CN118320184A - Injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel and preparation method and application thereof - Google Patents
Injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an injectable silk fibroin microsphere-carboxymethyl cellulose composite gel loaded with exosomes, and a preparation method and application thereof, wherein the composite gel comprises the following raw materials: mesenchymal stem cell exosomes, silk fibroin and carboxymethyl cellulose, the mesenchymal stem cell exosomes are loaded on the silk fibroin to form the silk fibroin microsphere loaded with exosomes. The invention prepares the sericin protein microsphere-carboxymethyl cellulose composite gel loaded with exosomes, which has the advantages of better biocompatibility, high safety, uniform and controllable microsphere particle size, better viscosity of the composite gel and good needle penetrating property. The exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel prepared by the method is suitable for injection application, can play a role in instantaneous filling and long-acting repairing, and has huge application market and development prospect in medical beauty filling market.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an injectable silk fibroin microsphere-carboxymethyl cellulose composite gel loaded with exosomes, and a preparation method and application thereof.
Background
The medical cosmetic filling refers to filling local tissues of a human body by using a filling agent, thereby having the effects of modifying five sense organs, filling tissue contour pits and creating contour lines. Currently, more and more soft tissue fillers are introduced into the cosmetic market, where medical cosmetic fillers are classified by filler type, and medical cosmetic fillers can be classified into injection type and surgical type. The injection mode has the advantages of small risk, flexible and simple operation, no incision, short treatment time, small pain of patients and the like, so the injection mode becomes the main mode of facial filling.
Hydrogels are a class of solid soft materials that are physically or chemically crosslinked from natural or synthetic polymers to form unique three-dimensional network structures and that can adjust physical and chemical properties. The hydrogel three-dimensional network has high water retention, elasticity and biocompatibility, so that the hydrogel three-dimensional network is very suitable for being used as a filling agent for medical cosmetic filling. The injectable hydrogel can be formed in situ under environmental stimulus, can avoid blocking syringe needle, improve drug delivery efficiency, and exhibit single or multiple responses to temperature, pH, ions, electric field, magnetic field, light irradiation, etc. Therefore, the hydrogel has a plurality of advantages suitable for medical filling, and is a good choice from a plurality of filling materials, so that the hydrogel is a pet in the medical filling industry. The injectable hydrogel commonly used at present comprises gelatin hydrogel, hyaluronic acid hydrogel, carboxymethyl cellulose hydrogel and the like, wherein the gelatin hydrogel has the defects of weak mechanical property, complex molecular structure and poor controllability; the hyaluronic acid hydrogel has the problem of light transmission after injection; compared with the two hydrogels, the carboxymethyl cellulose hydrogel has good viscoelasticity and strong mechanical property, and has no light transmission problem, thus being a better hydrogel material. Carboxymethyl cellulose (CMC) is a polysaccharide-based water-soluble cellulose ether, which is widely used in biomedical, food industry, cosmetics and other fields as a drug carrier, a coating material, a thickener and the like due to its good biocompatibility, biodegradability and viscoelasticity, and is also an excellent hydrogel matrix.
Silk fibroin is natural high molecular fibrin extracted from silk, contains 18 amino acids, has the characteristics of excellent mechanical property, biodegradability, biocompatibility, low inflammation, extremely low immunogenicity, capability of promoting cell proliferation and the like, and is considered as a promising material for tissue engineering; it is also possible to promote the wound healing process by modulating NF-kB and reduce scar formation. The ability of silk fibroin to be processed into various material forms and its versatile chemical functionalization options (e.g., films, fibers, gels, sponges, and microspheres) make such biomaterials interesting candidates for biomedical applications. Wherein, the size of the microsphere particles prepared by the silk fibroin has adjustability and can adapt to different transmission barriers in organisms; the antigenicity of the modified collagen is lower than that of other common biodegradable polymers, such as high molecular polylactic acid, natural macromolecular collagen and the like. The injectable microspheres currently on the market comprise polymethyl methacrylate PMMA microspheres, polycaprolactone PCL microspheres and the like. Wherein the PMMA microspheres are not degraded and metabolized, and can remain at the injection site for a long time, and the risk of forming nodules by aggregation of the microspheres exists; although the polycaprolactone PCL microsphere can be degraded by human body, the degradation time is uncontrollable, the naked eye nodule is easy to cause, and the uncontrollable side effect of PCL on patients with immune diseases is great. Compared with the prior art, the silk fibroin is slow to degrade and controllable, is beneficial to cell repair, ensures the cell growth space, reduces the material consumption, and the degradation products can remarkably promote the skin tissue healing, so that the silk fibroin microsphere is an ideal filling microsphere material.
Exosomes are small vesicles secreted by cells and containing multiple RNAs and proteins, which can enhance the proliferation capacity of fibroblasts, have concentration dependence, can reverse ultraviolet-induced aging of fibroblasts, have the potential to reduce ROS production, reduce oxidative stress damage of skin by inhibiting ROS, and thereby improve aging-related cell damage. Matrix Metalloproteinases (MMPs) are one of the main targets for regulating skin photoaging, and exosomes can regulate MMPs, reduce the damage of dermis elasticity and collagen fibers, and improve dermis aging. Besides the anti-aging function, the exosomes have lower immunogenicity, so that immune rejection reaction can be avoided, and the safety is greatly improved. However, exosomes are difficult to transport and store and are easily inactivated when stored alone at normal temperature.
