CN115920126B - Polyhydroxyalkanoate microsphere loaded with plant exosomes and preparation method thereof - Google Patents
Polyhydroxyalkanoate microsphere loaded with plant exosomes and preparation method thereof Download PDFInfo
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- 239000004005 microsphere Substances 0.000 title claims abstract description 146
- 210000001808 exosome Anatomy 0.000 title claims abstract description 128
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 title claims abstract description 78
- 229920000903 polyhydroxyalkanoate Polymers 0.000 title claims abstract description 77
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 61
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- 239000000843 powder Substances 0.000 claims abstract description 32
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 8
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
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- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 7
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- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 2
- FMRLDPWIRHBCCC-UHFFFAOYSA-L Zinc carbonate Chemical compound [Zn+2].[O-]C([O-])=O FMRLDPWIRHBCCC-UHFFFAOYSA-L 0.000 description 2
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- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 2
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- 206010057249 Phagocytosis Diseases 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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- 150000003904 phospholipids Chemical group 0.000 description 1
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- 229920001610 polycaprolactone Polymers 0.000 description 1
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- 229920000728 polyester Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- 230000005808 skin problem Effects 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
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Classifications
-
- 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
Landscapes
- Medicinal Preparation (AREA)
Abstract
The invention discloses a polyhydroxyalkanoate microsphere loaded with plant exosomes and a preparation method thereof. The preparation method of the polyhydroxyalkanoate microsphere loaded with plant exosomes comprises the following steps: purifying the polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder; dissolving the purified polyhydroxyalkanoate powder in dichloromethane to prepare a first oil phase, preparing Kong Jishui solution as a first water phase, mixing the first water phase with the first oil phase, and emulsifying in a homogenizer to prepare emulsion; taking the emulsion as a second oil phase, dissolving polyvinyl alcohol in water to prepare a second water phase for secondary emulsification, dripping the second oil phase solution into the second water phase for secondary emulsification, and stirring to obtain the polyhydroxy fatty acid ester microsphere with rough porous surface; and re-dispersing the prepared polyhydroxyalkanoate microsphere in sterile water, mixing the dispersion liquid with a plant exosome solution in an equal volume, and centrifuging to obtain the polyhydroxyalkanoate microsphere loaded with the plant exosome.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a polyhydroxyalkanoate microsphere loaded with plant exosomes and a preparation method thereof.
Background
The biodegradable medical material is widely applied to the fields of surgical sutures, artificial organs, drug sustained release agents and other medical instruments due to good biocompatibility and degradability, wherein the biodegradable organic polymer is widely applied due to wide sources of raw materials, reproducibility, good processability and good biocompatibility.
The injectable soft tissue filler is used for minimally invasive therapy for skin wrinkles and depressions, and the soft tissue filler appearing on the market mainly comprises 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 the body to exert a long-acting effect, and the condition that 50 microns or more of microspheres easily cause needle tube blockage during injection is not beneficial to practical operation.
Polyhydroxyalkanoate (PHA) is a natural polymer biomaterial, is intracellular polyester synthesized by microorganisms, and has good biocompatibility. It can be made into microsphere, and can be used as soft tissue filler by needle injection.
The exosomes are microvesicles secreted by cells and containing complex RNA and protein for information exchange, and are widely applied to pharmacological active substances due to the advantages of stable property, easy reaction, no immunogenicity, easy acquisition, transformation and transformation, and the like, and the exosomes of some plant sources can automatically release nutrient substances after entering soft tissues, so that the cell environment is continuously improved, and the skin problem is repaired.
The invention discloses a drug-loaded sustained-release microsphere based on exosomes and a preparation method thereof (CN 114432260A), and discloses the drug-loaded sustained-release microsphere based on exosomes and the preparation method thereof, wherein the exosomes and polymers are dissolved in an organic solvent and then electrosprayed to prepare the microsphere loaded with exosomes. However, common organic solvents such as N, N-dimethylformamide, dichloromethane, chloroform and the like can damage the phospholipid structure on the surface of the exosomes, resulting in the damage of the final exosome structure and the early release of the contents.
