CN113248772A - Microfluidic preparation method of porous polyhydroxyalkanoate polymer microspheres, porous polymer microspheres prepared by microfluidic preparation method and application of porous polyhydroxyalkanoate polymer microspheres - Google Patents
Microfluidic preparation method of porous polyhydroxyalkanoate polymer microspheres, porous polymer microspheres prepared by microfluidic preparation method and application of porous polyhydroxyalkanoate polymer microspheres Download PDFInfo
- Publication number
- CN113248772A CN113248772A CN202110572734.7A CN202110572734A CN113248772A CN 113248772 A CN113248772 A CN 113248772A CN 202110572734 A CN202110572734 A CN 202110572734A CN 113248772 A CN113248772 A CN 113248772A
- Authority
- CN
- China
- Prior art keywords
- polymer
- microspheres
- porous
- solution
- pha
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000004005 microsphere Substances 0.000 title claims abstract description 168
- 229920000642 polymer Polymers 0.000 title claims abstract description 108
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 title abstract description 44
- 229920000903 polyhydroxyalkanoate Polymers 0.000 title description 43
- 239000000243 solution Substances 0.000 claims abstract description 70
- 238000000034 method Methods 0.000 claims abstract description 48
- 238000004945 emulsification Methods 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 239000007864 aqueous solution Substances 0.000 claims abstract description 23
- 238000004108 freeze drying Methods 0.000 claims abstract description 23
- 239000004088 foaming agent Substances 0.000 claims abstract description 20
- 238000002156 mixing Methods 0.000 claims abstract description 13
- 239000003960 organic solvent Substances 0.000 claims abstract description 13
- 238000002347 injection Methods 0.000 claims abstract description 10
- 239000007924 injection Substances 0.000 claims abstract description 10
- 239000006185 dispersion Substances 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 42
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- 239000011148 porous material Substances 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 19
- 238000003756 stirring Methods 0.000 claims description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 16
- 239000002245 particle Substances 0.000 claims description 14
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 11
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 9
- 239000003361 porogen Substances 0.000 claims description 8
- JWEBFZPSIPMEFI-UHFFFAOYSA-M sodium acetonitrile hydroxide Chemical compound [OH-].[Na+].CC#N JWEBFZPSIPMEFI-UHFFFAOYSA-M 0.000 claims description 8
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 7
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 7
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 7
- 239000001099 ammonium carbonate Substances 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- 238000004381 surface treatment Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 229920000520 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polymers 0.000 claims description 3
- FHUDZSGRYLAEKR-UHFFFAOYSA-N 3-hydroxybutanoic acid;4-hydroxybutanoic acid Chemical compound CC(O)CC(O)=O.OCCCC(O)=O FHUDZSGRYLAEKR-UHFFFAOYSA-N 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims 1
- 238000012377 drug delivery Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 70
- 210000004027 cell Anatomy 0.000 description 36
- 239000007787 solid Substances 0.000 description 22
- 239000011521 glass Substances 0.000 description 20
- 229920003023 plastic Polymers 0.000 description 20
- 239000004033 plastic Substances 0.000 description 20
- 210000004761 scalp Anatomy 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000007710 freezing Methods 0.000 description 7
- 230000008014 freezing Effects 0.000 description 7
- 229910021642 ultra pure water Inorganic materials 0.000 description 7
- 239000012498 ultrapure water Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 210000001185 bone marrow Anatomy 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 230000017423 tissue regeneration Effects 0.000 description 6
- 230000005514 two-phase flow Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 5
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- IECPWNUMDGFDKC-UHFFFAOYSA-N Fusicsaeure Natural products C12C(O)CC3C(=C(CCC=C(C)C)C(O)=O)C(OC(C)=O)CC3(C)C1(C)CCC1C2(C)CCC(O)C1C IECPWNUMDGFDKC-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000001804 emulsifying effect Effects 0.000 description 4
- IECPWNUMDGFDKC-MZJAQBGESA-N fusidic acid Chemical compound O[C@@H]([C@@H]12)C[C@H]3\C(=C(/CCC=C(C)C)C(O)=O)[C@@H](OC(C)=O)C[C@]3(C)[C@@]2(C)CC[C@@H]2[C@]1(C)CC[C@@H](O)[C@H]2C IECPWNUMDGFDKC-MZJAQBGESA-N 0.000 description 4
- 229960004675 fusidic acid Drugs 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- -1 hydroxy fatty acids Chemical class 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 210000001912 transporting cell Anatomy 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- IMROMDMJAWUWLK-UHFFFAOYSA-N Ethenol Chemical compound OC=C IMROMDMJAWUWLK-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000037534 Progressive hemifacial atrophy Diseases 0.000 description 2
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000006727 cell loss Effects 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000012017 passive hemagglutination assay Methods 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- RRWWOSSPAGCJFO-UHFFFAOYSA-N 3-hydroxybutanoic acid;3-hydroxyhexanoic acid Chemical compound CC(O)CC(O)=O.CCCC(O)CC(O)=O RRWWOSSPAGCJFO-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010040844 Skin exfoliation Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003181 biological factor Substances 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000004656 cell transport Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000030944 contact inhibition Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000035618 desquamation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004146 energy storage Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000008384 inner phase Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000010558 suspension polymerization method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/16—Making expandable particles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/04—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof using blowing gases generated by a previously added blowing agent
- C08J9/06—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof using blowing gases generated by a previously added blowing agent by a chemical blowing agent
- C08J9/08—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof using blowing gases generated by a previously added blowing agent by a chemical blowing agent developing carbon dioxide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/36—After-treatment
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/096—Polyesters; Polyamides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2203/00—Foams characterized by the expanding agent
- C08J2203/02—CO2-releasing, e.g. NaHCO3 and citric acid
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2207/00—Foams characterised by their intended use
- C08J2207/10—Medical applications, e.g. biocompatible scaffolds
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
- C08J2367/04—Polyesters derived from hydroxy carboxylic acids, e.g. lactones
Abstract
The invention provides a microfluidic preparation method and application of Polyhydroxyalkanoate (PHA) porous microspheres, wherein the preparation method comprises the following steps: (A) dissolving Polyhydroxyalkanoate (PHA) polymer in an organic solvent to prepare a polymer organic solution, and dissolving a pore-foaming agent in water to prepare a pore-foaming agent aqueous solution; (B) mixing a polymer organic solution with a pore-foaming agent aqueous solution, and performing ultrasonic pre-emulsification to form a water-in-oil pre-emulsification system serving as a dispersion phase; (C) dissolving a water-soluble surface active polymer in water to obtain an aqueous solution serving as a continuous phase; (D) injecting samples by using an injection pump, mixing the two phases in a T-shaped microfluidic device, generating polymer droplets in a T-shaped pipe, and collecting the polymer droplets; (E) and removing the organic solvent in the polymer droplets, and solidifying and freeze-drying to obtain the porous polymer microspheres. The method can obtain the porous microspheres which have uniform size and controllable shape and are used as cell carrier scaffolds or drug delivery systems.
