHz to fully disperse the mixture, thereby obtaining a stem cell exosome repair agent.

[0081] Experimental Example 1- Determination of Purity and Biological Activity of Stem Cell Exosomes

[0082] Set up the following comparison ratio for comparative measurement:

[0083] Comparative Example 1

[0084] The difference between this comparative example and Example 3 is that the pulsed magnetic field segmented incubation is not used in the separation and purification method of stem cell exosomes, which is as follows:

[0085] S1-2, incubation: add superparamagnetic iron oxide beads to the above centrifuge tube and incubate in stages. Initial incubation: adjust the temperature to 5°C, stirring rate to 40 rpm, and incubate for 2 hours; intermediate incubation: temperature to 10°C, stirring rate to 80 rpm, and incubate for 5 hours; late incubation: temperature to 5°C, 50 rpm, and incubate for 3 hours.

[0086] Comparative Example 2

[0087] The difference between this comparative example and Example 3 is that the separation and purification method of stem cell exosomes does not involve segmented incubation, as follows:

[0088] S1-2, incubation: add superparamagnetic ferroferric oxide magnetic beads into the above centrifuge tube and incubate in a pulsed magnetic field, adjust the temperature to 5°C, stir at 50 rpm, and incubate for 10 hours. The pulsed magnetic field intensity of the above segmented incubation is 1300 mT; the pulse frequency is 50 Hz, and the number of pulses is 30.

[0089] Comparative Example 3

[0090] The difference between this comparative example and Example 3 is that the pulsed magnetic field segmented incubation is not used in the separation and purification method of stem cell exosomes, which is as follows:

[0091] S1-2, incubation: add superparamagnetic ferroferric oxide magnetic beads to the above centrifuge tube for incubation. Initial incubation: adjust the temperature to 5°C, stir at 50 rpm, and incubate for 10 hours.

[0092] Select the stem cell exosomes prepared in Example 3 and Comparative Examples 1-3 as samples;

[0093] 1. Particle size distribution range: measured by nanoparticle tracking analysis (NTA);

[0094] 2. TEM observation results: The morphology and membrane structure of exosomes were observed by transmission electron microscopy (TEM);

[0095] 3. Detection of specific protein markers: Detect specific protein markers on the surface of exosomes by Western Blotting: CD9, CD63, CD81, TSG101 and Alix markers;

[0096] 4. Result data:

[0097] Table 1:

[0098]

[0099] Note: The strongest expression of the protein marker (indicated by "++") indicates the highest purity; the second strongest expression (indicated by "+") indicates a higher purity; the weaker expression and uncertainty (indicated by "±") indicates a medium purity; the weaker expression and uncertainty (± indicates a weak positive or false positive) indicates a lower purity.

[0100] From the above results, it can be seen that the particle size distribution range of Example 3 of the present invention is the narrowest and most concentrated, the double-layer capsule structure is clear, the morphology is uniform, there are no impurities, the protein marker expression is the strongest, and the purity is the highest; among them, compared with Comparative Examples 1 and 3, the pulsed magnetic field in Example 3 has different forces on particles of different sizes, shapes, charges or components. Exosomes and impurity particles respond differently in the magnetic field, thereby achieving a certain degree of selective separation and reducing the presence of impurities; it promotes exosomes to aggregate more effectively, while impurities may be dispersed in the solution, making it easier to separate aggregated exosomes from impurities, thereby improving purity; it can also affect the membrane structure and properties of exosomes, enhance the interaction between exosomes, form a more uniform group, and reduce non-specific binding with impurities; the magnetic field induces the rearrangement of proteins, lipids and other molecules on the surface of exosomes, exposing certain specific sites, thereby enhancing the mutual recognition and binding between exosomes, and helping to separate a purer exosome population.