The exosome microsphere suspension and the preparation method thereof (CN 114921406A) disclose a preparation method of the exosome microsphere suspension, granular microspheres with different sizes are obtained by mixing exosome and polysaccharide suspension and then carrying out electrostatic spraying, however, polysaccharide such as chitosan and the like cannot be well dispersed in water, so that the exosome is not uniformly distributed after the exosome microsphere suspension is prepared, and meanwhile, the vesicle structure of the exosome is damaged to a certain extent by the electrostatic spraying to cause the advanced release of contents.
A purification method of plant exosomes (CN 114540271A) realizes the separation and purification of the exosomes in large scale and high purity by combining PEG8000 precipitation and gel filtration chromatography. The prepared exosomes are nano-sized and are not suitable for direct injection into the skin.
In summary, microspheres prepared by conventional methods do not have good fillability, which is detrimental to soft tissue filling applications. The existing polymer microsphere entrapped exosome cannot achieve the effect of not damaging the structure of the exosome, and whether the polymer aqueous suspension is used for mixing with the exosome or the polymer organic solution is used for mixing with the exosome, good dispersion and maintenance of the structural integrity of the exosome cannot be achieved at the same time.
Disclosure of Invention
The present invention provides polyhydroxyalkanoate microspheres loaded with plant exosomes and a method for producing the same.
In a first aspect, a method for preparing polyhydroxyalkanoate microsphere loaded with plant exosomes comprises:
Purifying the polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder;
dissolving the purified polyhydroxyalkanoate powder in dichloromethane to prepare a first oil phase, preparing Kong Jishui solution as a first water phase, mixing the first water phase with the first oil phase, and emulsifying in a homogenizer to prepare 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 to perform secondary emulsification, and stirring to obtain the polyhydroxy fatty acid ester microsphere with rough porous surface;
and (3) the prepared polyhydroxyalkanoate microsphere is subjected to irradiation sterilization and then is dispersed in sterile water again, the dispersion liquid is mixed with a plant exosome solution in an equal volume, the mixture is incubated at the room temperature of a shaking table, and the polyhydroxyalkanoate microsphere loaded with the plant exosome is obtained after centrifugation.
In one embodiment of the above technical solution, the polyhydroxyalkanoate is PHB, or PHBV, or a polymer or copolymer of the same family as PHBV.
The technical proposal is that in one embodiment of the present invention, a method for manufacturing a semiconductor device, the purification of polyhydroxyalkanoate comprises: taking polyhydroxyalkanoate, washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, dissolving by methylene dichloride to obtain a clear solution, and drying.
In one embodiment, 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, the plant exosome is a xiangyuan exosome or a polygonum cuspidatum exosome.
In a second aspect, a plant exosome-loaded polyhydroxyalkanoate microsphere is prepared by the method for preparing a plant exosome-loaded polyhydroxyalkanoate microsphere described in any one of the above.
In a third aspect, a use of polyhydroxyalkanoate microsphere loaded with plant exosomes, comprising: and adding the polyhydroxyalkanoate microsphere loaded with the plant exosomes into an injection solvent, stirring the mixture uniformly, and filling the mixture into a pre-filling and sealing injector to obtain the injectable polyhydroxyalkanoate microsphere preparation loaded with the plant exosomes.
In one embodiment of the above technical solution, the injection solvent includes hyaluronic acid solution, or carboxymethyl cellulose sodium solution, or sodium alginate solution, or collagen solution.
In a fourth aspect, a use of polyhydroxyalkanoate microsphere loaded with plant exosomes, comprising: adding the polyhydroxyalkanoate microsphere loaded with the plant exosomes into a solution containing a freeze-drying protective agent and an injection solvent, stirring the mixture uniformly, filling the mixture into a penicillin bottle, and freeze-drying the mixture to obtain the polyhydroxyalkanoate microsphere freeze-dried powder capable of being injected with the plant exosomes.
In one embodiment, the lyoprotectant comprises one or more of mannose, sucrose, lactose, dextran, and glucose;
The injection solvent comprises hyaluronic acid solution, sodium carboxymethyl cellulose solution, sodium alginate solution or collagen solution.
