Disclosure of Invention
To solve the above technical problems, according to one aspect of the present invention, there is provided a composition enriched in adipose stem cell exosomes.
The technical scheme provided by the invention for solving the technical problems is as follows:
a composition enriched in adipose-derived stem cell exosomes, the method of preparation comprising the steps of:
S1: extracting fat stem cell exosomes;
S2: encapsulation of stem cell exosomes;
S3: compounding an exosome interlayer structure;
s4: the microcapsule shell layer gel structure is loaded.
Preferably, the step of extracting the fat stem cell exosomes in step S1 is as follows:
C1: adding human fat stem cells into a DMEM/F12 culture medium for suspension, then dripping the suspension onto a standard tissue culture plate, culturing at 37 ℃ under the condition of 5% CO2, and replacing the DMEM/F12 culture medium every 1-2 days; culturing the adipose-derived stem cells to 80% of fusion degree, adding 0.25wt% of trypsin-EDTA solution to reduce the cell fusion degree for digestion and passage, and simultaneously culturing the cells by replacing a serum-free culture medium for 24-48 hours to obtain the adipose-derived stem cells;
C2: centrifuging the fat stem cells obtained in the step C1 at 2000-5000 rpm for 10min at a low speed, taking supernatant, performing ultrasonic treatment at 60W power for 20-30 min, and filtering with a filter membrane with the diameter of 0.22 mu m to obtain filtrate; putting the filtrate into a high-speed centrifuge, controlling the rotating speed to 8000-12000 rpm, centrifuging for 30min at the temperature of 4 ℃, filtering by using a 300-1000 kDa ultrafiltration membrane, washing by using PBS buffer solution, adding 3wt% of sucrose, and freeze-drying to obtain the fat stem cell exosome.
Preferably, the human adipose-derived stem cells in step C1 are washed with PBS buffer containing 1wt% penicillin, wherein the penicillin concentration is 90U/mL, before being added to the culture medium; the concentration of the human adipose-derived stem cells is 1X 106～3×106/mL.
Preferably, the step of encapsulating the stem cell exosomes in step S2 is as follows:
D1: dispersing fat stem cell exosomes in PBS buffer solution, and uniformly mixing to obtain 10mg/mL exosome solution; dissolving poly (acrylic acid-co-methacrylic acid) copolymer in deionized water to obtain 20mg/mL polymer solution;
D2: continuously and slowly adding the exosome solution prepared in the step D1 into the polymer solution prepared in the step D1, adding 1wt% of pentanediol, regulating the pH to 6.5-7.4 by using an HCl solution and a NaOH solution, stirring at a high speed of 10000rpm for 30-60 min at room temperature, centrifuging, washing by using PBS buffer solution, and performing vacuum freeze-drying at 0 ℃ for 12-24 h to obtain the exosome microcapsule.
Preferably, the poly (acrylic acid-co-methacrylic acid) copolymer in step D1 is prepared by free radical polymerization with benzoyl peroxide as initiator, and mixing acrylic acid and methacrylic acid in a molar ratio of 7:3.
Preferably, the ratio of the exosome solution to the polymer solution in step D2 is 1:3-10.
Preferably, the exosome interlayer structure compounding step in step S3 is as follows:
E1: dissolving N-isopropyl acrylamide, ammonium persulfate and N, N' -methylene bisacrylamide in deionized water, and uniformly dissolving to obtain a mixed solution; pre-charging nitrogen at the speed of 100mL/min for 10min, heating the mixed solution to 70 ℃, continuously charging nitrogen at the speed of 50-100 mL/min, reacting for 2-4 h, cooling to room temperature, centrifuging, and vacuum drying at the temperature of 20 ℃ for 12-24 h to obtain poly (N-isopropyl acrylamide);
E2: adding the exosome microcapsule prepared in the step D2 into deionized water to prepare microcapsule suspension of 10mg/mL, then adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, controlling the temperature to be 30 ℃, stirring at 100rpm for 10-30 min, adding lysine solution of 5mg/mL, controlling the temperature to be 30 ℃, stirring at 150rpm for 10-30 min, centrifuging, filtering, washing with deionized water, and vacuum drying at 30 ℃ for 12-16 h to obtain modified microcapsule;
E3: adding the poly (N-isopropyl acrylamide) prepared in the step E1 into deionized water to prepare a poly (N-isopropyl acrylamide) solution with the concentration of 2wt%, and adding the modified microcapsule prepared in the step E2 into the deionized water to prepare a modified microcapsule suspension with the concentration of 5 mg/mL; mixing poly (N-isopropyl acrylamide) solution with modified microcapsule suspension, adding nano silver particles with the total volume of 0.001% of the solution, heating in water bath at the temperature of 32-35 ℃, stirring at 200rpm for 30-60 min, cooling to room temperature after the reaction is finished, centrifuging, filtering, washing with deionized water, and vacuum drying at 30 ℃ for 12-24 h to obtain the exosome composite microcapsule.