[0101] Compared with Comparative Examples 2 and 3, the use of segmented incubation to control the incubation conditions separately is helpful to optimize the formation and maturation process of exosomes in cells. By simulating the gradual changes under physiological conditions, the exosomes can be made more stable and functional. By setting the conditions at different stages, the aggregation and dispersion state of the exosomes can be better controlled, so that the exosomes and impurities have different stability and solubility under different conditions, thereby achieving separation and improving purity.

[0102] Test Example 2- Encapsulation Efficiency and Targeted Release Characteristics Test

[0103] Set up the following comparison ratio for comparative measurement:

[0104] Comparative Example 4

[0105] The difference between this comparative example and Example 3 is that in the preparation method of the stem cell exosome repair agent, no complex enzyme is added in the preparation of the complex, which is as follows:

[0106] S3, preparing a complex: mixing the stem cell exosomes obtained in S1 and the liposome nanoparticles in S2, stirring and fusing at 35°C, and using intermittent electron beam irradiation to assist fusion during the stirring process, each irradiation lasts 12 minutes, with an interval of 20 minutes, the electron beam irradiation dose is 6 kGy, the electron beam energy is 4.0 MeV, and the beam current intensity is 6 mA, to obtain a complex;

[0107] Comparative Example 5

[0108] The difference between this comparative example and Example 3 is that the preparation of the complex in the preparation method of the stem cell exosome repair agent does not use electron beam intermittent irradiation to assist fusion, which is as follows:

[0109] S3. Preparation of a complex: Mix the stem cell exosomes obtained in S1 and the liposome nanoparticles in S2, add 0.9% of the mixture by weight of a complex enzyme, wherein the complex enzyme is α-glucosidase and phosphatase in a mass ratio of 2:5, stir and fuse at 35° C. for 5 hours to obtain a complex.

[0110] 1. Determination of Encapsulation Efficiency

[0111] 1. Sample preparation: The composites prepared in Example 3 and Comparative Examples 6-7 were selected as samples.

[0112] 2. Ultracentrifugation: Place the above samples in a centrifuge tube of an ultracentrifuge, set the centrifugal force to 200,000 g, and the centrifugation time to 2 hours.

[0113] 3. Collect the supernatant: After centrifugation, aspirate the supernatant, which contains unencapsulated exosomes.

[0114] 4. Exosome quantification: Quantify the exosomes in the supernatant:

[0115] Protein quantification: If using the BCA Protein Assay Kit, follow the instructions of the kit to determine the exosome protein content in the supernatant.

[0116] Marker detection: Exosome-specific markers can be detected by Western blot, ELISA and other methods to quantify exosomes.

[0117] 5. Calculate the encapsulation efficiency:

[0118] The total amount of exosomes initially added was measured (by the same quantification method).

[0119] Encapsulation efficiency = (initial total amount of exosomes - amount of exosomes in the supernatant) / initial total amount of exosomes × 100%.

[0120] 2. Determination of Targeted Release Characteristics

[0121] 1. Sample preparation: The complexes prepared in Example 3 and Comparative Examples 4-5 were selected as samples and labeled with a lipophilic fluorescent dye.

[0122] 2. Cultivate target cells: Cultivate human skin fibroblasts in a culture dish to a confluence of 80%.

[0123] 3. Establish a simulated skin environment: Add 100 μg collagen and 150 μg hyaluronic acid per ml into the culture system.

[0124] 4. Sample addition: Add the labeled sample to the cultured skin cells.

[0125] 5. Incubation: Place the culture dish in an incubator at 37°C and 5% CO 2 Incubate for 8 hours.

[0126] 6. Cell treatment: Aspirate the culture medium and gently wash the cells 3 times with PBS.

[0127] 7. Cell lysis and detection: Lyse the collected cells to release the fluorescent markers; use a fluorescence spectrophotometer to measure the fluorescence intensity in the lysate and calculate its targeted release efficiency;

[0128] Targeted release efficiency = (release amount in target cells / total input amount) × 100%

[0129] In the formula, the total input is the total amount of fluorescent markers added to the cell culture system;

[0130] The amount of release in the target cells is the amount of marker corresponding to the fluorescence intensity in the cell lysate.

