CN115920126A - Plant exosome-loaded polyhydroxyalkanoate microspheres and preparation method thereof - Google Patents
Plant exosome-loaded polyhydroxyalkanoate microspheres and preparation method thereof Download PDFInfo
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a plant exosome-loaded polyhydroxyalkanoate microsphere and a preparation method thereof. The preparation method of the plant exosome-loaded polyhydroxyalkanoate microspheres comprises the following steps: purifying the polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder; dissolving purified polyhydroxyalkanoate powder in dichloromethane to prepare a first oil phase, preparing a pore-foaming agent aqueous solution as a first water phase, mixing the first water phase and the first oil phase, and emulsifying in a homogenizer to prepare an emulsion; taking the emulsion as a second oil phase, dissolving polyvinyl alcohol in water to prepare a second water phase for secondary emulsification, dropwise adding the second oil phase solution into the second water phase for secondary emulsification, and stirring to obtain the polyhydroxy fatty acid ester microspheres with rough porous surfaces; and re-dispersing the prepared polyhydroxyalkanoate microspheres in sterile water, mixing the dispersion liquid and the plant exosome solution in equal volume, and centrifuging to obtain the polyhydroxyalkanoate microspheres loaded with plant exosomes.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a polyhydroxyalkanoate microsphere for loading plant exosomes and a preparation method thereof.
Background
Biodegradable medical materials are widely applied to the field of various medical instruments such as surgical sutures, artificial organs, drug sustained release agents and the like due to good biocompatibility and degradability, wherein biodegradable organic polymers are widely applied due to wide sources, renewability, good processability and good biocompatibility of raw materials.
The injectable soft tissue filler is a filler adopted by minimally invasive therapy aiming at skin wrinkle and depression filling, and the soft tissue fillers appearing in the market mainly comprise crosslinked sodium hyaluronate gel, collagen, polylactic acid, polycaprolactone, polylactic acid-polyethylene glycol copolymer, calcium hydroxyapatite and the like. The particle size of the microsphere-based injectable soft tissue filler product should be 20 microns or more to avoid phagocytosis of macrophages in vivo so as to exert long-acting effect, and meanwhile, the situation that the needle tube is blocked easily during the injection process of the microsphere of 50 microns or more is not favorable for practical operation.
Polyhydroxyalkanoate (PHA) is a natural high-molecular biomaterial, is an intracellular polyester synthesized by microorganisms, and has good biocompatibility. It can be made into microsphere, and can be used as soft tissue filler by injection through needle.
Exosomes are microvesicles which are secreted by cells and contain complex RNA and proteins and are used for information exchange, and are widely applied to pharmacologically active substances due to the advantages of stable property, easy reaction, no immunogenicity, easy acquisition, change, modification and the like.
A medicine-carrying slow-release microsphere based on exosome and a preparation method thereof (CN 114432260A) disclose a medicine-carrying slow-release microsphere based on exosome and a preparation method thereof, and the microsphere loaded with exosome is prepared by dissolving exosome and polymer in an organic solvent and then carrying out electrospray. However, common organic solvents such as N, N-dimethylformamide, dichloromethane and trichloromethane destroy phospholipid structures on the surface of exosomes, so that the final exosome structure is destroyed and the content is released in advance.
An exosome microsphere suspension and a preparation method thereof (CN 114921406A) disclose a preparation method of an exosome microsphere suspension, which is characterized in that exosomes and polysaccharide suspensions are mixed and then subjected to electrostatic spraying to obtain granular microspheres with different sizes, however, polysaccharides such as chitosan and the like cannot be well dispersed in water, so that the prepared exosomes are not uniformly distributed, and meanwhile, the electrostatic spraying can also damage the vesicle structure of the exosomes to a certain extent to cause the early release of inclusions.
A method for purifying plant exosome (CN 114540271A) realizes large-scale and high-purity exosome separation and purification by combining PEG8000 precipitation and gel filtration chromatography. The prepared exosome has a nano-scale size and is not suitable for direct skin injection.
In summary, microspheres prepared using conventional methods do not have good fillability, which is not conducive to soft tissue filling applications. The existing polymer microsphere entrapped exosome cannot damage the structure of the exosome, and no matter the polymer water suspension is mixed with the exosome or the polymer organic solution is mixed with the exosome, good dispersion and the completeness of the structure of the exosome cannot be simultaneously realized.