Preferably, the dosage ratio of the N-isopropyl acrylamide, the ammonium persulfate, the N, N' -methylene bisacrylamide and the deionized water in the step E1 is 1g to 0.01-0.02 g to 0.01g to 10mL.
Preferably, the microcapsule suspension, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and lysine are used in a ratio of 1mL:0.5 mg:0.4-0.5 mg in step E2: 10mg.
Preferably, the volume ratio of poly (N-isopropylacrylamide) solution to modified microcapsule suspension in step E3 is 1-5:1.
Preferably, the microcapsule shell layer gel structure loading step in step S4 is as follows:
h1: respectively dissolving carboxymethyl chitosan and sodium alginate in deionized water to prepare carboxymethyl chitosan solution and sodium alginate solution with concentration of 2 wt%; adding the exosome composite microcapsule in the step E3 into deionized water to prepare exosome composite microcapsule suspension with the concentration of 1 mg/mL;
H2: mixing the carboxymethyl chitosan solution prepared in the step H1 and the sodium alginate solution according to the volume ratio of 1:1, controlling the temperature to be 4 ℃, stirring at 300rpm for 15-25 min, adding 20wt% of the exosome composite microcapsule suspension prepared in the step H1 and 2 times of 0.05mol/L KOH solution, continuously stirring for 30min, dropwise adding 2wt% of CaCl2 solution at the speed of 0.1-1 mL/min in the process, keeping the pH value to be neutral by using NaOH solution and HCl solution, finishing the reaction, washing by deionized water, and freeze-drying at-40 ℃ for 24H to obtain the exosome microcapsule composition.
Preferably, another aspect of the present invention provides the use of a composition enriched in adipose stem cell exosomes as a gel directly applied to the cleaned wound surface twice daily for a time interval of more than 8 hours.
It should be noted that the application of the stem cell exosome composition gel prepared by the invention is mainly used for providing wound coverage, providing a protective layer for the wound, being incapable of directly curing the wound as a medicament, and mainly used for assisting the natural recovery of the wound and providing an environment favorable for cell proliferation.
The methods used in the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available.
The beneficial effects of the invention are as follows:
In the process of preparing the exosome microcapsule, the poly (acrylic acid-co-methacrylic acid) copolymer creates an encapsulation environment, and the environment not only protects the exosome from physical and chemical damages, but also has the characteristic of pH sensitivity, can trigger the controlled release of the exosome under the specific pH condition (such as when the pH value is increased), and can more accurately release the exosome to act on the specific part of the skin. Specifically, during inflammation, a large number of inflammatory cells (e.g., leukocytes) accumulate on the skin, and the metabolic products released by these cells cause an increase in the local environmental pH, as well as the metabolic activity of certain bacteria causing a further increase in the pH of the wound area; specifically, the invention uses the polymer containing carboxyl (-COOH) to be non-ionized in an acidic environment and has lower hydrophilicity; in an alkaline environment, carboxyl is ionized into carboxylate (-COO-), so that the hydrophilic characteristic of the material is increased, the swelling and the porosity of the material are increased, and further the gradual release of exosomes is effectively realized.
In the process of preparing the intermediate layer structure of the exosome microcapsule, positive charge is introduced by adding lysine, and meanwhile, the stability of the microcapsule is enhanced by utilizing poly (N-isopropyl acrylamide) and nano silver particles, and the capability of targeting cells is provided; the positive charge also helps the microcapsules interact with negatively charged biological membranes, thereby increasing adsorption and permeation capacity. Compared with the method of directly modifying the surface of the microcapsule, the method can more effectively realize the uniformity of modification by preparing the intermediate layer and then packaging the exosome, greatly simplify the preparation process and lead the preparation of the whole system to be more direct and efficient.