[0131] 3. Result data:

[0132] Table 2:

[0133] Group Encapsulation rate (%) Targeted release efficiency (%) Example 3 85.2 75 Comparative Example 4 62.5 55 Comparative Example 5 71.8 61

[0134] 8. Result analysis: The above results show that the method for preparing the complex of the present invention can improve the encapsulation rate and targeting of exosomes. Compared with Comparative Example 4, it is shown that the composite enzyme used in the present invention can decompose the impurities such as proteins and nucleic acids that may exist in liposome nanoparticles and exosomes; specifically modify the surface components of liposomes or exosomes, thereby optimizing their composite performance and targeting; help break the membrane structure barriers of liposomes and exosomes, promote more effective fusion of the two, and form a more stable and uniform composite structure; activate the functional sites on the surface of liposomes or exosomes, and enhance their binding ability or biological activity with receptors; change the surface charge distribution, and use electrostatic attraction to enhance the interaction and encapsulation efficiency between them, which is conducive to the composite process and the stability of the structure after composite, and can improve the encapsulation efficiency of liposomes;

[0135] Compared with Comparative Example 5, the use of electron beam intermittent irradiation to assist the fusion complex can increase the membrane permeability of liposomes and exosomes, so that the substances inside them can be exchanged and fused more effectively, promoting the mixing of the contents of the two; inducing changes in the membrane structure, making the membrane more fluid, increasing the chances of liposomes and exosomes contacting and fusing with each other; activating intracellular molecules related to membrane fusion, thereby improving the efficiency and success rate of fusion.

[0136] Comparative Example 6

[0137] The difference between this comparative example and Example 3 is that the stem cell exosome repair agent includes the following components in weight: 3 parts of stem cell exosomes, 6 parts of hyaluronic acid, 1 part of collagen, 0.2 parts of vitamin E mixture, 1.5 parts of glutathione, 1 part of anti-inflammatory agent, 12 parts of liposome nanoparticles, and 90 parts of sterile water.

[0138] Comparative Example 7

[0139] The difference between this comparative example and Example 3 is that the components of the stem cell exosome repair agent do not contain an anti-inflammatory agent.

[0140] Comparative Example 8

[0141] The difference between this comparative example and Example 3 is that the components of the stem cell exosome repair agent do not contain liposome nanoparticles.

[0142] Test Example 1-Tissue Repair Ability

[0143] 1. Animal preparation: 180 healthy C57BL / 6 experimental mice were selected, male, weighing 25 g, 6 weeks old, and adaptively raised for 2 weeks;

[0144] 2. Create skin injuries: Use a sterilized scalpel to create a 1 cm circular wound in the full-thickness skin on the back or flank of the mouse, keeping the wound size consistent.

[0145] 3. Grouping treatment: The mice were randomly divided into an experimental group and a control group, with 20 mice in each group. The experimental groups corresponded to Examples 1-5 and Comparative Examples 6-8 of the present invention, respectively, and 100 μL of stem cell exosome repair agent was injected into the wound, and the control group was given an equal amount of normal saline. After treatment, the wound was gently covered with sterile gauze to avoid infection.

[0146] 4. Observation and measurement: At specific time points after surgery (3rd day, 7th day, 14th day), observe the wound. Use a vernier caliper or ruler to measure the length and width of the wound and calculate the wound area.

[0147] 5. Data analysis: Based on the measured wound area data and histological observation results, analyze the effect of stem cell exosome repair agent on skin damage repair and calculate the wound healing rate;

[0148] Wound healing rate = (initial wound area - wound area at a specific time point) / initial wound area × 100%

[0149] 6. Data results:

[0150] Table 3:

[0151]

[0152]

[0153] Test Example 2-Anti-inflammatory Effect Test

[0154] 1. Experimental animals: C57BL / 6 mice were selected as experimental animals, male weighing 25 g and 6 weeks old;

[0155] 2. Experimental groups:

[0156] (1) Normal control group: no treatment.