Compared with the prior art, the invention can control the synthesis of the porous polyhydroxyalkanoate microsphere with the size of about 50 mu m, and the size of the porous polyhydroxyalkanoate microsphere is not too small to be phagocytosed by macrophages in advance, or too large to block a needle head in injection, thus being applicable to the injectable soft tissue filling agent. Meanwhile, the porous and rough structure of the polyhydroxyalkanoate microsphere endows the polyhydroxyalkanoate microsphere with larger specific surface area, so that the loading of an exosome is facilitated, the damage of the exosome structure and the advanced leakage of the content of the exosome can be effectively prevented in the loading process, the good dispersibility is realized, the structural integrity of the exosome is kept, and meanwhile, the plant exosome with the repairing function is also beneficial to tissue repair in the soft tissue filling process.
For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.
Drawings
FIG. 1 is a scanning electron micrograph (scale 6 μm) of the PHBV microsphere prepared.
FIG. 2 is a scanning electron micrograph (scale 6 μm) of PHBV microspheres prepared with aromatic exosomes.
FIG. 3 is a zeta potential bar graph of the incense burner, PHBV blank microsphere and PHBV microsphere loaded incense burner.
FIG. 4 is a photomicrograph (scale 50 μm) of the PHBV smooth microspheres prepared.
FIG. 5 is a bar graph of cell proliferation rate of the control, PHBV blank microspheres and PHBV microsphere-loaded incense burner exosomes over time.
FIG. 6 is a graph of experiments on repair of HSF cell scratches from PHBV blank microspheres and PHBV microsphere-loaded incense park exosomes.
Detailed Description
Terms of orientation such as up, down, left, right, front, rear, front, back, top, bottom, etc. mentioned or possible mentioned in this specification are defined with respect to their construction, and they are relative concepts. Therefore, the position and the use state of the device may be changed accordingly. These and other directional terms should not be construed as limiting terms.
The implementations described in the following exemplary examples are not representative of all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with some aspects of the disclosure as detailed in the accompanying 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 or all possible combinations of one or more of the associated listed items.
The porous polyhydroxyalkanoate microsphere is prepared by utilizing the polyhydroxyalkanoate which is easy to obtain and adopting a secondary emulsification method, and the rough surface and the porous structure can endow the polymer microsphere with larger specific surface area, so that the polymer microsphere is favorable for further exosome adsorption, and the exosome loading can be realized through the co-incubation of the microsphere and the plant exosome.
In a first aspect, a method for preparing polyhydroxyalkanoate microsphere loaded with plant exosomes comprises:
S1, purifying polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder.
Optionally, the polyhydroxyalkanoate is PHB, or PHBV, or a cognate polymer or copolymer of PHBV.
Specifically, the purification of polyhydroxyalkanoate comprises: taking polyhydroxyalkanoate, washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, dissolving by methylene dichloride to obtain a clear solution, adopting Soxhlet extraction or filtration, and drying.
S2, dissolving the purified polyhydroxyalkanoate powder in methylene dichloride to prepare a first oil phase, preparing Kong Jishui solution as a first water phase, mixing the first water phase with the first oil phase, and emulsifying in a homogenizer to prepare emulsion.
Optionally, the pore-forming agent aqueous solution comprises one of ammonium bicarbonate, sodium bicarbonate, ammonium carbonate, zinc carbonate, hydrogen peroxide, albumin and cyclodextrin.
S3, taking the emulsion as a second oil phase, dissolving polyvinyl alcohol in water to prepare a second water phase for secondary emulsification, dripping the second oil phase solution into the second water phase for secondary emulsification under the magnetic stirring at a lower speed, and stirring until the organic solvent is completely volatilized, thus obtaining the polyhydroxy fatty acid ester microsphere with a rough porous surface.
S4, the prepared polyhydroxyalkanoate microsphere is subjected to irradiation sterilization and then is redispersed in sterile water, the dispersion liquid is mixed with a plant exosome solution in an equal volume, and the mixture is incubated at the room temperature of a shaking table and centrifuged to obtain the polyhydroxyalkanoate microsphere loaded with the plant exosome.