Description
Technical Field
The invention belongs to the field of biological materials, and relates to a microfluidic preparation method of porous Polyhydroxyalkanoate (PHA) polymer microspheres, the prepared porous polymer microspheres and application thereof.
Background
In recent years, cell therapy has been receiving much attention in the fields of tissue engineering and regeneration. At present, most cell therapies adopt a direct injection method, namely, a cell suspension is directly injected into a body, cells are likely to die in the injection process due to insufficient protection, and only a small amount of surviving cells are remained in an injection site and are not enough to play a role in effectively repairing tissues. The biological material is used as a carrier to carry cells, so that the cells can be protected in the injection process, the survival rate of the cells is improved, most of the cells can be reserved in the injection site, the cell loss is reduced, and if the stem cells are loaded, the subsequent differentiation process can be regulated and controlled, so that the tissue regeneration effect is improved.
The microspheres are excellent cell transport carriers, can be directly injected to tissue sites needing to be repaired due to small volume, have small wound caused by injection and can reduce the pain of patients, so the microspheres have obvious advantages compared with macroscopic-size scaffold materials. The microsphere carrier with the porous structure can be loaded with cells, the cells are protected in the injection process, the subsequent cell loss is reduced, the diffusion of nutrients and metabolites is facilitated by the porous structure, and a larger space is provided for cell proliferation and tissue regeneration. The microspheres can be used as a carrier for transporting cell types related to tissues needing to be repaired, and can also be used for carrying cells capable of secreting biological factors with therapeutic effects, so that the cells can continuously secrete signal factors required by tissue regeneration in vivo, and the differentiation of stem cells and the regeneration and repair of tissues are assisted to be regulated.
Polyhydroxyalkanoates (PHA) is a linear polyester composed of hydroxy fatty acids synthesized by microorganisms, and is used as a carbon source and an energy storage substance of bacteria under the condition of metabolic imbalance, so that the viability of the bacteria in the adverse environment is improved. Until now, it has been discovered that there are at least 150 different monomer structures for PHAs, and that PHA's with new monomer structures are also being continuously explored. The diversity of the monomer brings the diversification of physicochemical properties to PHAs, so that PHA has good biocompatibility and biodegradability, and degradation products of PHA are nontoxic and harmless, and the PHA has good long-term safety when used as an in vivo transplantation stent. The PHA has obvious advantages in the application field of biodegradable materials, has good application prospects in the fields of vascular tissue engineering, cartilage tissue engineering, artificial nerve conduits and the like, and is a potential excellent material for preparing biodegradable microspheres.
Common preparation methods of biopolymer microspheres include emulsification methods, spray drying methods, suspension polymerization methods and the like, which can be used for mass production, but the preparation steps are more, the obtained microspheres have uneven size distribution, the product state is difficult to control, and the repeatability is poor. The micro-fluidic technology provides a new strategy for the production and preparation of the polymer microspheres, the method can be used for accurately controlling the preparation process of the microspheres, and the obtained microspheres have narrow particle size distribution, adjustable size and high repeatability.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a microfluidic preparation method of porous PHA polymer microspheres, the prepared porous polymer microspheres and application thereof. The preparation method of the invention can overcome the problems of uneven grain diameter, uncontrollable property and poor repeatability of the porous microspheres prepared by the traditional method, and can obtain the porous microspheres which have uniform size and controllable appearance and are used as cell carrier scaffolds.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a microfluidic method for preparing porous PHA polymer microspheres, the method comprising the steps of:
(A) dissolving PHA polymer in organic solvent to prepare polymer organic solution, and dissolving pore-foaming agent in water to prepare pore-foaming agent water solution;
(B) mixing a PHA polymer organic solution with a pore-foaming agent aqueous solution for ultrasonic pre-emulsification to form a water-in-oil pre-emulsification system as a dispersion phase;
(C) dissolving a water-soluble surface active polymer in water to obtain an aqueous solution serving as a continuous phase;
(D) injecting samples by using an injection pump, mixing the two phases in a T-shaped microfluidic device, generating polymer droplets in a T-shaped pipe, and collecting the polymer droplets;
(E) and removing the organic solvent in the PHA polymer liquid drop, solidifying and freeze-drying to obtain the porous polymer microsphere.
In the preparation method, when two-phase liquids which are not mutually soluble are mixed in a microfluidic device, an inner phase with a lower flow speed is broken under the shearing action of a two-phase interface to form liquid drops, so that the flow channel structure, the two-phase flow speed, the fluid viscosity and the use of a surfactant of the microfluidic device can influence the formation and the properties of the liquid drops. The invention selects the T-shaped structure flow channel, so that the size of the microsphere is uniform and stable. In addition, the micro-fluidic technology is utilized to accurately control the preparation process of the microspheres by adjusting the flow rate of two phases, the viscosity of fluid and the interfacial tension, so that the property of the microsphere product can be regulated and controlled.
Preferably, the PHA polymer of step (A) is poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (P3HB3HV3HHx), poly (3-hydroxybutyrate-3-hydroxyhexanoate) (PHBHHx), poly (3-hydroxybutyrate-4-hydroxybutyrate) (P3HB4HB), poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).
Preferably, the concentration of the PHA polymer organic solution of step (A) is from 0.1 to 20% (e.g., 0.3%, 1.5%, 5%, 10%, 20%). If the concentration of the polymer is lower than 0.05 percent, a uniform water-in-oil pre-emulsification system cannot be formed, and the formation of a porous structure of the microsphere is influenced; if the concentration of the polymer is higher than 20%, the viscosity of the dispersed phase is too high, the shearing force of the continuous phase is insufficient, and the micro-fluidic control method cannot be used for effectively forming liquid drops.
Preferably, the organic solvent in step (a) is any one or a combination of at least two of dichloromethane, chloroform or acetonitrile.