[0157] (2) Inflammation model group: An inflammation model was established, but no repair agent was given.

[0158] (3) Experimental group (Examples 1-5): After the inflammatory model was established, 100 μL of the stem cell exosome repair agent of different examples was administered respectively.

[0159] (4) Comparative Example Group (Comparative Examples 6-8): After the inflammation model was established, 100 μL of the repair agent of different comparative examples were administered for treatment.

[0160] 3. Establishment of inflammation model: LPS-induced mouse skin inflammation model was used by intraperitoneal injection of LPS; 4. Sample collection: Blood and inflammatory tissues of animals were collected 24 hours after modeling;

[0161] 5. Detection of inflammatory factors:

[0162] (1) Detection of inflammatory factors in serum: ELISA kits were used to measure the levels of TNF-α, IL-1β, and IL-6 in serum.

[0163] (2) Detection of inflammatory factors in tissues: The mRNA expression levels of TNF-α, IL-1β, and IL-6 in tissues were detected by qPCR.

[0164] 6. Data analysis: Compare the levels of inflammatory factors in the serum and tissues of each group of animals to evaluate the anti-inflammatory effect of stem cell exosome repair agents.

[0165] 7. Data results:

[0166] Table 4:

[0167]

[0168] From the results in Tables 3 and 4 above, it can be seen that the stem cell exosome repair agent of the present invention has certain tissue repair ability and anti-inflammatory effect. Compared with Comparative Example 6, the scientific proportion of the repair agent components can achieve better results;

[0169] Compared with Comparative Example 7, adding a certain mass of Prunella Vulgaris extract, aloe extract and licorice extract as anti-inflammatory agents can synergize with stem cell exosomes, enhance the overall anti-inflammatory ability, more effectively inhibit the inflammatory response, and exert anti-inflammatory effects through different molecular mechanisms and targets, thereby covering a wider range of inflammatory pathways and improving the comprehensive anti-inflammatory effect of the repair agent. Each extract usually also has antioxidant properties, which can reduce the damage to tissues caused by oxidative stress and create a favorable environment for the repair of inflammation.

[0170] Compared with Comparative Example 8, it is shown that liposome nanoparticles can help protect exosomes, enhance their stability, achieve targeted transport and controlled release, etc., so as to improve the effect of stem cell exosomes in treatment and repair.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A stem cell exosome repair agent, characterized in that: The invention comprises the following components in parts by weight: 5-15 parts of stem cell exosomes, 1-5 parts of hyaluronic acid, 2-8 parts of collagen, 0.5-2 parts of vitamin E, 0.2-1 parts of glutathione, 3-6 parts of anti-inflammatory agent, 20-28 parts of liposome nanoparticles, and 50-80 parts of sterile water; the concentration of the stem cell exosomes is greater than 5.0×10 10 particles / mL, and the protein ratio of stem cell exosomes was higher than 4.17×10 8 particles / μg.

2. The stem cell exosome repair agent according to claim 1, characterized in that: The invention comprises the following components in parts by weight: 10 parts of stem cell exosomes, 3 parts of hyaluronic acid, 5 parts of collagen, 1 part of vitamin E, 0.6 parts of glutathione, 5 parts of anti-inflammatory agent, 24 parts of liposome nanoparticles and 60 parts of sterile water.

3. The stem cell exosome repair agent according to claim 1, characterized in that: The anti-inflammatory agent is a selfheal extract, an aloe extract and a liquorice extract in a mass ratio of (2-5):(3-8):(1-3).