Therefore, the existing injectable hydrogel microsphere still has the problems of general mechanical property, poor supporting property, poor particle size controllability, poor safety, poor biocompatibility and the like. For example, patent CN201210314947.0 adopts a chemical crosslinking method to prepare silk fibroin/hyaluronic acid composite hydrogel, and the crosslinking agent is one or more than two of divinyl sulfone, ethylene glycol diglycidyl ether, butanediol diglycidyl ether and polypropylene glycol diglycidyl ether, wherein part of the crosslinking agent has high toxicity, and the preparation process has the problems of unsafety, easiness in residue and the like. Patent CN 15970444A uses singly crosslinked hyaluronic acid to prepare a hyaluronic acid gel. The crosslinked hyaluronic acid may have unreacted terminal groups exposed on the surface of gel particles, which may cause red swelling of skin, incomplete crosslinking, and easy crosslinking agent retention, thereby generating potential safety hazard. The single hyaluronic acid gel is used as a soft tissue filling material, has softer texture, poorer viscoelasticity, shorter residence time in tissues, quicker metabolic decomposition and absorption and can not play a role in long-term filling. The patent CN 102836456A discloses a silk fibroin hyaluronic acid composite gel, and compared with the traditional gel with pure cross-linked hyaluronic acid, the silk fibroin particles are added to effectively slow down the degradation speed of the composite gel and prolong the repair filling time, but the mechanical property of the hyaluronic acid is poor, only the occupying effect can be achieved, and a better subcutaneous supporting effect cannot be formed. Patent CN101502676a mixes polymethyl methacrylate (PMMA) microspheres and crosslinked hyaluronic acid gel. Because PMMA is a high polymer which is not degradable by human body, foreign matter stimulation can be generated on local tissues, once the PMMA is difficult to take out after injection, the biocompatibility is poor, and a certain danger exists. The patent CN202010659791.4 provides an injectable silk fibroin/gelatin hydrogel, which is prepared into silk fibroin microspheres with the particle size of 250 nm-400 nm by a method of emulsifying polyethylene glycol and then freezing, and is mixed with gelatin particles with opposite charges under the condition of neutral pH, and the injectable silk fibroin gel is obtained by utilizing electrostatic force. Wherein, the gelatin-based hydrogel has the defects of lower gel strength and poor mechanical property, resulting in poor filling effect; and the silk fibroin microsphere has too small particle size, is easy to be phagocytized by macrophages to cause inflammatory reaction and is easy to generate systemic displacement, and has certain danger.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides the silk fibroin microsphere-carboxymethyl cellulose composite gel which can be injected in situ, has good mechanical property, good supporting property, controllable microsphere particle size, narrow particle size distribution, good needle penetration, high biocompatibility and safety, can promote cell proliferation, has long action effect, can stimulate collagen regeneration, is suitable for subcutaneous injection implantation or filling and is used for loading exosomes, and solves the problems that the existing injectable gel has common mechanical property, poor supporting property, effectively controlled microsphere particle size, poor safety, biocompatibility and the like.
The invention also provides a preparation method and application of the injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel.
The technical scheme is as follows: in order to achieve the scheme, the invention provides the injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel, which comprises the following raw materials: the mesenchymal stem cell exosomes are loaded on the silk fibroin to form exosome-loaded silk fibroin microspheres, and the mass ratio of the carboxymethyl cellulose to the exosome-loaded silk fibroin microspheres is 5-8:1-4.
Wherein the silk fibroin is obtained by degumming silkworm cocoon shells to remove sericin, and the molecular weight is 8000-14000Da.
Wherein the viscosity of the carboxymethyl cellulose is 800-1200 mPa.s.
Wherein the exosome-loaded silk fibroin microspheres are uniformly dispersed in carboxymethyl cellulose hydrogel, and the particle size of the exosome-loaded silk fibroin microspheres is 10-60 mu m; the microsphere particle size D10 is 10-20 μm, preferably 15 μm, 19 μm, D50 is 30-40 μm, preferably 37 μm, 38 μm, D90 is 50-60 μm, preferably 56 μm, 58 μm, and the exosome-loaded silk fibroin microsphere is an injection implant.
Wherein the span value of the exosome-loaded silk fibroin microsphere is 1.0-1.4, preferably 1.017, and the span value is (D90-D10)/D50.
The preparation method of the injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel comprises the following steps:
(1) Extraction and separation of mesenchymal stem cell exosomes: culturing mesenchymal stem cells, extracting mesenchymal stem cell exosomes by an ultracentrifugation method, dispersing the mesenchymal stem cell exosomes in PBS buffer solution to form exosome solution, and storing the exosomes for later use;
(2) Preparation of exosome-loaded silk fibroin microspheres: mixing and incubating the mesenchymal stem cell exosome solution and the silk fibroin solution in the step (1), taking liquid paraffin and Span 80, stirring and mixing, slowly dripping the mixture into the incubated mixed solution, stirring to form uniform and stable water-in-oil emulsion, slowly dripping a cross-linking agent solution, stirring and centrifuging at a low speed, pouring out supernatant, washing and drying until the organic solvent is completely evaporated to obtain the silk fibroin microsphere loaded with exosome, and dispersing in PBS buffer to form suspension for later use;
(3) Preparation of carboxymethyl cellulose hydrogel: adding carboxymethyl cellulose into PBS buffer solution, stirring, dissolving uniformly, cooling for standby, and preparing carboxymethyl cellulose hydrogel;
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
Adding carboxymethyl cellulose hydrogel into an exosome-loaded silk fibroin microsphere suspension, and continuously stirring the mixture at room temperature to obtain exosome-loaded silk fibroin-carboxymethyl cellulose composite gel;
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection:
Dialyzing the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel material, homogenizing, extruding, sieving, and sterilizing with high-temperature and high-pressure steam to obtain the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection.
Wherein, the number of exosomes per milliliter in the intermediate mesenchymal stem cell exosome solution in the step (1) is 10 6~107; the concentration of the silk fibroin solution in the step (2) is 1-60 mg/ml; the two solutions were mixed in equal volumes.
Preferably, the concentration of the silk fibroin solution is 30mg/ml.
Preferably, in the step (2), the silkworm cocoon shells are cut into small pieces, the small pieces are soaked in sodium carbonate solution according to a certain bath ratio for degumming, deionized water is taken out and washed until no greasy feeling exists, and the degummed silk is obtained after the silkworm cocoon shells are baked in an oven until the quality is constant. The degummed silk is immersed in a lithium bromide solution according to a certain bath ratio, heated, stirred and dissolved, and the silk fibroin-lithium bromide solution is obtained. Centrifuging, injecting into a dialysis bag, dialyzing with distilled water for a certain time to obtain silk fibroin solution, centrifuging, and collecting supernatant to obtain silk fibroin solution.
Wherein, the cross-linking agent in the step (2) is one or more than two of glutaraldehyde, genipin, divinyl sulfone, ethylene glycol diglycidyl ether, butanediol diglycidyl ether or polypropylene glycol diglycidyl ether, and most preferably glutaraldehyde.
Preferably, the crosslinking agent is a 50% glutaraldehyde solution when preparing the microspheres.
Wherein the mass-volume ratio of the carboxymethyl cellulose in the carboxymethyl cellulose hydrogel in the step (3) is 0.025-0.09 g/mL. .
Preferably, in the step (5), the above-mentioned silk fibroin microsphere-carboxymethyl cellulose composite gel material loaded with exosomes is added into a dialysis bag with MW 8,000-14,000, PBS buffer with pH value of 7.4 is used as dialysis liquid, dialysis treatment is carried out under magnetic stirring, the gel in the dialysis bag after dialysis treatment is homogenized for 10min by using a homogenizer at 24000rpm/min, and then the gel is placed into a syringe to be extruded to make the gel pass through a 60-mesh sieve, and after high-temperature high-pressure steam sterilization for 15min, aseptic split charging is carried out, and the obtained silk fibroin microsphere-carboxymethyl cellulose composite gel loaded with exosomes for injection is filled into a disposable syringe, and can be used as a subcutaneous injection product.