The invention also utilizes carboxymethyl chitosan and sodium alginate to prepare a hydrogel loaded framework, and effectively avoids stress reaction by virtue of excellent biocompatibility and degradability. Through the use of CaCl2 for crosslinking, a stable three-dimensional network structure with certain strength is formed, so that the biocompatibility of the three-dimensional network structure is improved, and rejection is effectively avoided. This structure provides physical protection to the exosome from rapid degradation or inactivation in the external environment, thereby achieving sustained release in vivo. The localized drug release provided by the hydrogel system can maximize the drug concentration in the target area while reducing the impact on surrounding healthy tissue. This system can continue to function effectively when combined with microcapsule structures that have both temperature and pH responsiveness. The exosome microcapsules are loaded on the hydrogel, so that the hydrogel absorbs heat when water evaporates, a cool feel is provided for skin, the skin temperature is effectively reduced, pain and burning feel are relieved, meanwhile, the skin moisture is kept, and the skin moisture-keeping gel has a good moisturizing effect on dry and damaged skin.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: a composition enriched in adipose-derived stem cell exosomes, the method of preparation comprising the steps of:
s1: extracting exosomes of human adipose-derived stem cells;
C1: obtaining waste adipose tissue of human abdomen under the aseptic condition, flushing for three times by using sterile saline, immersing the waste adipose tissue into PBS buffer solution added with 1wt% of streptomycin, repeatedly flushing to remove blood and other visible impurities, simultaneously removing massive tissues and fibers in the adipose tissue, shearing the cleaned adipose tissue into small blocks of about 1mm by using surgical scissors, adding 30mg of adipose tissue into 100mL of PBS buffer solution, adding 2mL of collagenase, controlling the temperature to 37 ℃, slightly shaking at 40rpm for 12h,6000rpm for centrifugation for 10min, and removing supernatant to obtain adipose stem cells; washing fat stem cells with PBS buffer solution containing 1wt% penicillin, transferring into DMEM/F12 culture medium for suspension, preparing cell concentration of 1×106/mL, dripping 10mL onto standard tissue culture plate, culturing at 37 deg.C under 5% CO2, and changing DMEM/F12 culture medium every 1 day; when the cell fusion degree exceeds 80%, adding 80 mu L of 2.5wt% trypsin-EDTA solution to reduce the cell fusion degree for digestion and passage, and simultaneously culturing cells for 24 hours by replacing a serum-free culture medium to obtain adipose-derived stem cells;
C2: adding 60mg of the adipose-derived stem cells obtained in the step C1 into deionized water to prepare 60mL of suspension, adding 2mL of physiological saline, centrifuging at 2000rpm for 10min at a low speed, taking supernatant, performing ultrasonic treatment at 60W power for 20min, and filtering with a 0.22 mu m filter membrane to obtain filtrate; placing the filtrate into a high-speed centrifuge, controlling the rotation speed to 8000rpm, centrifuging at 4 ℃ for 30min, filtering with a 300kDa ultrafiltration membrane, washing with PBS buffer, adding 1.8g of sucrose, and freeze-drying to obtain the fat stem cell exosomes.
S2: encapsulation of stem cell exosomes:
D1: dispersing fat stem cell exosomes in PBS buffer solution, and uniformly mixing to obtain 10mg/mL exosome solution; dissolving poly (acrylic acid-co-methacrylic acid) copolymer in deionized water to obtain 20mg/mL polymer solution;
D2: continuously and slowly adding 10mL of the exosome solution prepared in the step D1 into 30mL of the polymer solution prepared in the step D1, adding 0.4mL of pentanediol, adjusting the pH to 6.5 by using an HCl solution and a NaOH solution, stirring at a high speed of 10000rpm at room temperature for 30min, centrifuging, washing by using PBS buffer solution, and performing vacuum freeze-drying at 0 ℃ for 12h to obtain the exosome microcapsule.
S3: exosome intermediate layer structure is compound:
E1: 5g of N-isopropyl acrylamide, 0.05g of ammonium persulfate and 0.05g of N, N' -methylene bisacrylamide are dissolved in 50mL of deionized water, and the solution is uniformly dissolved to obtain a mixed solution; pre-charging nitrogen at the speed of 100mL/min for 10min, heating the mixed solution to 70 ℃, continuously charging nitrogen at the speed of 50mL/min, reacting for 2h, cooling to room temperature, centrifuging, and vacuum drying at 20 ℃ for 12h to obtain poly (N-isopropyl acrylamide);
E2: adding the exosome microcapsule prepared in the step D2 into deionized water to prepare microcapsule suspension of 10mg/mL, then adding 2.5mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2mg of N-hydroxysuccinimide, controlling the temperature to be 30 ℃, stirring at 100rpm for 10min, adding 10mL of lysine solution of 5mg/mL, controlling the temperature to be 30 ℃, stirring at 150rpm for 10min, centrifuging, filtering, washing with deionized water, and drying at 30 ℃ in vacuum for 12h to obtain modified microcapsule;
e3: adding the poly (N-isopropyl acrylamide) prepared in the step E1 into deionized water to prepare a poly (N-isopropyl acrylamide) solution with the concentration of 2wt%, and adding the modified microcapsule prepared in the step E2 into the deionized water to prepare a modified microcapsule suspension with the concentration of 5 mg/mL; 50mL of poly (N-isopropyl acrylamide) solution is mixed with the modified microcapsule suspension, 0.001g of nano silver particles are added, the water bath heating is controlled at 32 ℃, the stirring is carried out at 200rpm for 30min, after the reaction is completed, the mixture is cooled to room temperature, centrifuged, filtered, washed by deionized water and dried at 30 ℃ in vacuum for 12h, and the exosome composite microcapsule is obtained.