4. A method for preparing a stem cell exosome repair agent according to claim 1 or 2, characterized in that: The following steps are involved: S1. Isolation and purification of stem cell exosomes: The collected stem cell supernatant is centrifuged to remove cells, cell debris and other large particles, and then magnetic beads are added for incubation. The incubated mixture is separated under a magnetic field, the supernatant is discarded, the exosomes on the magnetic beads are eluted, and the stem cell exosomes are purified by ultrafiltration; S2. Preparation of liposome nanoparticles: phospholipids and cholesterol are mixed in a mass ratio of 2-5:1, dissolved in chloroform to form a uniform solution, and chloroform is removed by rotary evaporation in a round-bottom flask at 30-40° C. to form a uniform lipid film, and then PBS buffer is added to hydrate the formed liposome suspension, and liposome nanoparticles are prepared by extrusion method; S3, preparing a complex: mixing the stem cell exosomes obtained in S1 and the liposome nanoparticles in S2, adding 0.3-1.5% of the mass of the complex enzyme to the mixture, stirring and fusing at 30-40°C for 3-6 hours, and using intermittent electron beam irradiation to assist fusion during the stirring process, each irradiation is 10-15 minutes, the interval time is 15-25 minutes, the electron beam irradiation dose is 2-8 kGy, the electron beam energy is 2.5-5.5 MeV, and the beam current intensity is 3-10 mA, to obtain a complex; S4. preparing a mixed solution: mixing hyaluronic acid, collagen, vitamin E, glutathione and sterile water in proportion, and stirring evenly to obtain a mixed solution; S5. Preparation of repair agent: adding the complex prepared in S3 and the anti-inflammatory agent to the mixed solution, ultrasonically treating for 10-30 minutes to fully disperse the mixture, and obtaining the stem cell exosome repair agent.

5. The method for preparing a stem cell exosome repair agent according to claim 4, characterized in that: The method for separating and purifying S1 stem cell exosomes comprises the following steps: S1-1, Centrifugation: Collect the stem cell supernatant into a sterile centrifuge tube and centrifuge at 3000-10000g for 10-25min to remove cells, cell debris and other large particles; S1-2, incubation: add the magnetic beads to the above centrifuge tube and incubate in segments using a pulsed magnetic field. Initial incubation: adjust the temperature of the mixture to 3-6°C, stir at 30-50rpm, and incubate for 1-3h; intermediate incubation: temperature is 8-12°C, stirring at 60-100rpm, and incubate for 3-6h; late incubation: temperature is 3-6°C, 40-60rpm, and incubate for 2-4h. The pulsed magnetic field strength of the above segmented incubation is 100-500mT; the pulse frequency is 30-80Hz, and the number of pulses is 10-50; S1-3, separation: the incubated mixture was placed in a magnetic field with a strength of 500-1000 Gauss for 3-5 hours, and the supernatant was aspirated and discarded; S1-4, elution: Use a phosphate buffer with a pH of 5.0 and a concentration of 0.1 mol / L to elute the exosomes bound to the magnetic beads, and transfer the eluted exosome solution to a new centrifuge tube; S1-5. Purification: The exosome solution is ultrafiltered 2-4 times using an ultrafiltration membrane with a pore size of 10-100 kDa, and the liquid in the lower chamber is collected to obtain stem cell exosomes.

6. The method for preparing a stem cell exosome repair agent according to claim 5, characterized in that: The stem cells are selected from any one of umbilical cord mesenchymal stem cells, human adipose mesenchymal stem cells, bone marrow mesenchymal stem cells and placental mesenchymal stem cells.

7. The method for preparing a stem cell exosome repair agent according to claim 5, characterized in that: The magnetic beads are superparamagnetic ferroferric oxide magnetic beads or ferric oxide nanoparticles.

8. The method for preparing a stem cell exosome repair agent according to claim 4, characterized in that: The S2 phospholipids include, but are not limited to, phosphatidylcholine or phosphatidylserine.

9. The method for preparing a stem cell exosome repair agent according to claim 4, characterized in that: The complex enzyme of S3 is α-glucosidase and phosphatase in a mass ratio of 1-3:

5.

10. The method for preparing a stem cell exosome repair agent according to claim 4, characterized in that: The ultrasonic power of the S5 is 200-500W, and the frequency is 20-40kHz.

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