Optionally, the plant exosome is a xiangyuan exosome or a polygonum cuspidatum exosome. 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 microsphere based on aromatic exosomes, and the preparation method is as follows:
s01, taking 5g PHBV powder (with the molecular weight of 40 ten thousand), washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, dissolving by methylene dichloride to obtain a clear solution, and carrying out Soxhlet extraction and drying to obtain the 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 of ammonium bicarbonate aqueous solution, and homogenizing for 3min at 10000rpm by using a homogenizer to obtain the colostrum.
S03, dripping 200mL of 5mg/mL aqueous solution of polyvinyl alcohol (Polyvinyl alcohol, mw=27000) into the colostrum, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, and centrifuging at 2000rpm to collect PHBV microspheres.
S04, cleaning the PHBV microsphere with deionized water for 3 times, and then re-dispersing the PHBV microsphere in 20mL of sterile deionized water to obtain a porous PHBV microsphere aqueous dispersion. 1mL of the porous PHBV microsphere aqueous dispersion liquid is taken, 1mL of the aromatic circular exosome (the protein content is 0.6 mg/mL) is added, after the mixture is mechanically stirred for 24h by a shaking table, the mixture is centrifuged at 2000rpm and is dispersed in sterile deionized water again, and the PHBV microsphere loaded with the aromatic circular exosome is obtained.
Example 2
Microscopic morphology observation and surface charge testing of PHBV microspheres.
Taking a certain amount of PHBV microspheres prepared in the example 1 and PHBV microspheres loaded with aromatic circular exosomes, placing the PHBV microspheres on a sample plate adhered with conductive double faced adhesive tape, and observing under a scanning electron microscope after gold plating to obtain the microsphere morphology shown in the figures 1 and 2.
From an examination of the picture, the PHBV microsphere prepared in example 1 has regular morphology, a size of about 50 microns, a rough surface and a plurality of holes randomly distributed. The prepared PHBV microsphere loaded with the aromatic circular exosomes retains the microscopic morphology and the microscopic dimension of the original porous PHBV microsphere.
Taking a certain amount of the PHBV microsphere prepared in the example 1, the PHBV microsphere loaded with the aromatic circular exosome and the aqueous dispersion of the aromatic circular exosome, and testing the surface charges on a laser particle analyzer to obtain the surface charges shown in the figure 3, wherein the surface charges of the aromatic circular exosome and the porous PHBV microsphere are negative charges, and the surface charges of the porous PHBV microsphere loaded with the aromatic circular exosome are slightly higher than the surface charges of the PHBV microsphere not loaded with the aromatic circular exosome, so that the successful loading of the aromatic circular exosome on the surface of the porous PHBV microsphere can be proved.
From the above analysis, it can be further seen that the present invention can be controlled to synthesize porous polyhydroxyalkanoate microspheres of about 50 μm, which are not too small in size to be engulfed by macrophages in advance, or too large to block the needle during injection, and are suitable for injectable soft tissue fillers. Meanwhile, the porous and rough structure of the polyhydroxyalkanoate microsphere endows the polyhydroxyalkanoate microsphere with larger specific surface area, so that the loading of an exosome is facilitated, the damage of the exosome structure and the advanced leakage of the content of the exosome can be effectively prevented in the loading process, the good dispersibility is realized, the structural integrity of the exosome is kept, and meanwhile, the plant exosome with the repairing function is also beneficial to tissue repair in the soft tissue filling process.
Example 3
And verifying the biological efficacy of PHBV porous microspheres loaded with the incense circle exosomes.
Human skin fibroblasts L929 were selected, 96-well plates were spread according to 100. Mu.l of a cell suspension having a density of 1X 10 5 cells/ml, after cells were attached to the wall for 12 hours, the culture solution was aspirated, and PHBV microspheres prepared in example 1 dispersed with the cell culture solution, PHBV microspheres loaded with aromatic circular exosomes and a control group (microsphere concentration was 50. Mu.g/ml) were added, incubated for different times and the cell proliferation rate was measured by MTT method. As shown in fig. 5, the PHBV microsphere loaded with the incense circle exosome has higher cell proliferation rate with time compared with the control group and the blank PHBV microsphere group, which indicates that the PHBV microsphere loaded with the incense circle exosome has good safety and has the effect of promoting cell proliferation.