S4: microcapsule shell layer gel structure loading:
h1: respectively dissolving carboxymethyl chitosan and sodium alginate in deionized water to prepare carboxymethyl chitosan solution and sodium alginate solution with concentration of 2 wt%; adding the exosome composite microcapsule in the step E3 into deionized water to prepare a suspension of 1 mg/mL;
h2: mixing the carboxymethyl chitosan solution prepared in the step H1 and the sodium alginate solution according to the proportion of 100mL, controlling the temperature to be 4 ℃, stirring at 300rpm for 15min, adding 40mL of the exosome composite microcapsule suspension prepared in the step H1 and 400mL of 0.05mol/L KOH solution, continuously stirring for 30min, dropwise adding 2wt% CaCl2 solution at the speed of 0.1mL/min in the process, keeping the pH value to be neutral by using NaOH solution and HCl solution, washing by deionized water, and freeze-drying at-40 ℃ for 24H to obtain the exosome microcapsule composition.
Example 2: a composition enriched in adipose-derived stem cell exosomes, the method of preparation comprising the steps of:
s1: extracting exosomes of human adipose-derived stem cells;
C1: obtaining waste adipose tissue of human abdomen under the aseptic condition, flushing for three times by using sterile saline, immersing the waste adipose tissue into PBS buffer solution added with 1wt% of streptomycin, repeatedly flushing to remove blood and other visible impurities, simultaneously removing massive tissues and fibers in the adipose tissue, shearing the cleaned adipose tissue into small blocks of about 1mm by using surgical scissors, adding 30mg of adipose tissue into 100mL of PBS buffer solution, adding 2mL of collagenase, controlling the temperature to 37 ℃, slightly shaking at 40rpm for 12h,6000rpm for centrifugation for 10min, and removing supernatant to obtain adipose stem cells; washing fat stem cells with PBS buffer solution containing 1wt% penicillin, transferring into DMEM/F12 culture medium for suspension, preparing cell concentration of 2×106/mL, dripping 10mL onto standard tissue culture plate, culturing at 37deg.C under 5% CO2, and changing DMEM/F12 culture medium every 2 days; when the cell fusion degree exceeds 80%, 85 mu L of 2.5wt% trypsin-EDTA solution is added to reduce the cell fusion degree for digestion and passage, and meanwhile, the serum-free culture medium is used for culturing cells for 36 hours to obtain adipose-derived stem cells;
C2: preparing 1mL of the fat stem cells obtained in the step C1 into 10mL of suspension, adding 2mL of physiological saline, centrifuging at 2500rpm for 10min at a low speed, taking supernatant, performing ultrasonic treatment at 60W power for 24min, and filtering with a 0.22 mu m filter membrane to obtain filtrate; putting the filtrate into a high-speed centrifuge, controlling the rotating speed to 9000rpm, centrifuging at 4 ℃ for 30min, filtering by using a 500kDa ultrafiltration membrane, washing by using PBS buffer solution, adding 1.8g of sucrose, and freeze-drying to obtain the fat stem cell exosome.
S2: encapsulation of stem cell exosomes:
D1: dispersing fat stem cell exosomes in PBS buffer solution, and uniformly mixing to obtain 10mg/mL exosome solution; dissolving poly (acrylic acid-co-methacrylic acid) copolymer in deionized water to obtain 20mg/mL polymer solution;
D2: continuously and slowly adding 10mL of the exosome solution prepared in the step D1 into 60mL of the polymer solution prepared in the step D1 in the stirring process, adding 0.7mL of pentanediol, adjusting the pH to 6.8 by using an HCl solution and a NaOH solution, stirring at a high speed of 10000rpm at room temperature for 40min, centrifuging, washing by using PBS buffer solution, and performing vacuum freeze-drying at 0 ℃ for 16h to obtain the exosome microcapsule.