Disclosure of Invention
Based on the above, the invention aims to provide a polyhydroxyalkanoate microsphere loaded with plant exosomes and a preparation method thereof.
In a first aspect, a preparation method of plant exosome-loaded polyhydroxyalkanoate microspheres comprises the following steps:
purifying the polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder;
dissolving purified polyhydroxyalkanoate powder in dichloromethane to prepare a first oil phase, preparing a pore-foaming agent aqueous solution as a first water phase, mixing the first water phase and the first oil phase, and emulsifying in a homogenizer to prepare an emulsion;
taking the emulsion as a second oil phase, dissolving polyvinyl alcohol in water to prepare a second water phase for secondary emulsification, dropwise adding the second oil phase solution into the second water phase under magnetic stirring for secondary emulsification, and stirring to obtain the polyhydroxy fatty acid ester microspheres with rough porous surfaces;
and (3) performing irradiation sterilization on the prepared polyhydroxyalkanoate microspheres, then re-dispersing the polyhydroxyalkanoate microspheres in sterile water, mixing the dispersion liquid and the plant exosome solution in equal volume, incubating the mixture in a shaking table at room temperature, and centrifuging the mixture to obtain the polyhydroxyalkanoate microspheres loaded with plant exosomes.
In one embodiment of the above technical means, the polyhydroxyfatty acid ester is PHB, or PHBV, or a family polymer or copolymer of PHBV.
In one embodiment, the purification of polyhydroxyalkanoate comprises: taking polyhydroxyalkanoate, washing water-soluble impurities and alcohol-soluble impurities in the polymer by using deionized water and ethanol respectively, dissolving by using dichloromethane to obtain a clear solution, and then drying.
In an embodiment of the above technical solution, the pore-forming agent aqueous solution includes one or more of ammonium bicarbonate, sodium bicarbonate, ammonia carbonate, zinc carbonate, hydrogen peroxide, albumin, and cyclodextrin.
In one embodiment of the above technical solution, the plant exosomes are fragrant round exosomes or giant knotweed exosomes.
In a second aspect, the plant exosome-loaded polyhydroxyalkanoate microspheres are prepared by any one of the preparation methods of the plant exosome-loaded polyhydroxyalkanoate microspheres.
In a third aspect, the application of the plant exosome-loaded polyhydroxyalkanoate microspheres comprises the following steps: adding the polyhydroxyalkanoate microspheres loaded with the plant exosomes into an injection solvent, stirring and uniformly mixing, and filling into a pre-filled syringe to obtain the injectable polyhydroxyalkanoate microsphere preparation loaded with the plant exosomes.
In one embodiment, the injection solvent includes a hyaluronic acid solution, a sodium carboxymethylcellulose solution, a sodium alginate solution, or a collagen solution.
In a fourth aspect, a use of polyhydroxyalkanoate microspheres loaded with plant exosomes, comprises: adding the polyhydroxyalkanoate microspheres loaded with the plant exosomes into a solution containing a freeze-drying protective agent and an injection solvent, stirring and uniformly mixing, filling into a penicillin bottle, and freeze-drying to obtain the polyhydroxyalkanoate microsphere freeze-dried powder capable of being injected with the plant exosomes.
In one embodiment, the lyoprotectant includes one or more of mannose, sucrose, lactose, dextran, and glucose;
the injection solvent comprises a hyaluronic acid solution, a sodium carboxymethylcellulose solution, a sodium alginate solution or a collagen solution.
Compared with the prior art, the invention can control and synthesize the porous polyhydroxyalkanoate microspheres with the size of about 50 microns, the size of the porous polyhydroxyalkanoate microspheres is not too small to be phagocytized by macrophages in advance, or too large to block a needle head in injection, and the porous polyhydroxyalkanoate microspheres are suitable for injectable soft tissue fillers. Meanwhile, the porous and rough structure of the polyhydroxyalkanoate microspheres endows the exosome with a larger specific surface area, the loading of the exosome is facilitated, the loading process can effectively prevent the structure of the exosome from being damaged and the content of the exosome from leaking in advance, good dispersibility is realized, the integrity of the exosome structure is kept, and meanwhile, the plant exosome with the repairing function is beneficial to tissue repair in the soft tissue filling process.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a scanning electron micrograph of the PHBV microspheres (ruler: 6 μm).