The invention relates to application of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel in promoting cell proliferation and preparing medical cosmetic filling materials.
The design principle of the invention is as follows: the existing injectable filling materials on the market have the defects of common mechanical property, poor supporting property, incapability of effectively controlling microsphere particle size, poor safety and controllability, need of pre-configuration and the like. Therefore, it is necessary to develop an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel as a subcutaneous tissue filler, and find a preparation method of the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite hydrogel, so that the exosome is wrapped in the silk fibroin microsphere to improve biocompatibility, the microsphere particle size is uniform and controllable, the distribution is narrower, the viscoelasticity of the CMC hydrogel is higher, the mechanical property is better, the pre-configuration is not needed, the biocompatibility is higher, and the medical subcutaneous injection implantation or filling application is facilitated. Compared with common organic high polymer fillers such as polycaprolactone, poly-L-lactic acid and the like, the silk fibroin from natural silk has the advantages of higher biocompatibility, safety, degradability, wide sources, flexible preparation and the like. The silk fibroin is prepared into microspheres, the particle size of the microspheres is uniform and controllable, the particle size is smaller, the push resistance is smaller, the hand feeling is better, and the controlled release capability is also provided. The exosomes have lower immunogenicity, can avoid generating immune rejection reaction, and greatly improve the safety. The exosomes are wrapped in the silk fibroin microspheres, so that the exosomes can be slowly released, and the long-acting repairing effect is achieved. Compared with gelatin and hyaluronic acid hydrogel, the carboxymethyl cellulose hydrogel has better stability and viscoelasticity. The invention prepares the silk fibroin microsphere-carboxymethyl cellulose composite gel loaded with exosomes, so as to improve the defects of uneven microsphere particle size, low viscoelasticity, large push injection resistance, poor filling effect and the like of the original filling material.
In the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel prepared by the invention, the particle size of the exosome-loaded silk fibroin microsphere is effectively controlled. The macrophages of the human body are 25-30 microns in size, and the volume of foreign matters which are up to 25% of the volume of the macrophages is taken up per hour, if the injected particles are smaller than 10 microns, the particles are immediately phagocytized by the macrophages after entering the human body, so that the effect of stimulating collagen production is not only achieved, but also the macrophages are mutually fused to form foreign matter granuloma, and the foreign matter granuloma is a common non-allergic chronic inflammatory reaction of the implantable skin filler, and the clinical treatment is quite difficult. Therefore, the invention controls the grain diameter of the silk fibroin microsphere loaded with exosomes within the range of more than 10 microns and less than 60 microns, prevents phagocytosis of macrophages, simultaneously avoids blocking an injection needle, and ensures better hand feeling of injection.
The invention adds the silk fibroin microsphere with proper and uniform controllable particle size and loaded exosomes into the carboxymethyl cellulose hydrogel to prepare the injectable silk fibroin carboxymethyl cellulose composite gel with the loaded exosomes, which has the advantages of high biocompatibility, controllable biodegradability, low bacterial attachment capability, lower immunogenicity, water retention and elasticity, better filling effect and higher safety, and is beneficial to repair and regeneration of skin tissues.
The invention combines the specific silk fibroin microsphere loaded with exosomes and the carboxymethyl cellulose hydrogel for the first time, and is characterized in that the silk fibroin is natural high molecular protein, has excellent biocompatibility, mechanical property and tissue repair function, and simultaneously the carboxymethyl cellulose is a biological source material, has high viscoelasticity modulus and better biocompatibility; compared with the published PCL, PLLA, PMMA and other artificially synthesized materials, the safety is higher, the biocompatibility is better, the mechanical property is stronger, the particle size of the microsphere is effectively controlled, the particle size distribution range is narrower, the microsphere can be uniformly distributed in gel, and the combined use effect of silk fibroin and carboxymethyl cellulose is obviously improved compared with the existing combined effect.
Beneficial results: compared with the prior art, the invention has the following advantages:
1. Most injectable filling materials require massaging after injection, are soft in texture and weak in supporting force. The carboxymethyl cellulose hydrogel with high viscoelasticity modulus and the silk fibroin microsphere which is beneficial to skin tissue regeneration and can be released in a controlled manner are used together, so that the effects of instantaneous filling and long-acting repair can be achieved, and a new strategy is provided for the medical beauty filling direction.
2. The silk fibroin selected by the invention is derived from natural silk with similar structure to human skin, 18 amino acids are contained in the silk fibroin, 11 amino acids are essential amino acids of human body, and the silk fibroin can accelerate cell metabolism, repair stratum corneum, delay skin aging and slow aging. The silk fibroin microsphere type repair agent is presented in a silk fibroin microsphere type, so that the silk fibroin microsphere type repair agent has the advantage of continuous repair.
3. The silk fibroin solution prepared by the method is clear and transparent and light yellow in character, has molecular weight close to that of natural silk fibroin molecules, has good dispersibility of silk fibroin molecules in the solution, does not have aggregation phenomenon or flocculent precipitate, has controllable concentration of 1-50mg/ml, has high content of high molecular silk fibroin and high protein purity, and can prepare silk fibroin solutions with different concentrations and different molecular weights;
4. The specific exosome-loaded silk fibroin microsphere prepared by the invention has the particle size of 20-60 mu m, uniform and controllable particle size and better morphology, can be prevented from being rapidly absorbed by human bodies (phagocytized by macrophages) and can stimulate and generate more collagen, and the metabolism period is longer than that of PCL, so that the effect is longer. The better properties and the optimal filling effect can be achieved by adjusting the mixture ratio of the used emulsifying agent, the dosage of the cross-linking agent, the mixture ratio of the microsphere and the gel, and the like.
5. The invention adopts exosomes loaded on specific silk fibroin microspheres to prepare composite gel, plays roles of resisting aging and promoting cell regeneration, and the exosomes and the silk fibroin microspheres have obvious synergistic effect in promoting cell proliferation and collagen regeneration, and have lower immunogenicity, can avoid generating immune rejection reaction and greatly improve safety. The silk fibroin microsphere in the composite gel has certain rigidity, takes long-acting filling effect, and the composite gel of the silk fibroin microsphere loaded with exosomes can effectively stimulate collagen regeneration and fill the concave part.
6. The invention effectively combines the silk fibroin microsphere with injection administration, and provides possibility for the application of the silk fibroin microsphere to wrap other active ingredients such as whitening, moisturizing and the like in injection administration.
Drawings
Fig. 1 is a transmission electron microscope characterization of exosomes.
FIG. 2 is a morphological characterization of silk fibroin blank microspheres by an inverted microscope.
FIG. 3 shows carboxymethyl cellulose gels at different concentrations.