Preferably, the porogen of step (a) is ammonium bicarbonate. As carbon dioxide and ammonia gas are generated after the ammonium bicarbonate is hydrolyzed, gas is removed when the organic solvent is volatilized and PHA polymer liquid drops are solidified to form the microspheres, and a pore structure is left in the microspheres, the porous microspheres prepared by the scheme do not need to be removed in the subsequent steps. The microspheres prepared by adopting the pore-forming agent have spongy pore structures, and the pores are communicated with one another.
Preferably, the aqueous porogen solution of step (a) has a concentration of 5-20% (e.g., 5%, 10%, 15%, 20%, etc.).
Preferably, the volume ratio of the PHA polymer organic solution to the porogen aqueous solution during the mixing in the step (B) is 10:1-2: 1. If the volume ratio of the PHA polymer organic solution to the pore-foaming agent aqueous solution is too large, the pore-forming efficiency is poor, and the prepared microspheres cannot effectively form an ideal porous structure; if the volume ratio is too small, a uniform pre-emulsification system cannot be formed, phase separation occurs in the subsequent preparation process, and microspheres with uniform appearance and a porous structure cannot be formed.
Preferably, the ultrasonic pre-emulsification in step (B) is pre-emulsification using a sonicator, and the emulsification is performed for 90s in one cycle, with the number of cycles being 1 to 5 (e.g., 1, 2, 3, 4 or 5).
Preferably, the water-soluble surface-active polymer of step (C) is partially hydrolysed polyvinyl alcohol (PVA). The partially hydrolyzed polyvinyl alcohol is a copolymer of vinyl acetate and vinyl alcohol, wherein hydrophobic vinyl acetate groups can be adsorbed on the surface of oil phase droplets, hydrophilic vinyl alcohol groups are dissolved in a water phase, and the stabilizing effect on the oil phase droplets is achieved through the steric hindrance effect of a long-chain polymer. Meanwhile, polyvinyl alcohol is nontoxic and biodegradable, so that the polyvinyl alcohol is widely applied to the field of biological medicine.
Preferably, the concentration of the aqueous solution of step (C) is 0.1-0.5% (e.g., 0.1%, 0.25%, 0.5%, etc.).
Preferably, the specific method for mixing the two phases in the T-type microfluidic device in the step (D) is as follows: the continuous phase is firstly led into the device from one end port of the T-shaped pipe, after the whole pipeline is filled with the continuous phase, the dispersed phase is led into the device from the vertical port, so that the two phases are mixed in the T-shaped microfluidic device, polymer liquid drops are generated in the T-shaped pipe, and the polymer liquid drops are collected at the other end port.
Preferably, the flow rate ratio of the dispersed phase to the continuous phase when the two phases are mixed in the T-type microfluidic device in step (D) is 1:3-1:10, such as 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc., under the flow rate ratio, PHA polymer microspheres with good monodispersity and uniform spherical morphology can be stably prepared, and the particle size of the microspheres can be ensured to be 100-500 μm, and the surface pore size is 1-10 μm.
Preferably, the flow rate of the dispersed phase is 300 μ L/min and the flow rate of the continuous phase is 1500 μ L/min; when the flow rate ratio of the two phases is 1:6, the porous microspheres with high monodispersity and uniform appearance can be stably and efficiently prepared, and the average particle size is 253 mu m.
Preferably, the method for removing the organic solvent in the polymer droplets in the step (E) adopts a mode of stirring to volatilize the organic solvent.
Preferably, the curing of step (E) is at a temperature of 4 to 60 deg.C (e.g., 4 deg.C, 25 deg.C, 35 deg.C, 60 deg.C) for a period of 2 to 18 hours (e.g., 2 hours, 4 hours, 8 hours, 12 hours, 18 hours).
Preferably, the temperature of the lyophilization of step (E) is-50 to-60 ℃ (e.g., -50 ℃, -53 ℃, -55 ℃, -58 ℃ or-60 ℃), and the time of the lyophilization is 8 to 18h (e.g., 8h, 10h, 12h, 15h or 18 h).
On the other hand, the invention provides the porous polymer microspheres prepared by the preparation method, wherein the particle size of the porous polymer microspheres is 100-500 mu m, and the surface pore size is 1-10 mu m.
The invention utilizes the microfluidic technology to obtain stable and uniform porous polymer microspheres, the size of the microspheres is adjustable, and a large number of pore structures are arranged on the surface and inside of the microspheres.
In another aspect, the present invention provides a method for performing surface optimization treatment on the porous polymer microspheres, which comprises: and (3) carrying out surface treatment on the porous microspheres by using a sodium hydroxide solution or a sodium hydroxide-acetonitrile mixed solution.
In the invention, after the surface treatment is carried out on the porous microspheres by using the sodium hydroxide-acetonitrile mixed solution, the number of pores on the surfaces of the microspheres is increased, the pore diameter is increased, the surface area is increased, the porous microspheres can be used for loading the drugs to be delivered more efficiently, and when the pore diameter is increased to allow cells to pass, the porous microspheres can also be used for loading and transporting cells and stem cells, so that protection is provided for the cells in the delivery process, and the porous microspheres are used for tissue engineering repair application.
Preferably, the sodium hydroxide-acetonitrile mixed solution is a mixed solution obtained by mixing a sodium hydroxide aqueous solution with the concentration of 0.25M and acetonitrile according to the volume ratio of 3: 7.
Preferably, the surface treatment time is 2-16min, such as 2min, 5min, 8min, 10min, 13min or 16min, preferably the treatment time is 8-16 min.
In another aspect, the present invention provides a porous scaffold material comprising porous PHA polymer microspheres as described above.
In another aspect, the present invention provides a porous scaffold loaded with cells, comprising the porous polymeric microspheres as described above, and cells loaded on the porous polymeric microspheres.
The porous scaffold material provided by the invention is used for loading and transporting cells, wherein when the porous polymer microspheres are used for loading the cells, the specific scheme is that the cells and the porous microspheres prepared by the micro-fluidic control method are co-cultured to form a cell-microsphere compound, and the cell-microsphere compound can be injected into tissues needing to be repaired to realize local tissue repair. In one embodiment of the invention, the cell is a mouse bone marrow mesenchymal stem cell.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method can prepare the porous microspheres with uniform particle size distribution and controllable morphology and size, and overcomes the defects of nonuniform particle size distribution and poor repeatability of the microspheres prepared by the traditional double-emulsification method. The invention also provides a porous scaffold material, and the porous polymer microspheres are used as carriers for cell transportation, can be used for loading various cells such as stem cells, can be locally injected, and can be used for tissue regeneration and repair.