FIG. 2 is a scanning electron micrograph (ruler: 6 μm) of the prepared PHBV microspheres loaded with aromatase exosomes.
FIG. 3 is a zeta potential histogram of the fragrant garden exosomes, PHBV blank microspheres and PHBV microspheres loaded fragrant garden exosomes.
FIG. 4 is a photomicrograph (50 μm ruler) of the prepared PHBV smooth microspheres.
FIG. 5 is a bar graph showing the change of cell proliferation rate over time for control group, PHBV blank microspheres and PHBV microspheres loaded with fragrance garden exosomes.
FIG. 6 is a drawing of an HSF cell scratch repair experiment of PHBV blank microspheres and PHBV microspheres loaded with Garden exosomes.
Detailed Description
The directional terms upper, lower, left, right, front, rear, front, back, top, bottom, etc. referred to or which may be referred to in this specification are defined relative to their construction and are relative concepts. Therefore, it may be changed according to different positions and different use states. Therefore, these and other directional terms should not be construed as limiting terms.
The implementations described in the exemplary embodiments below are not intended to represent all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.
The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
According to the invention, the porous polyhydroxyalkanoate microspheres are prepared by using the easily obtained polyhydroxyalkanoate through a secondary emulsification method, the rough surface and the porous structure can endow the polymer microspheres with a larger specific surface area, so that the further exosome adsorption is facilitated, and the loading of exosomes can be realized through the co-incubation of the microspheres and plant exosomes.
In a first aspect, a method for preparing a plant exosome-loaded polyhydroxyalkanoate microsphere comprises the following steps:
s1, purifying the polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder.
Optionally, the polyhydroxyalkanoate is PHB, or PHBV, or a family polymer or copolymer of PHBV.
Specifically, the purification of the polyhydroxyalkanoate comprises the following steps: taking polyhydroxyalkanoate, washing water-soluble impurities and alcohol-soluble impurities in the polymer by using deionized water and ethanol respectively, dissolving by using dichloromethane to obtain a clear solution, performing Soxhlet extraction or filtration, and then drying.
S2, dissolving the purified polyhydroxyalkanoate powder in dichloromethane to prepare a first oil phase, preparing a pore-foaming agent aqueous solution to serve as a first water phase, mixing the first water phase with the first oil phase, and emulsifying in a homogenizer to prepare the emulsion.
Optionally, the porogen aqueous solution includes one of ammonium bicarbonate, sodium bicarbonate, ammonia carbonate, zinc carbonate, hydrogen peroxide, albumin, and cyclodextrin.
And S3, taking the emulsion as a second oil phase, dissolving polyvinyl alcohol in water to prepare a second water phase for secondary emulsification, dropwise adding the second oil phase solution into the second water phase for secondary emulsification under magnetic stirring at a slow speed, and stirring until the organic solvent is completely volatilized to obtain the polyhydroxy fatty acid ester microspheres with rough porous surfaces.
And S4, performing irradiation sterilization on the prepared polyhydroxyalkanoate microspheres, then re-dispersing the polyhydroxyalkanoate microspheres in sterile water, mixing the dispersion liquid and the plant exosome solution in equal volume, incubating the mixture in a shaking table at room temperature, and centrifuging the mixture to obtain the polyhydroxyalkanoate microspheres loaded with plant exosomes.
Optionally, the plant exosomes are myrtle exosomes or polygonum cuspidatum exosomes. The entrapped plant exosomes can be slowly released, and are also beneficial to tissue repair in the soft tissue filling process.
Example 1
The embodiment is PHBV porous microspheres based on fragrant round exosomes, and the preparation method comprises the following steps:
s01, 5g of PHBV powder (molecular weight 40 ten thousand) is taken to respectively wash away water-soluble impurities and alcohol-soluble impurities in the polymer by using deionized water and ethanol, dichloromethane is used for dissolving to obtain clear solution, and Soxhlet extraction and drying are carried out to obtain purified PHBV powder.
S02, weighing 200mg of purified PHBV powder, adding 8mL of dichloromethane to dissolve the PHBV powder, adding 2.4mL of 5mg/mL ammonium bicarbonate aqueous solution, and homogenizing for 3min at 10000rpm by using a homogenizer to obtain colostrum.