Fig. 4 is a graph of cell proliferation (representing p < 0.0001).
Fig. 5 is a graph of inflammatory factor release (representing p < 0.0001).
Fig. 6 is a comparison of the back skin treatment effect of mice before and after injection. Hollow silk fibroin microsphere-carboxymethyl cellulose composite gel set (left); exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel set (right).
FIG. 7 shows the results of three-color staining of skin HE and Marathon in a physiological saline group, a hollow silk fibroin microsphere-carboxymethyl cellulose composite gel group and an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel group after three months of injection. Saline group (left); hollow silk fibroin microsphere-carboxymethyl cellulose composite gel group (medium); exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel set (right).
Fig. 8 shows the increase in collagen for each group three months after injection (p < 0.0001).
Detailed Description
In order that the invention may be more readily understood, it is further described below in connection with specific examples which are intended to be in no way limiting, but are intended to be in any way limiting, and any modifications or alterations which would be readily apparent to a person of ordinary skill in the art without departing from the technical solutions of the present invention will fall within the scope of the claims of the present invention.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
The silkworm cocoon shell is derived from northwest silkworm mulberry base, and is commercially available. Glutaraldehyde 50%, aladdin (aledine); carboxymethyl cellulose, available from Nanjing-one-base Biochemical technology Co., ltd, has a viscosity of 800-1200 mPa.s.
Human umbilical cord-derived mesenchymal stem cells were purchased from Shanghai ATCC cell bank.
Poly-L-lactic acid (PLLA) with molecular weight of 34000g/mol, intrinsic viscosity [ eta ] =3.8 d L/g, purac.
Example 1
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes:
Culturing mesenchymal stem cells of human umbilical cord source with DMEM containing 10% fetal bovine serum as culture medium at 37deg.C under 5% CO 2 until generation 4, culturing at 37deg.C for 24 hr (about 1× 7 cells/dish) with DMEM culture medium without serum, collecting culture medium, centrifuging at 4deg.C for 10min with ultra-high speed centrifuge, filtering supernatant with 0.22 μm filter membrane, centrifuging for 70min to obtain exosomes, dispersing in PBS (pH 7.4) buffer solution to form exosome solution with concentration of 10 6 cells/ml, and storing at-80deg.C for use.
(2) Preparation of exosome-loaded silk fibroin microspheres:
cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4, soaking the mixture in 9.3M lithium bromide solution in a mass ratio, and heating and dissolving the mixture to obtain the silk fibroin-lithium bromide solution. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80mL of liquid paraffin, stirring and mixing with 0.8mL of Span 80 and 400r/min for 40min, slowly dripping 8mL of the incubated mixed solution, stirring for 1h at 400r/min to form uniform and stable water-in-oil emulsion, slowly dripping 4mL of 50% glutaraldehyde solution, centrifuging for 30min at 10000r/min at 4 ℃ after continuously stirring for 3h at 400r/min, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/mL.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10mL of PBS (pH 7.4) buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel. The exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite hydrogel is shown in fig. 3.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: adding the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel material into a dialysis bag with MW of 8,000-14,000, taking PBS buffer with pH value of 7.4 as dialysis liquid, performing dialysis treatment for 24 hours under magnetic stirring, homogenizing the composite gel material in the dialysis bag after the dialysis treatment for 10 minutes by using a homogenizer at 24000rpm/min, placing the homogenized composite gel material in a syringe to squeeze the gel to pass through a 60-mesh sieve, sterilizing the gel by using high-temperature high-pressure steam at 120 ℃ for 15 minutes, performing aseptic split charging, and loading the gel into a disposable syringe to obtain the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection, which can be used as a subcutaneous injection product.
Example 2
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 400r/min for 40min, slowly dripping 8ml of the incubated mixed solution, stirring for 1h to form uniform and stable water-in-oil emulsion, slowly dripping 8ml of 50% glutaraldehyde solution, continuously stirring for 3h at 400r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS (pH 7.4) buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 3
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 400r/min for 40min, slowly dripping 8ml of the incubated mixed solution, stirring for 1h to form uniform and stable water-in-oil emulsion, slowly dripping 12ml of 50% glutaraldehyde solution, continuously stirring for 3h at 400r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 4
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 400r/min for 40min, slowly dripping 8ml of the incubated mixed solution, stirring for 1h to form uniform and stable water-in-oil emulsion, slowly dripping 2ml of 50% glutaraldehyde solution, continuously stirring for 3h at 400r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 5
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 300r/min for 40min, slowly dripping 8ml of the incubated mixed solution, stirring for 1h to form uniform and stable water-in-oil emulsion, slowly dripping 4ml of 50% glutaraldehyde solution, continuously stirring for 3h at 300r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 6
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 500r/min for 40min, slowly dripping 8ml of the incubated mixed solution, stirring for 1h for forming uniform and stable water-in-oil emulsion, slowly dripping 4ml of 50% glutaraldehyde solution, continuously stirring for 3h at 500r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 7
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 700r/min for 40min, slowly dripping 8ml of the incubated mixed solution, stirring for 1h for forming uniform and stable water-in-oil emulsion, slowly dripping 4ml of 50% glutaraldehyde solution, continuously stirring for 3h at 700r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 8
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of span 80 and 900r/min for 40min, slowly dropwise adding 8ml of the incubated mixed solution, stirring for 1h to form uniform and stable water-in-oil emulsion, slowly dropwise adding 4ml of 50% glutaraldehyde solution, continuously stirring for 3h at 900r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 6ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
2.75mL of the exosome-loaded silk fibroin microsphere suspension with the concentration of 0.04g/mL is taken and added into the CMC gel matrix in the step (3), wherein the final concentration of CMC is 0.08g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 9
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 400r/min for 40min, slowly dripping 4ml of the incubated mixed solution, stirring for 1h to form uniform and stable water-in-oil emulsion, slowly dripping 4ml of 50% glutaraldehyde solution, continuously stirring for 3h at 400r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Example 10
Preparation of injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
(1) Extraction and separation of mesenchymal stem cell exosomes are the same as in example 1;
(2) Preparation of exosome-loaded silk fibroin microspheres:
Cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, and measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30 mg/ml.
Mixing and incubating the mesenchymal stem cell exosome solution in the step (1) and the silk fibroin solution in the step (2) according to the volume ratio of 1:1, taking 80ml of liquid paraffin, stirring and mixing with 0.8ml of Span 80 and 400r/min for 40min, slowly dripping 4ml of 50% glutaraldehyde solution after stirring for 1h to form uniform and stable water-in-oil emulsion, slowly dripping 400r/min for 3h, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, and then freeze-drying the product for 24h to obtain the silk fibroin microspheres loaded with exosomes, and dispersing the silk fibroin microspheres in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml.