Drawings
FIG. 1 is a schematic diagram of the structure of a microfluidic device for preparing porous PHA microspheres of the present invention.
FIG. 2 is a SEM photograph of the surface of the PHA microspheres obtained after lyophilization in step (11) of example 1 (concentration of the polymer solution of 1.5%), wherein the scale on the A-graph is 500 μm and the scale on the B-graph is 200 μm.
FIG. 3 is a SEM photograph of the cross-section of the PHA microspheres obtained after lyophilization in step (11) of example 1 (concentration of the polymer solution of 1.5%), wherein the scale on the A-diagram is 100 μm and the scale on the B-diagram is 200 μm.
FIG. 4 is an SEM image (concentration of polymer solution is 1.5%) of the surface of the porous microspheres subjected to the surface optimization treatment in example 1, wherein the scale of the A image is 100 μm and the scale of the B image is 300 μm.
FIG. 5 is a graph showing the cell survival rate of the porous microspheres co-cultured with mouse bone marrow mesenchymal stem cells for 10 days in example 1.
FIG. 6 is a laser confocal image of the cell microspheres obtained on day 7 of co-culture of the porous microspheres with mouse bone marrow mesenchymal stem cells in example 1.
FIG. 7 is a surface SEM picture (polymer solution concentration of 1.5%) of porous P3HB3HV3HHx microspheres prepared using a stirred pre-emulsification method, with scale bar 300 μm for graph A and 400 μm for graph B.
FIG. 8 is a cross-sectional SEM picture (1.5% concentration of polymer solution) of porous P3HB3HV3HHx microspheres after 30min treatment with 1M sodium hydroxide reagent, with the scale on panel A being 200 μ M and the scale on panel B being 100 μ M.
FIG. 9 is a cross-sectional SEM picture (polymer solution concentration 1.5%) of porous P3HB3HV3HHx microspheres treated with 1M sodium hydroxide reagent for 60min, with the scale on panel A being 100 μ M and the scale on panel B being 100 μ M.
FIG. 10 is a cross-sectional SEM picture (polymer solution concentration 1.5%) of porous P3HB3HV3HHx microspheres after 90min treatment with 1M sodium hydroxide reagent, where the scale on the A plot is 100 μ M and the scale on the B plot is 100 μ M.
FIG. 11 is a surface SEM picture (polymer solution concentration 0.3%) of porous P3HB3HV3HHx microspheres prepared using the ultrasonic pre-emulsification method with scale bar 50 μm and scale bar 100 μm for B.
FIG. 12 is a surface SEM picture (polymer solution concentration of 1.5%) of porous P3HB4HB microspheres prepared using the ultrasonic pre-emulsification method, with the scale on graph A being 100 μm and the scale on graph B being 40 μm.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
In this example, the polyhydroxyalkanoate microspheres are prepared by the following method, specifically including the following steps:
1) polymer solution a: adding 0.3g P3HB3HV3HHx polymer solid into 20ml dichloromethane, stirring and dissolving to prepare a colorless and transparent polymer organic solution;
2) pore-forming agent solution B: 1.5g of ammonium bicarbonate solid is added into 10ml of water to be dissolved to prepare a pore-foaming agent aqueous solution;
3) dispersed phase C: taking 7.5ml of the prepared polymer solution A, adding 2.5ml of the prepared pore-foaming agent solution B, and pre-emulsifying in an ice bath by using an ultrasonic crusher to obtain a dispersed phase emulsification system;
4) continuous phase D: adding 2g of polyvinyl alcohol solid into 400ml of water, stirring and dissolving to prepare a solution which is used as a continuous phase for preparing microspheres by a micro-fluidic control method;
5) and (3) collecting a phase E: the same continuous phase D;
6) a glass syringe F: 10ml glass syringe (with metal needle);
7) a plastic syringe G: 60ml plastic syringe (scalp needle);
8) the disperse phase C was drawn into a glass syringe F and the continuous phase D was drawn into a plastic syringe G;
9) t-type microfluidic device: connecting a plastic syringe G into one end port of a T-shaped pipe by using a scalp needle, directly connecting a needle head of a glass syringe F into a vertical port (the two-phase flow direction is shown in figure 1) to form a T-shaped microfluidic device for generating liquid drops, wherein the generated liquid drops flow out from the other end port of the T-shaped pipe and enter a collecting phase E;
10) collecting the polymer droplets generated in the microfluidic device in the collecting phase E, stirring to volatilize the dichloromethane solvent, and solidifying the polymer droplets to form solid polyhydroxyalkanoate microspheres;
11) washing the obtained microspheres with ultrapure water for 3 times, placing the microspheres at-80 ℃ for pre-freezing, and then freeze-drying the microspheres in a freeze dryer at-50 to-60 ℃ for 18 hours;
12) sodium hydroxide-acetonitrile reagent H: mixing 0.25M sodium hydroxide solution with acetonitrile according to the volume ratio of 3: 7;
13) collecting the freeze-dried microspheres, and treating the surfaces of the microspheres with a sodium hydroxide-acetonitrile reagent H for 2min, 4min, 8min and 16 min;
14) washing the treated microspheres with ultrapure water for 3 times, placing the microspheres at-80 ℃ for pre-freezing, and then freeze-drying the microspheres in a freeze dryer at-50 to-60 ℃ for 18 hours;
collecting the freeze-dried microspheres, taking a small amount of samples for analysis by a scanning electron microscope, co-culturing the rest samples and the bone marrow mesenchymal cells of the mice for 10 days, and inspecting the survival and growth conditions of the cells at a fixed time point.
Surface SEM (scanning Electron microscope for Cold field emission, Hitachi, S-4800) characterization is carried out on the P3HB3HV3HHx microspheres obtained after freeze-drying in the step (11), and the result is shown in figure 2, wherein the scale of the graph A is 500 μm, the scale of the graph B is 200 μm, and as can be seen from figure 2, the microspheres have the size of 200-500 μm, the surfaces of the microspheres are in the shape of fluctuated folds, a large number of holes are distributed, and the hole diameters are about 1-10 μm.