Human skin fibroblasts were seeded at a cell density of 2 x 10 5/ml in 6-well plates and cultured in an incubator for 24h; drawing a horizontal line straight by using a gun head, washing cells for 3 times by using PBS, removing the drawn cells, and adding a serum-free culture medium; a blank group and a test group were set up, (concentration) respectively, placed in a 37 ℃ 5% co 2 incubator, incubated, and photographed under observation at different time points. As shown in the figure 6, compared with the blank PHBV microsphere group, the PHBV microsphere loaded with the incense circle exosome has more remarkable scratch repairing effect, which further proves that the PHBV microsphere loaded with the incense circle exosome has good cell repairing promoting performance. Example 4
The present embodiment is a PHBV porous microsphere based on Polygonum cuspidatum exosomes, and the preparation method is the same as that of embodiment 1, and the main difference is that the exosomes selected are Polygonum cuspidatum exosomes, which are specifically as follows:
S001, taking 5g PHBV powder (with the molecular weight of 40 ten thousand), washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, dissolving by methylene dichloride to obtain a clear solution, and carrying out Soxhlet extraction and drying to obtain the purified PHBV powder.
S002, weighing 200mg of purified PHBV powder, adding 8mL of dichloromethane, adding 2.4mL of 5mg/mL of ammonium bicarbonate aqueous solution after the PHBV powder is dissolved, and homogenizing for 3 minutes at 10000rpm by using a homogenizer to obtain the colostrum.
S003, dropping 200mL of 5mg/mL aqueous solution of polyvinyl alcohol (Polyvinyl alcohol, mw=27000) into the colostrum, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, centrifuging at 2000rpm and collecting to obtain PHBV microspheres.
S004, cleaning the PHBV microsphere with deionized water for 3 times, and then re-dispersing the PHBV microsphere in 20mL of sterile deionized water to obtain the porous PHBV microsphere aqueous dispersion. 1mL of the porous PHBV microsphere aqueous dispersion liquid is taken, 1mL of the giant knotweed exosome (with the protein content of 0.4 mg/mL) is added, after the giant knotweed exosome is mechanically stirred for 24 hours by a shaking table, the mixture is centrifuged at 2000rpm and is dispersed in sterile deionized water again, and the PHBV microsphere loaded with the giant knotweed exosome is obtained.
Example 5
The present embodiment is PHBVHHX porous microsphere based on aromatic exosomes, and the preparation method is the same as that of embodiment 1, and the main difference is that the microsphere polymer selected is PHBVHHX, and the specific steps are as follows:
S0001, taking 5g PHBVHHX powder (with the molecular weight of 50 ten thousand), washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, dissolving by methylene dichloride to obtain a clear solution, and carrying out Soxhlet extraction and drying to obtain purified PHBVHHX powder.
S0002 after 200mg of purified PHBVHHX mg of powder was weighed and added to 8mL of methylene chloride to dissolve the powder, 2.4mL of 5mg/mL of ammonium bicarbonate aqueous solution was added, and the mixture was homogenized at 10000rpm for 3 minutes by a homogenizer to obtain colostrum.
S0003 dripping 200mL of 5mg/mL aqueous solution of polyvinyl alcohol (Polyvinyl alcohol, mw=27000) into the colostrum, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, centrifuging at 2000rpm, and collecting the microspheres to obtain PHBVHHX microspheres
S0004, cleaning with deionized water for 3 times, and then re-dispersing in 20mL of sterile deionized water to obtain the porous PHBVHHX microsphere aqueous dispersion. 1mL of the porous PHBVHHX microsphere aqueous dispersion is taken, 1mL of the aromatic circular exosome (the protein content is 0.6 mg/mL) is added, after the mixture is mechanically stirred for 24 hours by a shaking table, the mixture is centrifuged at 2000rpm and is dispersed in sterile deionized water again, and PHBVHHX microsphere loaded with the aromatic circular exosome is obtained.