S3: exosome intermediate layer structure is compound:
E1: 5g of N-isopropyl acrylamide, 0.06g of ammonium persulfate and 0.05g of N, N' -methylene bisacrylamide are dissolved in 50mL of deionized water, and the solution is uniformly dissolved to obtain a mixed solution; pre-charging nitrogen at the speed of 100mL/min for 10min, heating the mixed solution to 70 ℃, continuously charging nitrogen at the speed of 70mL/min, reacting for 3h, cooling to room temperature, centrifuging, and vacuum drying at 20 ℃ for 16h to obtain poly (N-isopropyl acrylamide);
E2: adding the exosome microcapsule prepared in the step D2 into deionized water to prepare a microcapsule suspension of 5mL of 10mg/mL, then adding 2.5mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2.2mg of N-hydroxysuccinimide, controlling the temperature to be 30 ℃, stirring at 100rpm for 15min, adding 10mL of lysine solution of 5mg/mL, controlling the temperature to be 30 ℃, stirring at 150rpm for 15min, centrifuging, filtering, washing with deionized water, and vacuum-drying at 30 ℃ for 14h to obtain a modified microcapsule;
e3: adding the poly (N-isopropyl acrylamide) prepared in the step E1 into deionized water to prepare a poly (N-isopropyl acrylamide) solution with the concentration of 2wt%, and adding the modified microcapsule prepared in the step E2 into the deionized water to prepare a modified microcapsule suspension with the concentration of 5 mg/mL; 150mL of poly (N-isopropyl acrylamide) solution and 50mL of modified microcapsule suspension are mixed, 0.002g of nano silver particles are added, the water bath heating temperature is controlled at 33 ℃, stirring is carried out at 200rpm for 40min, after the reaction is completed, cooling to room temperature, centrifuging, filtering, washing with deionized water, and vacuum drying at 30 ℃ for 16h, thus obtaining the exosome composite microcapsule.
S4: microcapsule shell layer gel structure loading:
h1: respectively dissolving carboxymethyl chitosan and sodium alginate in deionized water to prepare carboxymethyl chitosan solution and sodium alginate solution with concentration of 2 wt%; adding the exosome composite microcapsule in the step E3 into deionized water to prepare a suspension of 1 mg/mL;
h2: mixing the carboxymethyl chitosan solution prepared in the step H1 and the sodium alginate solution according to the proportion of 100mL, controlling the temperature to be 4 ℃, stirring at 300rpm for 20min, adding 40mL of the exosome composite microcapsule suspension prepared in the step H1 and 400mL of 0.05mol/L KOH solution, continuously stirring for 30min, dropwise adding 2wt% CaCl2 solution at the speed of 0.4mL/min in the process, keeping the pH value to be neutral by using NaOH solution and HCl solution, washing by deionized water, and freeze-drying at-40 ℃ for 24H to obtain the exosome microcapsule composition.
Example 3: a composition enriched in adipose-derived stem cell exosomes, the method of preparation comprising the steps of:
s1: extracting exosomes of human adipose-derived stem cells;
C1: obtaining waste adipose tissue of human abdomen under the aseptic condition, flushing for three times by using sterile saline, immersing the waste adipose tissue into PBS buffer solution added with 1wt% of streptomycin, repeatedly flushing to remove blood and other visible impurities, simultaneously removing massive tissues and fibers in the adipose tissue, shearing the cleaned adipose tissue into small blocks of about 1mm by using surgical scissors, adding 30mg of adipose tissue into 100mL of PBS buffer solution, adding 2mL of collagenase, controlling the temperature to 37 ℃, slightly shaking at 40rpm for 12h,6000rpm for centrifugation for 10min, and removing supernatant to obtain adipose stem cells; washing fat stem cells with PBS buffer solution containing 1wt% penicillin, transferring into DMEM/F12 culture medium for suspension, preparing cell concentration of 3×106/mL, dripping 10mL onto standard tissue culture plate, culturing at 37deg.C under 5% CO2, and changing DMEM/F12 culture medium every 2 days; when the cell fusion degree exceeds 80%, 85 mu L of 2.5wt% trypsin-EDTA solution is added to reduce the cell fusion degree for digestion and passage, and meanwhile, the serum-free culture medium is used for culturing cells for 48 hours to obtain adipose-derived stem cells;
C2: preparing 1mL of the fat stem cells obtained in the step C1 into 10mL of suspension, adding 2mL of physiological saline, centrifuging at 4000rpm for 10min at a low speed, taking supernatant, performing ultrasonic treatment at 60W power for 28min, and filtering with a 0.22 mu m filter membrane to obtain filtrate; putting the filtrate into a high-speed centrifuge, controlling the rotating speed to 11000rpm, centrifuging at 4 ℃ for 30min, filtering by an ultrafiltration membrane of 800kDa, washing by a PBS buffer solution, adding 1.8g of sucrose, and freeze-drying to obtain the fat stem cell exosome.
S2: encapsulation of stem cell exosomes:
D1: dispersing fat stem cell exosomes in PBS buffer solution, and uniformly mixing to obtain 10mg/mL exosome solution; dissolving poly (acrylic acid-co-methacrylic acid) copolymer in deionized water to obtain 20mg/mL polymer solution;
D2: continuously and slowly adding 10mL of the exosome solution prepared in the step D1 into 80mL of the polymer solution prepared in the step D1, adding 0.8mL of pentanediol, adjusting the pH to 7.2 by using an HCl solution and a NaOH solution, stirring at a high speed of 10000rpm at room temperature for 50min, centrifuging, washing by using PBS buffer solution, and performing vacuum freeze-drying at 0 ℃ for 20h to obtain the exosome microcapsule.