(3) Preparation of carboxymethyl cellulose hydrogel:
0.7g of carboxymethyl cellulose is gradually added into 10ml of PBS buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling for standby.
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
7.5mL of the exosome-loaded silk fibroin microsphere suspension with a concentration of 0.04g/mL was added to the CMC gel matrix in step (3), wherein the final CMC concentration was 0.04g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel.
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection: as in example 1.
Test example 1
Morphological characterization of blank silk fibroin microspheres
Cutting silkworm cocoon shells into small pieces of about 1cm 2 according to the method of example 8, soaking 10g of silkworm cocoon slices in 1L of sodium carbonate solution with the concentration of 0.02mol/L, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into a dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36h, centrifuging at 10000rpm for 10min, collecting supernatant, measuring Nanodrop protein concentration to obtain silk fibroin solution with concentration of 30mg/ml, and diluting with PBS solution with pH of 7.4 twice. Taking 80ml of liquid paraffin and 0.8ml of Span 80, stirring and mixing for 40min at 900r/min, slowly dripping 8ml of silk fibroin solution, stirring for 1h at 900r/min to form uniform and stable water-in-oil emulsion, slowly dripping 4ml of 50% glutaraldehyde solution, continuously stirring for 3h at 900r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, mixing and incubating the product with rhodamine dye solution for 20min, preparing a sample, beating and inverting a microscope, and clearly observing the appearance of the silk fibroin microsphere loaded with exosomes, wherein the microsphere is round and has good appearance, as shown in figure 2.
Test example 2
Particle size detection of exosome-loaded silk fibroin microspheres
(1) The exosome-loaded silk fibroin microsphere suspensions prepared in examples 1 to 10 were each taken at 3ml (0.04 g/ml), sonicated for 10min, and then measured with a particle size analyzer, and the results are shown in Table 1.
TABLE 1 detection of the particle size of exosome-loaded silk fibroin microspheres
The experimental results of table 1 show that factors affecting the variation of the particle size of the microspheres include glutaraldehyde amount, stirring rotation speed, and amount of silk fibroin solution (water-oil ratio), wherein the particle size shows a tendency of ascending and then descending as glutaraldehyde amount increases, and the particle size also increases as rotation speed increases and increases as water-oil ratio increases.
The invention adjusts the optimal proportion by controlling the dosage of glutaraldehyde, the stirring rotation speed and the water-oil ratio, controls the grain diameter to be 20-60 mu m, the grain diameter span value to be 1.0-1.4, and the grain diameter distribution is narrower and more uniform, and is most preferable in the embodiment 8. The composite gel prepared by the invention has controllable particle size and narrower particle size distribution.
Test example 3
Viscosity test of carboxymethyl cellulose hydrogels and gelatin hydrogels at different concentrations
Carboxymethyl cellulose and gelatin with different mass numbers are respectively added into water at 60 ℃ and stirred, and dissolved uniformly to prepare carboxymethyl cellulose hydrogel and gelatin hydrogel with the concentration of 0.0025g/ml, 0.005g/ml, 0.01g/ml, 0.02g/ml, 0.04g/ml and 0.08g/ml, and the viscosity is measured after cooling, as shown in table 2. Various concentrations of carboxymethyl cellulose hydrogels were prepared as shown in figure 3.
TABLE 2 viscosity comparison of carboxymethylcellulose hydrogels and gelatin hydrogels at different concentrations
Table 2 shows that the viscosity of carboxymethyl cellulose and gelatin hydrogels increases with increasing concentration; compared with gelatin hydrogel, the carboxymethyl cellulose hydrogel has higher viscosity, stronger plastic shape, better mechanical property and better filling effect under the same concentration, and is more suitable for being used as a carrier matrix for subcutaneous filling, so that the composite gel prepared by the invention has good mechanical property and supporting property.
Test example 4
Cell proliferation assay
The cell proliferation experiment is to examine the safety of the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel.
The final concentration composition of the cell culture fluid is as follows: 10% FBS,90% HI-DMEM, ultrapure water as solvent and pH value of 7.0-7.2. The test cells are L929 fibroblasts, and the test cells are cells with vigorous growth after passage for 48-72 h.
(1) Preparation of blank silk fibroin microsphere suspension: cutting silkworm cocoon shells into small blocks of about 1cm 2, soaking 10g of silkworm cocoon slices in 1L of 0.02mol/L sodium carbonate solution, boiling for degumming for 30min, taking out, washing with deionized water until no greasy feeling exists, and drying in an oven until the quality is constant to obtain degummed silk. Degumming silk according to a weight ratio of 1:4 bath ratio is immersed in 9.3M lithium bromide solution, heated and dissolved, and silk fibroin-lithium bromide solution is obtained. Injecting into dialysis bag with MW 8,000-14,000, dialyzing with distilled water for 36 hr, centrifuging at 10000rpm for 10min, collecting supernatant, measuring Nanodrop protein content to obtain silk fibroin solution with concentration of 30mg/ml, and diluting with PBS solution with pH of 7.4. Taking 80ml of liquid paraffin and 0.8ml of Span 80, stirring and mixing for 40min at 900r/min, slowly dripping 8ml of the silk fibroin solution, stirring for 1h at 900r/min to form uniform and stable water-in-oil emulsion, slowly dripping 4ml of 50% glutaraldehyde solution, continuously stirring for 3h at 900r/min, centrifuging for 30min at 10000r/min at 4 ℃, pouring out supernatant, washing with isopropanol, drying until the organic solvent is completely evaporated, then freeze-drying the product for 24h to obtain hollow silk fibroin microspheres, preparing a mixed suspension of 1mg/ml by using a cell culture solution, and incubating for 24h at 37 ℃;
(2) Preparation of exosome-loaded silk fibroin microsphere suspension: preparing a mixed suspension of 1mg/ml by using a cell culture solution for the exosome-loaded silk fibroin microsphere prepared in the example 8, and incubating at 37 ℃ for 24 hours;
(3) Preparation of PLLA microsphere suspension: dissolving poly-L-lactic acid (PLLA) in chloroform to prepare a solution with the concentration of 1 g/L; sodium carboxymethyl cellulose and mannitol (mass ratio of 1:20) are dissolved in distilled water to prepare a solution with concentration of 0.5 percent. The PLLA chloroform solution was slowly added dropwise to the sodium carboxymethylcellulose/mannitol aqueous solution with stirring at a volume ratio of 1:100. Continuously stirring for 12h, vacuum-filtering for 5h to obtain poly-L-lactic acid microspheres, preparing a mixed suspension of 1mg/ml by using a cell culture solution, and incubating at 37 ℃ for 24h;
(4) Preparation of exosome-loaded PLLA microsphere suspension: dissolving poly-L-lactic acid (PLLA) in chloroform to prepare a solution with the concentration of 1g/L, mixing and incubating the mesenchymal stem cell exosome solution in the step (1) in the example 8 with the chloroform solution of PLLA according to the volume ratio of 1:1, and dissolving sodium carboxymethyl cellulose and mannitol (the mass ratio is 1:20) in distilled water to prepare a solution with the concentration of 0.5%. The exosomes and PLLA in chloroform solution were slowly added dropwise to the sodium carboxymethylcellulose/mannitol aqueous solution with stirring, the volume ratio of the two solutions being 1:100. Continuously stirring for 12h, vacuum-filtering for 5h to obtain exosome-loaded poly-L-lactic acid microspheres, preparing 1mg/ml mixed suspension by using a cell culture solution, and incubating at 37 ℃ for 24h;