Example 6
The embodiment is PHBV porous microsphere based on aromatic exosomes, and the preparation method is the same as that of embodiment 1, and the main difference is that the selected pore-foaming agent is hydrogen peroxide, and the preparation method is as follows:
S00001, taking 5g PHBV powder (with the molecular weight of 40 ten thousand), washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, dissolving by methylene dichloride to obtain a clear solution, and carrying out Soxhlet extraction and drying to obtain the 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 5mg/mL aqueous solution of polyvinyl alcohol (Polyvinyl alcohol, mw=27000) into the colostrum, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, and centrifuging at 2000rpm to obtain PHBV microspheres.
S00004, cleaning with deionized water for 3 times, and then re-dispersing in 20mL of sterile deionized water to obtain the porous PHBV microsphere aqueous dispersion. 1mL of the porous PHBV microsphere aqueous dispersion liquid is taken, 1mL of the aromatic circular exosome (the protein content is 0.6 mg/mL) is added, after the mixture is mechanically stirred for 24h by a shaking table, the mixture is centrifuged at 2000rpm and is dispersed in sterile deionized water again, and the PHBV microsphere loaded with the aromatic circular exosome is obtained.
Comparative example 1
The preparation method of the smooth microsphere with PHBV as comparative example is as follows:
Step1, taking 5g PHBV powder (with the molecular weight of 40 ten thousand), washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, and carrying out Soxhlet extraction and drying to obtain purified PHBV powder which can be dissolved in dichloromethane.
Step 2, weighing 200mg of purified PHBV powder, adding 8mL of dichloromethane, dropwise adding 200mL of 5mg/mL of polyvinyl alcohol (Polyvinyl alcohol, mw=27000) aqueous solution after the PHBV powder is dissolved, stirring for 4 hours at 400rpm, volatilizing to remove dichloromethane, centrifugally collecting microspheres at 2000rpm, washing 3 times with deionized water, and then re-dispersing in 20mL of sterile deionized water to obtain the PHBV smooth microspheres.
Comparative example 2
Morphology observation of smooth microspheres of PHBV
A certain amount of the smooth microsphere of PHBV prepared in comparative example 1 was placed in a glass slide, and observed under an optical microscope, to obtain the microsphere morphology as shown in fig. 4.
The PHBV smooth microsphere prepared in comparative example 1 has regular morphology, a size of about 50 microns and a smooth surface. Compared with example 1 (fig. 1), the surface is smooth and void-free, has a smaller specific surface area and the absence of pores is detrimental to further entrapment of plant exosomes.
In a second aspect, a plant exosome-loaded polyhydroxyalkanoate microsphere is prepared by the method for preparing the plant exosome-loaded polyhydroxyalkanoate microsphere.
In a third aspect, a use of polyhydroxyalkanoate microsphere loaded with plant exosomes, comprising: and adding the polyhydroxyalkanoate microsphere loaded with the plant exosomes into an injection solvent, stirring the mixture uniformly, filling the mixture into a pre-filling and sealing syringe, and carrying out aseptic operation in the whole process to obtain the injectable polyhydroxyalkanoate microsphere preparation loaded with the plant exosomes.
Optionally, the injection solvent comprises hyaluronic acid solution, or sodium carboxymethyl cellulose solution, or sodium alginate solution, or collagen solution.
Example 7
This example is an injectable formulation based on the PHBV porous microspheres loaded with the Polygonum cuspidatum exosomes of example 4, prepared as follows:
and adding 1g of PHBV microspheres loaded with the giant knotweed external body prepared in the example 4 into 20mL of 2% hyaluronic acid solution, stirring at 300rpm for 6h, and filling into a prefilled syringe after uniformly mixing, thus obtaining the injectable PHBV porous microsphere preparation loaded with the giant knotweed external body.
In a fourth aspect, a use of polyhydroxyalkanoate microsphere loaded with plant exosomes, comprising: 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 into a penicillin bottle after uniformly mixing, and obtaining the polyhydroxyalkanoate microsphere freeze-dried powder capable of loading the plant exosomes after freeze-drying in a whole process without sterile operation.
Optionally, the lyoprotectant comprises one or more of mannose, sucrose, lactose, dextran, glucose.
Optionally, the injection solvent comprises hyaluronic acid solution, or sodium carboxymethyl cellulose solution, or sodium alginate solution, or collagen solution.