S3: exosome intermediate layer structure is compound:
E1: dissolving N-isopropyl acrylamide, ammonium persulfate and N, N' -methylene bisacrylamide in deionized water, and uniformly dissolving to obtain a mixed solution; pre-charging nitrogen at the speed of 100mL/min for 10min, heating the mixed solution to 70 ℃, continuously charging nitrogen at the speed of 80mL/min, reacting for 3.5h, cooling to room temperature, centrifuging, and vacuum drying at 20 ℃ for 20h to obtain poly (N-isopropyl acrylamide);
E2: adding the exosome microcapsule prepared in the step D2 into deionized water to prepare microcapsule suspension of 10mg/mL, then adding 2.5mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2.4mg of N-hydroxysuccinimide, controlling the temperature to be 30 ℃, stirring at 100rpm for 25min, adding 10mL of lysine solution of 5mg/mL, controlling the temperature to be 30 ℃, stirring at 150rpm for 25min, centrifuging, filtering, washing with deionized water, and vacuum-drying at 30 ℃ for 15h to obtain modified microcapsule;
E3: adding the poly (N-isopropyl acrylamide) prepared in the step E1 into deionized water to prepare a poly (N-isopropyl acrylamide) solution with the concentration of 2wt%, and adding the modified microcapsule prepared in the step E2 into the deionized water to prepare a modified microcapsule suspension with the concentration of 5 mg/mL; 200mL of poly (N-isopropyl acrylamide) solution is mixed with 50mL of modified microcapsule suspension, nano silver particles with the total volume of 0.0025g are added, the temperature is controlled by heating in a water bath at 34 ℃, stirring is carried out at 200rpm for 50min, after the reaction is completed, cooling to room temperature, centrifuging, filtering, washing with deionized water, and vacuum drying at 30 ℃ for 20h, thus obtaining the exosome composite microcapsule.
S4: microcapsule shell layer gel structure loading:
h1: respectively dissolving carboxymethyl chitosan and sodium alginate in deionized water to prepare carboxymethyl chitosan solution and sodium alginate solution with concentration of 2 wt%; adding the exosome composite microcapsule in the step E3 into deionized water to prepare a suspension of 1 mg/mL;
H2: mixing the carboxymethyl chitosan solution prepared in the step H1 and the sodium alginate solution according to the proportion of 100mL, controlling the temperature to be 4 ℃, stirring at 300rpm for 20min, adding 40mL of the exosome composite microcapsule suspension prepared in the step H1 and 400mL of 0.05mol/L KOH solution, continuously stirring for 30min, dropwise adding 2wt% CaCl2 solution at the speed of 0.8mL/min in the process, keeping the pH value to be neutral by using NaOH solution and HCl solution, washing by deionized water, and freeze-drying at-40 ℃ for 24H to obtain the exosome microcapsule composition.
Example 4: a composition enriched in adipose-derived stem cell exosomes, the method of preparation comprising the steps of:
s1: extracting exosomes of human adipose-derived stem cells;
C1: obtaining waste adipose tissue of human abdomen under the aseptic condition, flushing for three times by using sterile saline, immersing the waste adipose tissue into PBS buffer solution added with 1wt% of streptomycin, repeatedly flushing to remove blood and other visible impurities, simultaneously removing massive tissues and fibers in the adipose tissue, shearing the cleaned adipose tissue into small blocks of about 1mm by using surgical scissors, adding 30mg of adipose tissue into 100mL of PBS buffer solution, adding 2mL of collagenase, controlling the temperature to 37 ℃, slightly shaking at 40rpm for 12h,6000rpm for centrifugation for 10min, and removing supernatant to obtain adipose stem cells; washing fat stem cells with PBS buffer solution containing 1wt% penicillin, transferring into DMEM/F12 culture medium for suspension, preparing cell concentration of 3×106/mL, dripping 10mL onto standard tissue culture plate, culturing at 37deg.C under 5% CO2, and changing DMEM/F12 culture medium every 2 days; when the cell fusion degree exceeds 80%, adding 90 mu L of 2.5wt% trypsin-EDTA solution to reduce the cell fusion degree for digestion and passage, and simultaneously culturing cells for 48 hours by replacing a serum-free culture medium to obtain adipose-derived stem cells;
C2: preparing 1mL of the fat stem cells obtained in the step C1 into 10mL of suspension, adding 2mL of physiological saline, centrifuging at 5000rpm for 10min at a low speed, taking supernatant, performing ultrasonic treatment at 60W power for 30min, and filtering with a 0.22 mu m filter membrane to obtain filtrate; putting the filtrate into a high-speed centrifuge, controlling the rotation speed to 12000rpm, centrifuging at 4 ℃ for 30min, filtering with a 1000kDa ultrafiltration membrane, washing with PBS buffer solution, adding 1.8g of sucrose, and freeze-drying to obtain the fat stem cell exosome.