(5) Preparation of exosome-loaded silk fibroin sponge suspension: mixing and incubating the mesenchymal stem cell exosome solution and the silk fibroin solution according to the volume ratio of 1:1 in the embodiment 8, freezing overnight in a refrigerator at the temperature of minus 20 ℃, rapidly moving in the refrigerator at the temperature of minus 3 ℃ to minus 5 ℃ for low-temperature annealing treatment, and freeze-drying to obtain the silk fibroin sponge scaffold carrying exosomes. Preparing a mixed suspension of 1mg/ml by using a cell culture solution, and incubating at 37 ℃ for 24 hours;
(6) Preparation of small particle size exosome-loaded silk fibroin microsphere suspension: an equal proportion of mixed solution of 20% wt% PEG6000, TWEEN20 and silk fibroin and exosomes prepared in example 8 was prepared at 1:2:4, after the mixture is mixed and stirred for 10min, the mixture is stored at-20 ℃ for 24h, thawed, centrifuged at 12000rpm for 15min, supernatant is removed, and the mixture is washed for three times and freeze-dried to obtain the small-particle-size exosome-loaded silk fibroin microspheres. The particle size of the microsphere is 0.1-10 μm. Preparing a cell culture solution for the prepared silk fibroin microsphere with small particle size and loaded with exosomes into a mixed suspension of 1mg/ml, and incubating at 37 ℃ for 24 hours;
(7) Cell plating and dosing: 1X 10 4 L929 cell suspensions were prepared from the cell culture solution and were dispensed into 96-well plates, and a blank group, a positive control group, an SFM group (blank silk fibroin microspheres), an EXO-loaded SFM group (exosome-loaded silk fibroin microspheres), a PLLA microsphere group, an EXO-loaded silk fibroin sponge group, and an EXO-loaded SFM group having a small particle size were set. Culturing in an incubator containing 5% (V/V) carbon dioxide air at 37deg.C for 24 hr. After 24h, the original culture medium was discarded, fresh cell culture medium was added to the blank control group, positive control solution, i.e., glutaraldehyde (50%), was added to the positive control group, and suspension of blank SFM, EXO-loaded SFM, PLLA microspheres, EXO-loaded silk fibroin sponge, small particle size EXO-loaded SFM, 3 wells per group, 100. Mu.L per well, was placed in the above culture environment and maintained for 72h.
(8) 20 Mu L of MTT solution with the mass concentration of 5g/L is added to each hole of the culture plate in the step (3), the culture is continued for 4 hours under the same condition, stock solution is sucked off, and 150 mu L/hole of DMSO is added. The results of the evaluation of cell proliferation by shaking for 10min and measuring the absorbance at 490nm wavelength on an enzyme-labeled instrument (infinite 200, tecan) are shown in fig. 4.
From the cell proliferation diagram, the difference between the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel group and other groups is P <0.0001, and the difference is extremely remarkable. Therefore, the hollow silk fibroin microsphere and the exosome-loaded silk fibroin microsphere prepared by the method have no cytotoxicity, and can promote cell proliferation. Compared with the hollow silk fibroin microspheres, the silk fibroin microspheres loaded with exosomes can obviously promote cell proliferation, which indicates that the exosomes loaded with the silk fibroin microspheres have obvious promotion effect on cell growth, and obviously improve the activity of cells; further, compared with the hollow PLLA microspheres, the hollow silk fibroin has stronger cell activity, which indicates that silk fibroin has better biocompatibility than PLLA; compared with the PLLA microspheres loaded with exosomes, the silk fibroin microspheres loaded with exosomes can obviously promote cell proliferation, and the silk fibroin microspheres combined with exosomes are obviously stronger than hollow silk fibroin microspheres, so that the synergistic effect of the exosomes and the silk fibroin microspheres in the silk fibroin microspheres loaded with exosomes is generated in promoting cell growth, and the effect is better than that of singly using the silk fibroin and replacing the exosomes and the PLLA microspheres. The results show that the combined application of the silk fibroin microsphere and the exosome has higher biocompatibility, can obviously promote cell proliferation, and has excellent synergistic effect. Meanwhile, after the silk fibroin microsphere prepared by adopting the specific method is loaded on an exosome, the effect of the silk fibroin microsphere is obviously better than that of the silk fibroin sponge loaded on the exosome and the silk fibroin microsphere loaded on the exosome with small particle size.
Test example 5
Inflammatory factor Release test
Cell plating and dosing: 1X 10 4 pieces/ml of RAW 249.7 cell suspension was prepared from the cell culture solution, and the suspension was dispensed into 96-well plates, and a blank group, an SFM group (blank silk fibroin microspheres), an EXO-loaded SFM group (exosome-loaded silk fibroin microspheres), a PLLA microsphere group, an EXO-loaded silk fibroin sponge group, and small-particle-size EXO-loaded SFM suspension were prepared in accordance with test example 4 except for the positive control group. Culturing in an incubator containing 5% (V/V) carbon dioxide air at 37deg.C for 24 hr. After 24h, the original culture medium was discarded, fresh cell culture medium was added to the blank control group, blank SFM, EXO-loaded SFM, PLLA microsphere, EXO-loaded suspension of silk fibroin sponge and small particle size EXO-loaded SFM suspension were added to the other wells, each 3 wells were connected with 100. Mu.L of each well, and the culture medium was kept in the above culture environment for 72h, and the supernatant was evaluated for the release degree of inflammatory factors in each group by ELISA kit. The results are shown in FIG. 5, where there was a significant difference between the EXO-loaded SFM group and the other groups, and where IL-6 secretion by the stimulated RAW246.7 cells was significantly lower than that of the other groups except the blank group, indicating less inflammatory response and lower short-term immunogenicity; compared with the hollow PLLA microspheres, the hollow silk fibroin microspheres generate fewer inflammatory factors, which indicates that silk fibroin has better biocompatibility than PLLA; compared with the PLLA microspheres loaded with exosomes, the silk fibroin microspheres loaded with exosomes can generate fewer inflammatory factors, and the inflammatory factors combined with the silk fibroin microspheres loaded with exosomes are obviously lower than those of the hollow silk fibroin microspheres, so that the synergistic effect of the exosomes and the silk fibroin microspheres in the silk fibroin microspheres loaded with exosomes is proved to be better than that of the silk fibroin alone and replaced by the exosomes and the PLLA microspheres; the small-particle-size EXO-loaded SFM group has higher inflammatory factor release amount compared with other groups, and the microspheres with smaller particle sizes prove that the microspheres can stimulate macrophages to release inflammatory factors and promote inflammation. The experiment successfully proves that the 10-60 mu m exosome-loaded silk fibroin microsphere prepared by the invention has smaller inflammatory reaction after injection, and has lower risk of generating inflammation compared with other PLLA microspheres or PCL microspheres or exosome-loaded silk fibroin sponge and the like.