S03, dripping 200mL of Polyvinyl alcohol (Mw = 27000) water solution of the colostrum into the colostrum, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, centrifuging at 2000rpm, and collecting the PHBV microspheres.
And S04, washing the PHBV microspheres for 3 times by using deionized water, and dispersing the PHBV microspheres in 20mL of sterile deionized water again to obtain the porous PHBV microsphere water dispersion. And (3) taking 1mL of the porous PHBV microsphere aqueous dispersion, adding 1mL of a fragrant round exosome (with the protein content of 0.6 mg/mL), mechanically stirring for 24 hours in a shaking table, centrifuging at 2000rpm, and re-dispersing in sterile deionized water to obtain the PHBV microspheres loaded with the fragrant round exosome.
Example 2
Observing the micro morphology of the PHBV microspheres and testing the surface charge.
A certain amount of the PHBV microspheres prepared in example 1 and the PHBV microspheres loaded with the fragrant round exosomes were placed on a sample plate adhered with a conductive double-sided adhesive, and observed under a scanning electron microscope after gold plating to obtain the microsphere morphology shown in figures 1 and 2.
As can be seen from the observation of the pictures, the PHBV microspheres prepared in example 1 have regular morphology, about 50 microns in size, rough surface and randomly distributed large number of pores. The prepared PHBV microsphere loaded with the fragrant round exosomes keeps the micro-morphology and the size of the original porous PHBV microsphere.
A certain amount of the PHBV microspheres prepared in example 1, the PHBV microspheres loaded with the fragrant circle exosomes and the aqueous dispersion of the fragrant circle exosomes were tested for surface charges on a laser particle analyzer to obtain the surface charges shown in figure 3, so that the surface charges of the fragrant circle exosomes and the porous PHBV microspheres are negative, the surface charges of the porous PHBV microspheres loaded with the fragrant circle exosomes are slightly higher than the surface charges of the PHBV microspheres not loaded with the fragrant circle exosomes, and the successful loading of the fragrant circle exosomes on the surfaces of the porous PHBV microspheres can be proved.
The analysis can further show that the invention can control and synthesize the porous polyhydroxyalkanoate microspheres with the size of about 50 mu m, the size of the microspheres is not too small to be phagocytized by macrophages in advance, or the microspheres are too large to block a needle head in injection, and the microspheres are suitable for injectable soft tissue fillers. Meanwhile, the porous and rough structure of the polyhydroxyalkanoate microspheres endows the exosome with a larger specific surface area, the loading of the exosome is facilitated, the loading process can effectively prevent the structure of the exosome from being damaged and the content of the exosome from leaking in advance, good dispersibility is realized, the integrity of the structure of the exosome is maintained, and meanwhile, the plant exosome with a repairing function is beneficial to tissue repair in the soft tissue filling process.
Example 3
And (3) verifying the biological efficacy of the PHBV porous microspheres loaded with the fragrant round exosomes.
Selecting human skin fibroblast L929 with density of 1 × 10 according to 100 μ L 5 Spreading the cell suspension per ml on a 96-well plate, sucking out the culture solution after the cells adhere to the wall for 12 hours, adding the PHBV microspheres prepared in the embodiment 1 dispersed by the cell culture solution, the PHBV microspheres loaded with the Xiangyuan exosomes and a control group (the concentration of the microspheres is 50 micrograms per ml), incubating for different time and measuring the cell proliferation rate by adopting an MTT method. As shown in figure 5, PHBV microspheres loaded with fragrant circle exosomes and PHBV microspheres loaded with fragrant circle exosomesCompared with the control group and the blank PHBV microsphere group, the cell proliferation rate is higher along with the change of time, which shows that the PHBV microspheres loaded with the fragrant orb exosomes not only have good safety, but also have the effect of promoting cell proliferation.