SEM representation is carried out on the section of the P3HB3HV3HHx microsphere obtained after freeze-drying in the step (11), the result is shown in figure 3, wherein the scale of the A diagram is 100 μm, the scale of the B diagram is 200 μm, and as can be seen from figure 3, the interior of the microsphere is in a sponge shape, has a large number of hole structures, is wide in hole diameter range distribution, and the holes are mutually communicated, so that good conditions can be provided for cell growth, nutrition and metabolite transportation.
Surface SEM characterization is carried out on the P3HB3HV3HHx microspheres obtained after freeze-drying in the step (14), the result is shown in figure 4, wherein the scale of the A picture is 100 μm, the scale of the B picture is 300 μm, and as can be seen from figure 4, the pore diameter of pores on the surfaces of the freeze-dried microspheres is slightly increased, and the pore diameter of part of the pores can reach 20-25 μm, so that cells can enter the interior of the microspheres to grow.
FIG. 5 shows the cell survival rate of mice cultured with solid microspheres surface-treated with NaOH-acetonitrile reagent H for 8min and bone marrow mesenchymal stem cells without porous structure for 10 days. It can be seen from the figure that the cell activity showed a growth trend within the first 7 days of inoculation, indicating that the cell growth state was good and the number was increased, and the cell activity was decreased in the 10 th day compared to the 7 th day, which may be a contact inhibition effect of the cells attached to the surface of the microspheres due to increased density, and the inner layer cells were less likely to obtain sufficient nutrients and partially apoptosis and desquamation.
FIG. 6 is a laser confocal image of cell microspheres obtained by co-culturing the porous microspheres subjected to surface treatment for 8min by using a sodium hydroxide-acetonitrile reagent H, solid microspheres without a porous structure and bone marrow mesenchymal stem cells of a mouse on day 7. It can be seen from the figure that, when the cells are cultured to day 7, a large number of cells are attached to the surface of the microsphere for growth, and some cells enter the interior of the microsphere for growth from the pores on the surface of the microsphere, which indicates that the internal pore structure of the porous microsphere of the embodiment can provide a space for cell shuttle entry and subsequent growth, and has a potential for application to cell loading and delivery.
Example 2
In this example, the polyhydroxyalkanoate microspheres are prepared by the following method, specifically including the following steps:
1) polymer solution a: adding 0.3g P3HB3HV3HHx solid into 20ml dichloromethane, stirring and dissolving to prepare a colorless and transparent polymer organic solution;
2) pore-forming agent solution B: 1.5g of ammonium bicarbonate solid is added into 10ml of water to be dissolved to prepare a pore-foaming agent aqueous solution;
3) dispersed phase C: taking 7.5ml of the prepared polymer solution A, adding 2.5ml of the prepared pore-forming agent solution B, and stirring and pre-emulsifying by using an overhead homogenizer at the rotating speed of 20000rpm for 2min to obtain a dispersed phase emulsification system;
4) continuous phase D: adding 2g of polyvinyl alcohol solid into 400ml of water, stirring and dissolving to prepare a solution which is used as a continuous phase for preparing microspheres by a micro-fluidic control method;
5) and (3) collecting a phase E: the same continuous phase D;
6) a glass syringe F: 10ml glass syringe (with metal needle);
7) a plastic syringe G: 60ml plastic syringe (scalp needle);
8) the disperse phase C was drawn into a glass syringe F and the continuous phase D was drawn into a plastic syringe G;
9) t-type microfluidic device: connecting a plastic syringe G into one end port of a T-shaped pipe by using a scalp needle, directly connecting a needle head of a glass syringe F into a vertical port (the two-phase flow direction is shown in figure 1) to form a T-shaped microfluidic device for generating liquid drops, wherein the generated liquid drops flow out from the other end port of the T-shaped pipe and enter a collecting phase E;
10) heating the collected phase E to 35 ℃, collecting polymer droplets generated in the microfluidic device, stirring to volatilize a dichloromethane solvent, and solidifying the polymer droplets to form solid polyhydroxyalkanoate microspheres;
11) washing the obtained microspheres with ultrapure water for 3 times, placing the microspheres at-80 ℃ for pre-freezing, and then freeze-drying the microspheres in a freeze dryer at-50 to-60 ℃ for 18 hours;
surface SEM representation is carried out on the P3HB3HV3HHx obtained after freeze-drying in the step (11), the result is shown in figure 7, wherein the scale of the A picture is 300 mu m, the scale of the B picture is 400 mu m, and as can be seen from figure 7, the particle size of the microsphere prepared by adopting the stirring pre-emulsification method is about 500 mu m, the surface holes are fewer, the pore diameter is 5-30 mu m, the maximum pore diameter can reach about 80 mu m, and the microsphere does not have an internally communicated network structure.
Example 3
In this example, the polyhydroxyalkanoate microspheres are prepared by the following method, specifically including the following steps:
1) polymer solution a: adding 0.3g P3HB3HV3HHx solid into 20ml dichloromethane, stirring and dissolving to prepare a colorless and transparent polymer organic solution;
2) pore-forming agent solution B: 1.5g of ammonium bicarbonate solid is added into 10ml of water to be dissolved to prepare a pore-foaming agent aqueous solution;
3) dispersed phase C: taking 7.5ml of the prepared polymer solution A, adding 2.5ml of the prepared pore-foaming agent solution B, and pre-emulsifying in an ice bath by using an ultrasonic crusher to obtain a dispersed phase emulsification system;
4) continuous phase D: adding 2g of polyvinyl alcohol solid into 400ml of water, stirring and dissolving to prepare a solution which is used as a continuous phase for preparing microspheres by a micro-fluidic control method;
5) and (3) collecting a phase E: the same continuous phase D;
6) a glass syringe F: 10ml glass syringe (with metal needle);
7) a plastic syringe G: 60ml plastic syringe (scalp needle);
8) the disperse phase C was drawn into a glass syringe F and the continuous phase D was drawn into a plastic syringe G;
9) t-type microfluidic device: connecting a plastic syringe G into one end port of a T-shaped pipe by using a scalp needle, directly connecting a needle head of a glass syringe F into a vertical port (the two-phase flow direction is shown in figure 1) to form a T-shaped microfluidic device for generating liquid drops, wherein the generated liquid drops flow out from the other end port of the T-shaped pipe and enter a collecting phase E;
10) collecting the polymer droplets generated in the microfluidic device in the collecting phase E, stirring to volatilize the dichloromethane solvent, and solidifying the polymer droplets to form solid polyhydroxyalkanoate microspheres;
11) washing the obtained microspheres with ultrapure water for 3 times, placing the microspheres at-80 ℃ for pre-freezing, and then freeze-drying the microspheres in a freeze dryer at-50 to-60 ℃ for 18 hours;
12) sodium hydroxide solution H: dissolving sodium hydroxide in purified water to prepare a sodium hydroxide solution with the concentration of 1M;
13) collecting the freeze-dried microspheres, and treating the surfaces of the microspheres with a sodium hydroxide solution H for 30min, 60min and 90 min;
14) washing the treated microspheres with ultrapure water for 3 times, placing the microspheres at-80 ℃ for pre-freezing, and then freeze-drying the microspheres in a freeze dryer at-50 to-60 ℃ for 18 hours;
surface SEM characterization of the P3HB3HV3HHx microspheres obtained after lyophilization in step (14) is shown in fig. 8, 9, and 10.