Test example 6
Needle penetration experiment of exosome-loaded silk fibroin-carboxymethyl cellulose composite gel
The compound gel prepared in examples 1-10 was thoroughly stirred and mixed, the viscosity of the system was measured, a 1mL syringe was used to suck the sample, a needle was used to cover and fix the sample, the needle was pushed out of the sample, the needle penetration was checked, the success rate of needle penetration was calculated, and the needle penetration = number of complete passes/number of attempts, and the results are shown in table 3.
TABLE 3 viscosity and needle penetration rate determination of exosome-loaded silk fibroin microsphere-carboxymethylcellulose composite gel
Numbering device | Viscosity/cP | 26G needle passing rate | 27G needle passing rate | 30G needle passing rate |
Example 1 | 144k | 50/50 | 41/50 | 34/50 |
Example 2 | 132k | 50/50 | 45/50 | 36/50 |
Example 3 | 145k | 50/50 | 47/50 | 33/50 |
Example 4 | 168k | 50/50 | 49/50 | 40/50 |
Example 5 | 137k | 50/50 | 46/50 | 36/50 |
Example 6 | 144k | 50/50 | 48/50 | 41/50 |
Example 7 | 159k | 50/50 | 45/50 | 34/50 |
Example 8 | 146k | 50/50 | 47/50 | 39/50 |
Example 9 | 142k | 50/50 | 40/50 | 32/50 |
Example 10 | 149k | 50/50 | 44/50 | 36/50 |
The invention controls the needle penetration rate of the composite gel by adjusting the viscosity and the particle size, the viscosity values are all in the range of 130-160cP, the needle penetration rates of the embodiments 1-10 are all good, and the embodiment 6 and the embodiment 8 are optimal.
Test example 6
Comparison of degradation rates for different composite gels
(1) 6 Parts of the silk fibroin microsphere-carboxymethyl cellulose composite gel loaded with exosomes in example 8 were weighed, and then mixed into an equal amount of distilled water, respectively; placing into a shaking table at 37 ℃ and shaking at 60r/min. The degradation period was measured for 48 weeks with 8 weeks as a degradation period. At the end of each degradation period, 1 sample is taken out, microspheres are collected by centrifugation and thoroughly washed by distilled water, and the microspheres are freeze-dried in vacuum to constant weight, so that the degradation rate of the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel is obtained.
(2) Dissolving poly-L-lactic acid (PLLA) in chloroform to prepare a solution with the concentration of 1g/L, mixing and incubating the mesenchymal stem cell exosome solution in the step (1) in the example 8 with the chloroform solution of PLLA according to the volume ratio of 1:1, and dissolving sodium carboxymethyl cellulose and mannitol (the mass ratio is 1:20) in distilled water to prepare a solution with the concentration of 0.5%. The exosomes and PLLA in chloroform solution were slowly added dropwise to the sodium carboxymethylcellulose/mannitol aqueous solution with stirring, the volume ratio of the two solutions being 1:100. Stirring is continued for 12h, vacuum filtration is carried out for 5h, and the poly-L-lactic acid microsphere loaded with exosomes is obtained and dispersed in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml. 0.7g of carboxymethyl cellulose is gradually added into 6mL of PBS (pH 7.4) buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling. 2.75mL of PLLA microsphere suspension having a concentration of 0.04g/mL was added to the resulting CMC gel matrix, wherein the final CMC concentration was 0.08g/mL. The mixture solution was continuously stirred at 600r/min for 30min at room temperature to obtain exosome-loaded PLLA microsphere-carboxymethyl cellulose composite gel. 6 parts of PLLA microsphere-carboxymethyl cellulose composite gel loaded with exosomes in an amount of 3g are weighed and respectively mixed into distilled water with equal amount; placing into a shaking table at 37 ℃ and shaking at 60r/min. The degradation period was measured for 48 weeks with 8 weeks as a degradation period. At the end of each degradation period, 1 sample was taken, microspheres were collected by centrifugation and thoroughly rinsed with distilled water, and lyophilized in vacuo to constant weight, thereby obtaining the degradation rate of the exosome-loaded PLLA microsphere-carboxymethyl cellulose composite gel.
TABLE 4 degradation rates of different composite gels
As can be seen from table 4 above, the degradation rate of the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel increased with time; compared with PLLA microsphere-carboxymethyl cellulose composite gel loaded with exosomes, the composite gel of the invention has slower degradation speed and longer duration of action.
In addition, the invention also constructs an exosome-loaded polycaprolactone microsphere-carboxymethyl cellulose composite gel, 10 g of Polycaprolactone (PCL) with MW 8k Da is dissolved in dichloromethane (DCM, 20 w/w%), the mesenchymal stem cell exosome solution of the step (1) in the example 8 and the dichloromethane solution of the Polycaprolactone (PCL) are mixed and incubated according to the volume ratio of 1:1, the solution is dispersed in distilled water, the extraction and the stirring are carried out, the obtained microsphere is filtered, washed and dried, and the obtained microsphere is dispersed in PBS (pH 7.4) buffer solution for standby at the concentration of 0.04 g/ml. 0.7g of carboxymethyl cellulose is gradually added into 6mL of PBS (pH 7.4) buffer solution at 60 ℃ and stirred, and the CMC gel matrix is prepared after uniform dissolution and cooling. 2.75mL of polycaprolactone microsphere suspension with the concentration of 0.04g/mL is taken and added into the prepared CMC gel matrix, wherein the final concentration of CMC is 0.08g/mL. Continuously stirring the mixture solution for 30min at room temperature of 600r/min to obtain the exosome-loaded polycaprolactone microsphere-carboxymethyl cellulose composite gel. The exosome-loaded polycaprolactone microsphere-carboxymethyl cellulose composite gel prepared by the method is similar to the exosome-loaded PLLA microsphere-carboxymethyl cellulose composite gel, so that the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel prepared by the method has longer action effect than the exosome-loaded PLLA microsphere or polycaprolactone microsphere composite gel.