S2: encapsulation of stem cell exosomes:
D1: dispersing fat stem cell exosomes in PBS buffer solution, and uniformly mixing to obtain 10mg/mL exosome solution; dissolving poly (acrylic acid-co-methacrylic acid) copolymer in deionized water to obtain 20mg/mL polymer solution;
D2: continuously and slowly adding 10mL of the exosome solution prepared in the step D1 into 100mL of the polymer solution prepared in the step D1, adding 1.1mL of pentanediol, adjusting the pH to 7.4 by using an HCl solution and a NaOH solution, stirring at a high speed of 10000rpm at room temperature for 60min, centrifuging, washing by using PBS buffer solution, and performing vacuum freeze-drying at 0 ℃ for 24h to obtain the exosome microcapsule.
S3: exosome intermediate layer structure is compound:
E1: 5g of N-isopropyl acrylamide, 0.1g of ammonium persulfate and 0.5g of N, N' -methylene bisacrylamide are dissolved in 50mL of deionized water, and the solution is uniformly dissolved to obtain a mixed solution; pre-charging nitrogen at the speed of 100mL/min for 10min, heating the mixed solution to 70 ℃, continuously charging nitrogen at the speed of 100mL/min, reacting for 4h, cooling to room temperature, centrifuging, and vacuum drying at 20 ℃ for 24h to obtain poly (N-isopropyl acrylamide);
E2: adding the exosome microcapsule prepared in the step D2 into deionized water to prepare microcapsule suspension of 10mg/mL, then adding 2.5mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 2.5mg of N-hydroxysuccinimide, controlling the temperature to be 30 ℃, stirring at 100rpm for 30min, adding 10mL of lysine solution of 5mg/mL, controlling the temperature to be 30 ℃, stirring at 150rpm for 30min, centrifuging, filtering, washing with deionized water, and vacuum-drying at 30 ℃ for 16h to obtain modified microcapsule;
E3: adding the poly (N-isopropyl acrylamide) prepared in the step E1 into deionized water to prepare a poly (N-isopropyl acrylamide) solution with the concentration of 2wt%, and adding the modified microcapsule prepared in the step E2 into the deionized water to prepare a modified microcapsule suspension with the concentration of 5 mg/mL; 250mL of poly (N-isopropyl acrylamide) solution is mixed with 50mL of modified microcapsule suspension, 0.003g of nano silver particles are added, the temperature is controlled at 35 ℃ by water bath heating, stirring is carried out for 60min at 200rpm, after the reaction is completed, cooling to room temperature, centrifuging, filtering, washing with deionized water, and vacuum drying is carried out at 30 ℃ for 24h, thus obtaining the exosome composite microcapsule.
S4: microcapsule shell layer gel structure loading:
h1: respectively dissolving carboxymethyl chitosan and sodium alginate in deionized water to prepare carboxymethyl chitosan solution and sodium alginate solution with concentration of 2 wt%; adding the exosome composite microcapsule in the step E3 into deionized water to prepare exosome composite microcapsule suspension with the concentration of 1 mg/mL;
h2: mixing the carboxymethyl chitosan solution and the sodium alginate solution prepared in the step H1 according to the proportion of 100mL, controlling the temperature to be 4 ℃, stirring at 300rpm for 25min, adding 40mL of the exosome composite microcapsule suspension prepared in the step H1 and 400mL of 0.05mol/L KOH solution, continuously stirring for 30min, dropwise adding 2wt% CaCl2 solution at the speed of 1mL/min in the process, keeping the pH value to be neutral by using NaOH solution and HCl solution, finishing the reaction, washing by deionized water, and freeze-drying at-40 ℃ for 24H to obtain the exosome microcapsule composition.
Performance test: 20 SCID mice (weighing about 200 g) were divided into 5 groups: blank, example 1, example 2, example 3, example 4, 4 of each group, after anesthesia, skin with back 1cm x 1cm was cut off and disinfected with iodophor, directly smeared with stem cell exosome gel at the wound, covered completely, smeared twice daily, 8 hours apart, with 50 μl of physiological saline each time, fixed with sterile patch, and wound healing was observed, wound area of mice was measured every two days, and healing rate (healing rate = measured area/initial area x 100%) was calculated, and the observation results are shown in table 1:
table 1. Exosome compositions have beneficial effects on skin wounds.