Human skin fibroblasts were treated with 2 x 10 5 Inoculating the cells in a 6-well plate at a cell density of one/ml, and culturing in an incubator for 24 hours; drawing a horizontal line straightly by using a gun head, washing cells for 3 times by using PBS, removing the drawn cells, and adding a serum-free culture medium; establishing blank control group and test group, respectively, (concentration), at 37 deg.C, 5% 2 And an incubator for culturing and observing and photographing at different time points. The result is shown in fig. 6, compared with the blank PHBV microsphere group, the PHBV microsphere loaded with the xiangyuan exosome has more remarkable scratch repair effect, and further shows that the PHBV microsphere loaded with the xiangyuan exosome has good cell repair promoting performance. Example 4
The embodiment is a PHBV porous microsphere based on polygonum cuspidatum exosomes, and the preparation method is the same as that in embodiment 1, and the main difference is that the selected exosomes are polygonum cuspidatum exosomes, which are specifically as follows:
s001, taking 5g of PHBV powder (molecular weight is 40 ten thousand), respectively washing out water-soluble impurities and alcohol-soluble impurities in the polymer by using deionized water and ethanol, dissolving by using dichloromethane to obtain a clear solution, and then performing Soxhlet extraction and drying to obtain purified PHBV powder.
S002, weighing 200mg of purified PHBV powder, adding 8mL of dichloromethane, adding 2.4mL of 5mg/mL ammonium bicarbonate aqueous solution after the PHBV powder is dissolved, and homogenizing for 3 minutes at 10000rpm by using a homogenizer to obtain colostrum.
And S003, dripping 200mL of Polyvinyl alcohol (Mw = 27000) water solution of 5mg/mL of the colostrum into the colostrum, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, and centrifuging at 2000rpm to collect the PHBV microspheres.
And S004, washing the PHBV microspheres for 3 times by using deionized water, and dispersing the PHBV microspheres in 20mL of sterile deionized water again to obtain a porous PHBV microsphere water dispersion. And (3) adding 1mL of polygonum cuspidatum exosome (with the protein content of 0.4 mg/mL) into 1mL of the porous PHBV microsphere aqueous dispersion, mechanically stirring for 24 hours in a shaking table, centrifuging at 2000rpm, and re-dispersing in sterile deionized water to obtain the PHBV microspheres loaded with polygonum cuspidatum exosomes.
Example 5
The embodiment is a PHBHVHHX porous microsphere based on a fragrant round exosome, and the preparation method is the same as that in embodiment 1, the main difference is that the selected microsphere polymer is PHBHHX, and the specific steps are as follows:
s0001, taking 5g of PHBHHX powder (molecular weight is 50 ten thousand), respectively washing water-soluble impurities and alcohol-soluble impurities in the polymer by using deionized water and ethanol, dissolving by using dichloromethane to obtain a clear solution, and performing Soxhlet extraction and drying to obtain the purified PHBHHX powder.
S0002, weighing 200mg of purified PHBHHX powder, adding 8mL of dichloromethane to dissolve the PHBHHX powder, adding 2.4mL of 5mg/mL ammonium bicarbonate aqueous solution, and homogenizing for 3 minutes at 10000rpm by using a homogenizer to obtain colostrum.
S0003, dripping the colostrum into 200mL of Polyvinyl alcohol (Mw = 27000) water solution of 5mg/mL, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, centrifuging at 2000rpm, and collecting microspheres to obtain PHBHHX microspheres
And S0004, cleaning for 3 times by using deionized water, and dispersing in 20mL of sterile deionized water again to obtain the porous PHBHHX microsphere aqueous dispersion. And (3) taking 1mL of the porous PHBHHX microsphere aqueous dispersion, adding 1mL of the coumarins exosome (with the protein content of 0.6 mg/mL), mechanically stirring for 24 hours in a shaking table, centrifuging at 2000rpm, and re-dispersing in sterile deionized water to obtain the PHBHVHHX microsphere loaded with the coumarins exosome.
Example 6
The embodiment is PHBV porous microspheres based on fragrant round exosomes, and the preparation method is the same as that in embodiment 1, and the main difference is that the selected pore-forming agent is hydrogen peroxide, and the specific steps are as follows:
s00001, washing 5g of PHBV powder (molecular weight 40 ten thousand) with deionized water and ethanol respectively to remove water-soluble impurities and alcohol-soluble impurities in the polymer, dissolving with dichloromethane to obtain a clear solution, and performing Soxhlet extraction and drying to obtain purified PHBV powder.
S00002, weighing 200mg of purified PHBV powder, adding 8mL of dichloromethane to dissolve the PHBV powder, adding 2.4mL of 1% hydrogen peroxide solution, and homogenizing for 3 minutes at 10000rpm by using a homogenizer to obtain colostrum.