Test example 7
Subcutaneous filling of composite microsphere gel and experiment for promoting collagen regeneration
(1) Healthy male BALB/c mice were taken, the experimental group photo-aged model was molded, the back was dehaired and disinfected, UVA (365 nm,20 w) was irradiated for 2h/d, 10% galactose was subcutaneously injected in the back of the neck (1000 mg/kg of the mice weight) for 10d. After successful molding, 0.05m L of physiological saline, EXO-loaded SFM-CMC composite gel (EXO-loaded silk fibroin-carboxymethyl cellulose composite gel), hollow SFM-CMC composite gel, hollow PLLA microsphere-CMC composite gel, and EXO-loaded PLLA microsphere-CMC composite gel (3 each) were subcutaneously injected at the same back site using a 1m L syringe, and the above were prepared into gels according to the method of example 8, according to the microspheres in test example 4. After injection, the growth status of mice in the experimental group is observed regularly, and whether symptoms such as erythema, edema and the like appear on the back skin or not. As shown in FIG. 6, the back photograph of the mice in the third month after injecting the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel and the hollow silk fibroin microsphere-carboxymethyl cellulose composite gel shows that the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel prepared by the invention has stronger subcutaneous filling effect, and the wrinkle filling effect is more obvious than that of injecting the hollow silk fibroin microsphere-carboxymethyl cellulose composite gel, thus indicating that the exosome combined silk fibroin has better effect than that of singly using the silk fibroin.
(2) The experimental group and the control group were sacrificed in the third month after cervical removal, the back skin was uncovered, and the skin and subcutaneous tissue were taken at the corresponding locations and immediately fixed with tissue fixative. After paraffin embedding, sections were stained with hematoxylin-eosin and masson trichromatic stain. As shown in the HE dyeing masson trichromatic dyeing result in FIG. 7, the filling part of the microsphere is clearly visible under the skin, new blue collagen fibers exist around the microsphere, and the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel has more blue collagen fibers and better effect than the hollow silk fibroin microsphere-carboxymethyl cellulose composite gel. Fig. 8 shows the increase in collagen at the injection site for each group, and the difference between the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel group and the other groups was P <0.0001, with very significant differences. Compared with PLLA microsphere-carboxymethyl cellulose composite gel and hollow silk fibroin microsphere-carboxymethyl cellulose composite gel loaded with exosomes, the injection of the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel can obviously promote collagen regeneration, and the exosome-loaded silk fibroin-carboxymethyl cellulose composite gel prepared by the invention can effectively stimulate collagen regeneration under the skin, and the combined use of exosomes and silk fibroin in the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel can play a better effect than that of single silk fibroin and substitution of exosomes and PLLA microspheres, and can also generate obvious synergistic effect in promoting collagen regeneration. Meanwhile, the specific surface area of the microspheres in the small-particle-size exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel is small, so that the production amount of collagen is small, and the exosome-loaded silk fibroin sponge-carboxymethyl cellulose composite gel is not suitable for injection, does not have a smooth surface, and is unfavorable for cell adhesion and regeneration. The composite gel prepared by the invention can better promote collagen regeneration and cell proliferation, generate smaller inflammatory reaction and have better filling effect, and can be effectively applied to medical filling materials.
Claims (10)
1. An injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel, characterized in that the composite gel comprises the following raw materials: the mesenchymal stem cell exosomes are loaded on the silk fibroin to form exosome-loaded silk fibroin microspheres, and the mass ratio of the carboxymethyl cellulose to the exosome-loaded silk fibroin microspheres is 5-8:1-4.
2. The injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel according to claim 1, wherein the silk fibroin is silk fibroin degummed from silkworm cocoon shells, and has a molecular weight of 8000-14000Da.
3. The injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel of claim 1, wherein the viscosity of the carboxymethyl cellulose is 800-1200 mPa-s.
4. The injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel of claim 1, wherein the exosome-loaded silk fibroin microsphere preferably has a span value of 1.0-1.4, and the exosome-loaded silk fibroin microsphere has a particle size of 10-60 μm.
5. A method of preparing an injectable exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel according to claim 1, comprising the steps of:
(1) Extraction and separation of mesenchymal stem cell exosomes: culturing mesenchymal stem cells, extracting mesenchymal stem cell exosomes by an ultracentrifugation method, dispersing the mesenchymal stem cell exosomes in PBS buffer solution to form exosome solution, and storing the exosomes for later use;
(2) Preparation of exosome-loaded silk fibroin microspheres: mixing and incubating the mesenchymal stem cell exosome solution and the silk fibroin solution in the step (1), taking liquid paraffin and Span 80, stirring and mixing, slowly dripping the mixture into the incubated mixed solution, stirring to form uniform and stable water-in-oil emulsion, slowly dripping a cross-linking agent solution, stirring and centrifuging at a low speed, pouring out supernatant, washing and drying until the organic solvent is completely evaporated to obtain the silk fibroin microsphere loaded with exosome, and dispersing in PBS buffer to form suspension for later use;
(3) Preparation of carboxymethyl cellulose hydrogel: adding carboxymethyl cellulose into water, stirring, dissolving uniformly, and cooling for later use to obtain carboxymethyl cellulose hydrogel;
(4) Preparation of exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel:
Adding carboxymethyl cellulose hydrogel into an exosome-loaded silk fibroin microsphere suspension, and continuously stirring the mixture at room temperature to obtain exosome-loaded silk fibroin-carboxymethyl cellulose composite gel;
(5) Preparation of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection:
Dialyzing the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel material, homogenizing, extruding, sieving, and sterilizing with high-temperature and high-pressure steam to obtain the exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel for injection.
6. The method according to claim 5, wherein the number of exosomes per ml in the exosome solution in step (1) is 10 6~107; the concentration of the silk fibroin solution in the step (2) is 1-60 mg/mL; the two solutions were mixed in equal volumes.
7. The method according to claim 5, wherein the crosslinking agent in the step (2) is one or more of glutaraldehyde, genipin, divinyl sulfone, ethylene glycol diglycidyl ether, butanediol diglycidyl ether, and polypropylene glycol diglycidyl ether.
8. The preparation method according to claim 5, wherein the mass-volume ratio of carboxymethyl cellulose in the carboxymethyl cellulose hydrogel in the step (3) is 0.025-0.09 g/mL.
9. An exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel prepared by the method of any one of claims 1-8.
10. Use of an exosome-loaded silk fibroin microsphere-carboxymethyl cellulose composite gel of claim 9 in promoting cell proliferation and injectable medical cosmetic filling material.
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