Taking the exosome compositions prepared in the examples 1 and 3 as samples, taking 1 cubic centimeter volume for testing, preparing three gradient phosphate buffer solutions with pH values of 5, 6.5 and 7.5, respectively placing the examples 1 and 3 into different three gradient buffers, controlling the temperature to 35 ℃, respectively observing the release conditions of the compositions at 0.5h, 1.5h and 3h, wherein the release conditions are shown in the table 2 (the release conditions are divided into less than 10 percent and regarded as unreleased, more than 10 percent and less than 50 percent and regarded as small, more than 50 percent and less than 90 percent and regarded as large, and more than 90 percent and regarded as whole):
table 2. Exosome composition release profile.
Cytotoxicity test: the exosome compositions prepared in examples 1 to 4 and the control group (DMEM medium) were uniformly distributed in 96-well plates with 16 wells per group of samples, then HaCaT epithelial cells were diluted to 1×105/mL and inoculated in 50 μl in 96-well plates, while 8 wells to which no sample was added were used as a negative control group, 8 wells to which HaCaT epithelial cell medium was added were used as a positive control group, the temperature was set at 37 ℃, CO2 at 5% concentration was added, after 24 hours of incubation, 20 μl of 5mg/mL MTT solution was added, the incubation was continued for 4 hours, the culture solution in the wells was aspirated, 100 μl of dimethyl sulfoxide was added to the wells, and the relative viability of cells [ cell viability (%) = (example OD value-negative control group OD value)/(positive control group OD value-negative control group OD value) ×100% ] was measured using an enzyme-marker instrument at 490nm, and the results are shown in fig. 1.
And (3) map measurement: SEM images of the outer hydrogel layer structures of the exosome-loaded compositions prepared in examples 1 and 3 were tested by using a scanning electron microscope, and the results are shown in fig. 2; SEM images of the exosome microcapsules prepared in example 3 were tested using a scanning electron microscope, and the results are shown in fig. 3.
Anti-inflammatory performance test: mouse macrophages were inoculated into a sterile medium (dmem+10% fetal bovine serum) at 37 ℃ and a CO2 concentration of 5%, after 24 hours of incubation, the cell solutions were diluted to a concentration of 1×105 cells/mL, the exosome compositions prepared in examples 1 to 4 and physiological saline were taken as control groups, these samples were sequentially added to the above cell solutions, 100 μl of each group was added to a TNF- α antibody-precoated ELISA plate after mixing, and allowed to stand at room temperature for 2 hours, after washing with PBS buffer three times, 100 μl of 1000-fold diluted secondary antibody labeled with horseradish peroxidase (HRP) was added, allowed to stand at room temperature for 1 hour, after washing with PBS buffer three times, 100 μl of 0.1mg/mL TMB solution was added, allowed to stand for 15 minutes, 100 μl of 1moL/L hydrochloric acid was added, absorbance was read at 450nm using a microplate reader, and anti-inflammatory = (TNF- α concentration of control group-TNF- α concentration-control group- α concentration-100%). As shown in fig. 4%.
Cell proliferation performance test: the exosome composition prepared in examples 1 to 4 was dissolved in physiological saline to obtain an exosome composition solution of 1mg/L, HDP cells were inoculated into 96-well plates at a rate of 104/well, DMEM medium (10% fetal bovine serum+1% penicillin-streptomycin solution) was added, the temperature was 37℃and CO2 concentration was 5%, after 24 hours of incubation, DMEM medium was replaced, 1mg/mL of the exosome composition solution prepared in examples 1 to 4 was added as a test group, physiological saline was added as a positive control group, wells without sample was used as a negative control group, the temperature was set at 37℃and CO2 at 5% concentration was added, after 72 hours of incubation, 20. Mu.L of MTT solution at 5mg/mL was added, the culture solution in the wells was aspirated, 100. Mu.L of dimethyl sulfoxide was added to the wells, and the absorbance (OD value) was measured at 570nm using a microplate reader [ cell proliferation rate= (OD value of example: positive group/OD value: negative control group). Times.). 100% ], as shown in FIG. 5.
As can be seen from the results of Table 1 and FIG. 1, the exosome composition prepared by the present invention has excellent biocompatibility, good adaptability to biological skin, no toxic damage to cells, and excellent growth promoting effect on cells; as can be seen from the image in FIG. 2, the loaded substance of the invention is uniformly distributed in the hydrogel, and the surface of the substance is smoother; as can be seen from the image in fig. 3, the exosome microcapsule prepared by the invention has complete microstructure and good surface shape characteristics; as can be seen from the results of table 1, fig. 4 and fig. 5, the exosome composition prepared by the present invention has a better promoting effect on wound healing, and simultaneously has an inhibiting effect on inflammatory cells, and can effectively assist wound healing while inhibiting inflammatory reactions; as can be seen from table 2, the compositions prepared according to the present invention are capable of exhibiting significant release differences at pH changes.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.