S00003, dripping 200mL of Polyvinyl alcohol (Mw = 27000) water solution of which the concentration is 200mL and is 5mg/mL into the colostrum, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, and centrifuging at 2000rpm to obtain the PHBV microspheres.
And S00004, washing for 3 times by using deionized water, and dispersing in 20mL of sterile deionized water again to obtain the porous PHBV microsphere aqueous dispersion. And (3) adding 1mL of the fragrant orb exosome (protein content is 0.6 mg/mL) into 1mL of the porous PHBV microsphere aqueous dispersion, mechanically stirring for 24 hours in a shaker, centrifuging at 2000rpm, and re-dispersing in sterile deionized water to obtain the PHBV microsphere loaded with the fragrant orb exosome.
Comparative example 1
The comparative example is a smooth microsphere of PHBV, and the preparation method is as follows:
step 1, 5g of PHBV powder (molecular weight 40 ten thousand) is taken to respectively wash out water-soluble impurities and alcohol-soluble impurities in the polymer by using deionized water and ethanol, and then purified PHBV powder which can be dissolved in dichloromethane is obtained by Soxhlet extraction and drying.
Step 2, weighing 200mg of purified PHBV powder, adding 8mL of dichloromethane to dissolve the PHBV powder, dripping 200mL of Polyvinyl alcohol (Mw = 27000) aqueous solution of 5mg/mL into the PHBV powder, stirring the mixture at 400rpm for 4h, volatilizing the dichloromethane, centrifugally collecting microspheres at 2000rpm, washing the microspheres for 3 times by using deionized water, and dispersing the microspheres in 20mL of sterile deionized water again to obtain the PHBV smooth microspheres.
Comparative example 2
Morphology observation of smooth microspheres of PHBV
A certain amount of the PHBV smooth microspheres prepared in the comparative example 1 were placed in a glass slide and observed under an optical microscope to obtain the microsphere morphology shown in FIG. 4.
The PHBV smooth microspheres prepared in comparative example 1 had regular morphology, a size of about 50 microns, and smooth surfaces. Compared to example 1 (fig. 1), the surface is smooth without voids, has a smaller specific surface area and is not provided with holes to be unfavorable for further entrapment of the plant exosomes.
In a second aspect, the polyhydroxyalkanoate microspheres loaded with the plant exosomes are prepared by the preparation method of the polyhydroxyalkanoate microspheres loaded with the plant exosomes.
In a third aspect, the application of the plant exosome-loaded polyhydroxyalkanoate microspheres comprises the following steps: adding the polyhydroxyalkanoate microspheres loaded with the plant exosomes into an injection solvent, stirring and uniformly mixing, filling into a pre-filled syringe, and performing aseptic operation in the whole process to obtain the polyhydroxyalkanoate microsphere preparation loaded with the plant exosomes.
Optionally, the injection solvent comprises a hyaluronic acid solution, or a sodium carboxymethylcellulose solution, or a sodium alginate solution, or a collagen solution.
Example 7
This example is an injectable preparation based on the PHBV porous microspheres loaded with the polygonum cuspidatum exosomes in example 4, and the preparation method thereof is as follows:
adding 1g of the PHBV microspheres loaded with the polygonum cuspidatum exosomes prepared in the example 4 into 20mL of 2% hyaluronic acid solution, stirring at 300rpm for 6h, and filling the mixture into a pre-filled syringe after uniform mixing to obtain the injectable PHBV porous microsphere preparation loaded with the polygonum cuspidatum exosomes.
In a fourth aspect, a use of polyhydroxyalkanoate microspheres loaded with plant exosomes, comprises: adding the polyhydroxyalkanoate microsphere loaded with the plant exosomes into a solution containing a freeze-drying protective agent and an injection solvent, stirring for 6 hours at 300rpm, filling the mixture into a penicillin bottle after uniform mixing, performing aseptic operation in the whole process, and performing freeze drying to obtain the polyhydroxyalkanoate microsphere freeze-dried powder capable of being injected with the plant exosomes.
Optionally, the lyoprotectant includes one or more of mannose, sucrose, lactose, dextran, glucose.
Optionally, the injection solvent comprises a hyaluronic acid solution, or a sodium carboxymethylcellulose solution, or a sodium alginate solution, or a collagen solution.