Example 8
This example is an injectable lyophilized powder based on PHBV porous microspheres loaded with aromatic exosomes in example 1, its preparation method is as follows:
The PHBV microsphere loaded with the incense circle exosome 1g prepared in the example 1 is added into 20mL of solution containing 5% mannose and 2% hyaluronic acid, stirred at 300rpm for 6h, and after being uniformly mixed, the solution is filled into a penicillin bottle, and the porous PHBV microsphere freeze-dried powder capable of being injected with the incense circle exosome is obtained after freeze drying.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (9)
1. The preparation method of the polyhydroxyalkanoate microsphere loaded with plant exosomes is characterized by comprising the following steps:
Purifying the polyhydroxyalkanoate to obtain purified polyhydroxyalkanoate powder;
200mg of purified polyhydroxyalkanoate powder is dissolved in 8mL of dichloromethane to prepare a first oil phase, kong Jishui solution is prepared as a first water phase, 2.4mL of the first water phase is mixed with the first oil phase, and emulsification is carried out in a homogenizer to prepare emulsion; the first water phase is sodium bicarbonate water solution with the concentration of 5mg/mL or hydrogen peroxide with the concentration of 1%;
Taking the emulsion as a second oil phase, dissolving polyvinyl alcohol in water to prepare a second water phase of which the second emulsification is 5 mg/mL, dropwise adding the second oil phase solution into 200mL of the second water phase under magnetic stirring to carry out secondary emulsification, and stirring to obtain the polyhydroxy fatty acid ester microsphere with a rough porous surface;
the prepared polyhydroxyalkanoate microsphere is subjected to irradiation sterilization and then is dispersed in sterile water again, the dispersion liquid is mixed with plant exosome solution in equal volume, and is incubated in a shaking table at room temperature, and the polyhydroxyalkanoate microsphere loaded with plant exosome is obtained after centrifugation;
The polyhydroxyalkanoate is PHBV.
2. The method for preparing polyhydroxyalkanoate microsphere loaded with plant exosomes according to claim 1, wherein the purifying the polyhydroxyalkanoate comprises: taking polyhydroxyalkanoate, washing water-soluble impurities and alcohol-soluble impurities in the polymer by deionized water and ethanol respectively, dissolving by methylene dichloride to obtain a clear solution, and drying.
3. The method for preparing polyhydroxyalkanoate microsphere loaded with plant exosomes according to claim 1 or 2, wherein the plant exosomes are xiangyuan exosomes or polygonum cuspidatum exosomes.
4. A polyhydroxyalkanoate microsphere loaded with plant exosomes is characterized in that, prepared by the method for preparing the polyhydroxyalkanoate microsphere loaded with plant exosomes according to any one of claims 1-3.
5. Use of polyhydroxyalkanoate microspheres loaded with plant exosomes, comprising: the polyhydroxyalkanoate microsphere loaded with plant exosomes according to claim 4 is added into an injection solvent, stirred and uniformly mixed, and then filled into a prefilled syringe, so as to obtain the injectable polyhydroxyalkanoate microsphere preparation loaded with plant exosomes.
6. The use of polyhydroxyalkanoate microspheres loaded with plant exosomes according to claim 5, wherein the injection solvent comprises hyaluronic acid solution, or sodium carboxymethyl cellulose solution, or sodium alginate solution, or collagen solution.
7. Use of polyhydroxyalkanoate microspheres loaded with plant exosomes, comprising: adding the polyhydroxyalkanoate microsphere loaded with plant exosomes according to claim 4 into a solution containing a freeze-drying protective agent and an injection solvent, stirring until the polyhydroxyalkanoate microsphere is uniformly mixed, filling the solution into a penicillin bottle, and freeze-drying the solution to obtain the freeze-dried polyhydroxyalkanoate microsphere powder capable of loading plant exosomes.
8. The use of polyhydroxyalkanoate microspheres loaded with plant exosomes according to claim 7, wherein the lyoprotectant comprises one or more of mannose, sucrose, lactose, dextran, glucose.
9. The use of polyhydroxyalkanoate microspheres loaded with plant exosomes according to claim 7, wherein the injection solvent comprises hyaluronic acid solution, or sodium carboxymethyl cellulose solution, or sodium alginate solution, or collagen solution.
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