FIG. 8 shows porous microspheres treated with 1M NaOH solution for 30min, where the scale on graph A is 200 μ M and the scale on graph B is 100 μ M. As can be seen from the graph A, the particle size of the microspheres after 30min treatment is 200-300 μm, the surface has a large number of pores, the pore size is about 1-10 μm, and no obvious change is observed compared with that before treatment. As can be seen from FIG. B, the surface of the partially treated microspheres was dissolved and irregular shapes appeared.
FIG. 9 shows porous microspheres treated with 1M NaOH solution for 60min, where the scale of graph A and graph B are both 100 μ M. The figure shows that the particle size of the microsphere after 60min treatment is 200-300 μm, the surface further has irregular shapes and spongy appearances, a large number of holes are distributed, the most of the hole diameters are 1-10 μm, and the partial hole diameters can reach about 15-20 μm.
FIG. 10 shows porous microspheres treated with 1M NaOH solution for 90min, where the scales of A and B are 100 μ M. As can be seen from the figure, the particle size of the microspheres after 90min treatment is 200-300 μm, the surface is further dissolved, a flocculent morphology appears, the surface pore size is 1-10 μm, part of the pore sizes can reach about 15-20 μm, and part of the microspheres can not maintain a regular spherical structure.
Example 4
In this example, the polyhydroxyalkanoate microspheres are prepared by the following method, specifically including the following steps:
1) polymer solution a: adding 0.06g P3HB3HV3HHx solid into 20ml dichloromethane, stirring and dissolving to prepare colorless transparent polymer organic solution;
2) pore-forming agent solution B: 1.5g of ammonium bicarbonate solid is added into 10ml of water to be dissolved to prepare a pore-foaming agent aqueous solution;
3) dispersed phase C: taking 7.5ml of the prepared polymer solution A, adding 2.5ml of the prepared pore-foaming agent solution B, and pre-emulsifying in an ice bath by using an ultrasonic crusher to obtain a dispersed phase emulsification system;
4) continuous phase D: adding 2g of polyvinyl alcohol solid into 400ml of water, stirring and dissolving to prepare a solution which is used as a continuous phase for preparing microspheres by a micro-fluidic control method;
5) and (3) collecting a phase E: the same continuous phase D;
6) a glass syringe F: 10ml glass syringe (with metal needle);
7) a plastic syringe G: 60ml plastic syringe (scalp needle);
8) the disperse phase C was drawn into a glass syringe F and the continuous phase D was drawn into a plastic syringe G;
9) t-type microfluidic device: connecting a plastic syringe G into one end port of a T-shaped pipe by using a scalp needle, directly connecting a needle head of a glass syringe F into a vertical port (the two-phase flow direction is shown in figure 1) to form a T-shaped microfluidic device for generating liquid drops, wherein the generated liquid drops flow out from the other end port of the T-shaped pipe and enter a collecting phase E;
10) collecting the polymer droplets generated in the microfluidic device in the collecting phase E, stirring to volatilize the dichloromethane solvent, and solidifying the polymer droplets to form solid polyhydroxyalkanoate microspheres;
11) washing the obtained microspheres with ultrapure water for 3 times, placing the microspheres at-80 ℃ for pre-freezing, and then freeze-drying the microspheres in a freeze dryer at-50 to-60 ℃ for 18 hours;
surface SEM representation is carried out on the P3HB3HV3HHx microspheres obtained after freeze-drying in the step (11), the result is shown in figure 2, wherein the scale of the graph A is 50 μm, the scale of the graph B is 100 μm, and as can be seen from figure 11, the particle size of the microspheres prepared by adopting 0.3% polymer concentration and an ultrasonic pre-emulsification method is about 50-150 μm, the microspheres have rich and interconnected network-shaped porous structures, the pore diameter is about 1-5 μm, the larger pores on the surface can reach 5-10 μm, and the maximum can reach about 10 μm.
Example 5
In this example, the polyhydroxyalkanoate microspheres are prepared by the following method, specifically including the following steps:
1) polymer solution a: adding 0.3g P3HB4HB solid into 20ml dichloromethane, stirring and dissolving to prepare a colorless transparent polymer organic solution;
4) continuous phase D: adding 2g of polyvinyl alcohol solid into 400ml of water, stirring and dissolving to prepare a solution which is used as a continuous phase for preparing microspheres by a micro-fluidic control method;
5) and (3) collecting a phase E: the same continuous phase D;
6) a glass syringe F: 10ml glass syringe (with metal needle);
7) a plastic syringe G: 60ml plastic syringe (scalp needle);
8) drawing polymer solution a into glass syringe F and continuous phase D into plastic syringe G;
9) t-type microfluidic device: connecting a plastic syringe G into one end port of a T-shaped pipe by using a scalp needle, directly connecting a needle head of a glass syringe F into a vertical port (the two-phase flow direction is shown in figure 1) to form a T-shaped microfluidic device for generating liquid drops, wherein the generated liquid drops flow out from the other end port of the T-shaped pipe and enter a collecting phase E;
10) collecting the polymer droplets generated in the microfluidic device in the collecting phase E, stirring to volatilize the dichloromethane solvent, and solidifying the polymer droplets to form solid polyhydroxyalkanoate microspheres;
11) washing the obtained microspheres with ultrapure water for 3 times, placing the microspheres at-80 ℃ for pre-freezing, and then freeze-drying the microspheres in a freeze dryer at-50 to-60 ℃ for 18 hours;
surface SEM representation is carried out on the P3HB4HB microsphere obtained after freeze-drying in the step (11), the result is shown in figure 2, wherein the scale of the A icon is 100 μm, the scale of the B icon is 40 μm, and as can be seen from figure 12, the particle size of the P3HB4HB microsphere prepared by the method of the embodiment is 50-100 μm, a certain number of holes are distributed on the surface, and the hole diameter is about 1-5 μm.