Example 8
The embodiment is injectable freeze-dried powder of PHBV porous microspheres based on the vector exosomes loaded in the embodiment 1, and the preparation method is as follows:
adding 1g of PHBV microspheres loaded with the coumarone exosomes prepared in the example 1 into 20mL of solution containing 5% of mannose and 2% of hyaluronic acid, stirring at 300rpm for 6h, filling the mixture into a penicillin bottle after uniform mixing, and freeze-drying to obtain the porous PHBV microsphere freeze-dried powder capable of being injected with the coumarone exosomes.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (10)
1. A preparation method of a polyhydroxyalkanoate microsphere for loading plant exosomes is characterized by comprising the following steps:
purifying the polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder;
dissolving purified polyhydroxyalkanoate powder in dichloromethane to prepare a first oil phase, preparing a pore-foaming agent aqueous solution as a first water phase, mixing the first water phase and the first oil phase, and emulsifying in a homogenizer to prepare an emulsion;
taking the emulsion as a second oil phase, dissolving polyvinyl alcohol in water to prepare a second water phase for secondary emulsification, dropwise adding the second oil phase solution into the second water phase under magnetic stirring for secondary emulsification, and stirring to obtain the porous polyhydroxyalkanoate microspheres with rough surfaces;
and (3) performing irradiation sterilization on the prepared polyhydroxyalkanoate microspheres, then re-dispersing the polyhydroxyalkanoate microspheres in sterile water, mixing the dispersion liquid and the plant exosome solution in equal volume, incubating the mixture in a shaking table at room temperature, and centrifuging the mixture to obtain the polyhydroxyalkanoate microspheres loaded with plant exosomes.
2. The method for preparing plant exosome-loaded polyhydroxyalkanoate microspheres of claim 1, wherein the polyhydroxyalkanoate is PHB, or PHBV, or a polymer or copolymer of the same family of PHBV.
3. The method for preparing plant exosome-loaded polyhydroxyalkanoate microspheres according to claim 1, wherein the purifying polyhydroxyalkanoate comprises: taking polyhydroxyalkanoate, washing off water-soluble impurities and alcohol-soluble impurities in the polymer by using deionized water and ethanol respectively, dissolving by using dichloromethane to obtain a clear solution, and then drying.
4. The method for preparing plant exosome-loaded polyhydroxyalkanoate microspheres according to claim 1, wherein the pore-forming agent aqueous solution comprises one or more of ammonium bicarbonate, sodium bicarbonate, ammonia carbonate, zinc carbonate, hydrogen peroxide, albumin, and cyclodextrin.
5. The method for preparing plant exosome-loaded polyhydroxyalkanoate microspheres according to any one of claims 1-4, wherein the plant exosomes are fragrant round exosomes or giant knotweed exosomes.
6. A plant exosome-loaded polyhydroxyalkanoate microsphere, which is prepared by the preparation method of the plant exosome-loaded polyhydroxyalkanoate microsphere according to any one of claims 1 to 5.
7. The application of the polyhydroxyalkanoate microspheres for loading plant exosomes is characterized by comprising the following components: the polyhydroxyalkanoate microspheres loaded with the plant exosomes of claim 6 are added into an injection solvent, stirred and mixed uniformly, and then filled into a pre-filled syringe to obtain the polyhydroxyalkanoate microsphere preparation loaded with the plant exosomes.
8. The method for preparing plant exosome-loaded polyhydroxyalkanoate microspheres according to claim 7, wherein the injection solvent comprises a hyaluronic acid solution, or a sodium carboxymethylcellulose solution, or a sodium alginate solution, or a collagen solution.
9. The application of the polyhydroxyalkanoate microspheres for loading plant exosomes is characterized by comprising the following components: adding the polyhydroxyalkanoate microspheres loaded with the plant exosomes of claim 6 into a solution containing a freeze-drying protective agent and an injection solvent, stirring, uniformly mixing, filling into a penicillin bottle, and freeze-drying to obtain the polyhydroxyalkanoate microsphere freeze-dried powder capable of being injected with the plant exosomes.
10. A method of preparing plant exosome-loaded polyhydroxyalkanoate microspheres as claimed in claim 9, wherein the lyoprotectant includes one or more of mannose, sucrose, lactose, dextran, glucose;
the injection solvent comprises a hyaluronic acid solution, a sodium carboxymethylcellulose solution, a sodium alginate solution or a collagen solution.
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