Example 6
According to the method described in example 1, except that 0.3g, 0.12g, 0.06g of P3HB3HV3HHx polymer with Mw 25000 is added into 20ml of dichloromethane, and the characteristics of a microscope and a scanning electron microscope show that the particle size of the formed microsphere is reduced from 200-500 μm to 50-100 μm by reducing the concentration of the polymer, which indicates that the method can effectively regulate and control the size of the microsphere in a larger range to adapt to different application requirements.
Comparative example 1
The procedure as described in example 1 was followed, except that 7.5% aqueous solution of gilidine was used as the porogen solution, the collected solidified P3HB3HV3HHx microspheres were washed 3 times with purified water, added to a 40 ℃ water bath and stirred for 2h to remove the gilidine, forming porous microspheres. Scanning electron microscope characterization shows that the polyhydroxy fatty acid ester microspheres adopting the Gelidine aqueous solution as the pore-foaming agent solution have small surface pore diameter and small quantity, and cannot effectively form a porous structure.
Comparative example 2
The method of example 1 was followed except that a 1% aqueous solution of tween 20 was used as the porogen solution. The ultrasonic pre-emulsification method finds that the Tween 20 aqueous solution as a pore-foaming agent solution cannot form a uniform and stable pre-emulsification system with the polymer organic solution. Scanning electron microscope characterization shows that the polyhydroxy fatty acid ester microspheres adopting the Tween 20 aqueous solution as the pore-foaming agent solution have small surface pore diameter and small quantity, and cannot effectively form a porous structure.
Comparative example 3
The process as described in example 1 is followed except that an organic solution with a total concentration of 1.5% is prepared as the dispersed phase using fusidic acid and polyhydroxyalkanoate solids dissolved in dichloromethane in a mass ratio of fusidic acid to polyhydroxyalkanoate of 2:3, 1:1, 3: 2. Scanning electron microscope characterization of the prepared microspheres shows that the fusidic acid cannot be uniformly subjected to phase separation in the polyhydroxyalkanoate, so that a porous structure cannot be obtained by removing the fusidic acid in the polyhydroxyalkanoate skeleton.
Comparative example 4
According to the method described in embodiment 6, except that 7.5ml of the prepared polymer solution a is adopted, 2.5ml of the prepared pore-forming agent solution B is added, and pre-emulsification is performed in an ice bath by using an ultrasonicator, it is found that the system after ultrasonic treatment has an obvious layering phenomenon, a stable and uniform pre-emulsification system cannot be formed, and the polyhydroxyalkanoate microspheres with a uniform porous structure are difficult to obtain.
The applicant states that the present invention is illustrated by the above examples to show the microfluidic preparation method of the porous polymer microspheres of the present invention, the prepared porous polymer microspheres and the application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. A microfluidic preparation method of porous polymer microspheres is characterized by comprising the following steps:
(A) dissolving PHA polymer in organic solvent to prepare polymer organic solution, and dissolving pore-foaming agent in water to prepare pore-foaming agent water solution;
(B) mixing a PHA polymer organic solution with a pore-foaming agent aqueous solution for ultrasonic pre-emulsification to form a water-in-oil pre-emulsification system as a dispersion phase;
(C) dissolving a water-soluble surface active PHA polymer in water to obtain an aqueous solution serving as a continuous phase;
(D) injecting samples by using an injection pump, mixing the two phases in a T-shaped microfluidic device, generating polymer droplets in a T-shaped pipe, and collecting the polymer droplets;
(E) and removing the organic solvent in the PHA polymer liquid drop, solidifying and freeze-drying to obtain the porous polymer microsphere.
2. The method as claimed in claim 1, wherein the PHA polymer in the step (A) is poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate), poly (3-hydroxybutyrate-4-hydroxybutyrate) or poly (3-hydroxybutyrate-co-3-hydroxyvalerate);
preferably, the concentration of the PHA polymer organic solution of step (A) is 0.05-20%;
preferably, the organic solvent in step (a) is any one or a combination of at least two of dichloromethane, chloroform or acetonitrile;
preferably, the porogen of step (a) is ammonium bicarbonate.
3. The method according to claim 1 or 2, wherein the concentration of the aqueous porogen solution in step (A) is 0.1-20%;
preferably, the volume ratio of the PHA polymer organic solution to the porogen aqueous solution during the mixing in the step (B) is 10:1-2: 1;
preferably, the ultrasonic pre-emulsification in the step (B) is pre-emulsification by using an ultrasonic crusher, the emulsification is performed for 90s in one cycle, and the cycle number is 1-5 times.
4. The method according to any one of claims 1 to 3, wherein the water-soluble surface-active polymer of step (C) is polyvinyl alcohol;
preferably, the concentration of the aqueous solution of step (C) is 0.1-0.5%;
preferably, the specific method for mixing the two phases in the T-type microfluidic device in the step (D) is as follows: the continuous phase is firstly introduced into the device from one end port of the T-shaped pipe, and after the whole pipeline is filled with the continuous phase, the dispersed phase is introduced from the vertical port, so that the two phases are mixed in the T-shaped microfluidic device.
5. The production method according to any one of claims 1 to 4, wherein the flow rate ratio of the dispersed phase to the continuous phase when the two phases are mixed in the T-type microfluidic device in the step (D) is 1:3 to 1: 10;
preferably, the flow rate of the dispersed phase is 300. mu.l/min and the flow rate of the continuous phase is 1500. mu.L/min.
6. The production method according to any one of claims 1 to 5, wherein, preferably, the method for removing the organic solvent in the polymer droplets in the step (E) adopts a mode of stirring to volatilize the organic solvent;
preferably, the curing temperature of the step (E) is 4-60 ℃ and the time is 2-18 h;
preferably, the temperature of the freeze-drying in the step (E) is-50 to-60 ℃, and the freeze-drying time is 8 to 18 hours.
7. PHA porous polymer microspheres produced by the production method according to any one of claims 1-5, wherein the particle size of the porous polymer microspheres is 100-500 μm, and the surface pore size is 1-10 μm.
8. A method for surface optimizing PHA cellular polymer microspheres according to claim 7, wherein the method comprises: carrying out surface treatment on the porous microspheres by using a sodium hydroxide-acetonitrile mixed solution;
preferably, the sodium hydroxide-acetonitrile mixed solution is a mixed solution obtained by mixing a sodium hydroxide aqueous solution with the concentration of 0.25M and acetonitrile according to the volume ratio of 3: 7;
preferably, the surface treatment time is 2-16min, and preferably, the treatment time is 8-16 min.
9. A porous scaffold material, comprising PHA porous polymer microspheres according to claim 7.
10. A cell-loaded porous scaffold material, comprising porous polymeric microspheres according to claim 7, and cells loaded on said porous polymeric microspheres.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110572734.7A CN113248772A (en) | 2021-05-25 | 2021-05-25 | Microfluidic preparation method of porous polyhydroxyalkanoate polymer microspheres, porous polymer microspheres prepared by microfluidic preparation method and application of porous polyhydroxyalkanoate polymer microspheres |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110572734.7A CN113248772A (en) | 2021-05-25 | 2021-05-25 | Microfluidic preparation method of porous polyhydroxyalkanoate polymer microspheres, porous polymer microspheres prepared by microfluidic preparation method and application of porous polyhydroxyalkanoate polymer microspheres |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113248772A true CN113248772A (en) | 2021-08-13 |
Family
ID=77184480
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110572734.7A Pending CN113248772A (en) | 2021-05-25 | 2021-05-25 | Microfluidic preparation method of porous polyhydroxyalkanoate polymer microspheres, porous polymer microspheres prepared by microfluidic preparation method and application of porous polyhydroxyalkanoate polymer microspheres |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113248772A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114010842A (en) * | 2021-10-28 | 2022-02-08 | 国科温州研究院(温州生物材料与工程研究所) | Microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate and preparation method thereof |
| CN114767929A (en) * | 2022-03-14 | 2022-07-22 | 上海理工大学 | Communicated porous PHBHHx bone filling microsphere material and preparation method thereof |
| CN114832743A (en) * | 2022-05-18 | 2022-08-02 | 西安交通大学 | Microfluidic preparation method of porous structure micron-sized polylactic acid microspheres |
| CN115920126A (en) * | 2022-11-30 | 2023-04-07 | 广州远想医学生物技术有限公司 | Plant exosome-loaded polyhydroxyalkanoate microspheres and preparation method thereof |
| WO2023061202A1 (en) * | 2021-10-13 | 2023-04-20 | 上海蓝晶生物科技有限公司 | Polyhydroxyalkanoate microspheres with narrow particle size distribution and preparation method therefor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101249077A (en) * | 2008-04-14 | 2008-08-27 | 西南交通大学 | Preparation of degradable pollutant polyalcohol stephanoporate microballoons and uses thereof |
| CN101279232A (en) * | 2008-01-11 | 2008-10-08 | 东南大学 | Preparation of microballoons based on microfluid |
| CN102211008A (en) * | 2011-03-23 | 2011-10-12 | 浙江大学 | Detachable T-shaped microchannel device and method for preparing monodisperse polymer microspheres by same |
| CN103374141A (en) * | 2012-04-25 | 2013-10-30 | 中国科学院大连化学物理研究所 | Method for preparing faveolate polymer microsphere on basis of micro-fluidic chip |
-
2021
- 2021-05-25 CN CN202110572734.7A patent/CN113248772A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101279232A (en) * | 2008-01-11 | 2008-10-08 | 东南大学 | Preparation of microballoons based on microfluid |
| CN101249077A (en) * | 2008-04-14 | 2008-08-27 | 西南交通大学 | Preparation of degradable pollutant polyalcohol stephanoporate microballoons and uses thereof |
| CN102211008A (en) * | 2011-03-23 | 2011-10-12 | 浙江大学 | Detachable T-shaped microchannel device and method for preparing monodisperse polymer microspheres by same |
| CN103374141A (en) * | 2012-04-25 | 2013-10-30 | 中国科学院大连化学物理研究所 | Method for preparing faveolate polymer microsphere on basis of micro-fluidic chip |
Non-Patent Citations (8)
| Title |
|---|
| OMAR QUTACHI ET AL.: "《Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature》", 《ACTA BIOMATERIALIA》 * |
| 孙立国等: "微流控装置制备聚合物微珠的研究", 《黑龙江大学工程学报》 * |
| 方国: "聚羟基脂肪酸酯三维多孔微球支架作为细胞载体在组织工程的应用", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 林月娟等: "聚羟基脂肪酸酯多孔微球的制备及扫描电镜观察", 《中国体视学与图像分析》 * |
| 罗婷婷等: "微流控技术制备吡柔比星聚乳酸微球及其体外释放研究", 《材料导报》 * |
| 赵广荣等: "《现代生命科学与生物技术》", 31 October 2008, 天津大学出版社 * |
| 邹文俊: "《有机磨具制造》", 30 September 2001, 中国标准出版社 * |
| 顾其胜: "《海藻酸盐基生物医用材料与临床医学》", 30 April 2015, 上海科学技术出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023061202A1 (en) * | 2021-10-13 | 2023-04-20 | 上海蓝晶生物科技有限公司 | Polyhydroxyalkanoate microspheres with narrow particle size distribution and preparation method therefor |
| CN114010842A (en) * | 2021-10-28 | 2022-02-08 | 国科温州研究院(温州生物材料与工程研究所) | Microfluidic 3D printing bionic skin stent based on polyhydroxyalkanoate and preparation method thereof |
| CN114767929A (en) * | 2022-03-14 | 2022-07-22 | 上海理工大学 | Communicated porous PHBHHx bone filling microsphere material and preparation method thereof |
| CN114832743A (en) * | 2022-05-18 | 2022-08-02 | 西安交通大学 | Microfluidic preparation method of porous structure micron-sized polylactic acid microspheres |
| CN115920126A (en) * | 2022-11-30 | 2023-04-07 | 广州远想医学生物技术有限公司 | Plant exosome-loaded polyhydroxyalkanoate microspheres and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113248772A (en) | Microfluidic preparation method of porous polyhydroxyalkanoate polymer microspheres, porous polymer microspheres prepared by microfluidic preparation method and application of porous polyhydroxyalkanoate polymer microspheres | |
| Zhang et al. | Nanofiber-based delivery of bioactive agents and stem cells to bone sites | |
| Woods et al. | Materials processing in supercritical carbon dioxide: surfactants, polymers and biomaterials | |
| Kumar Giri et al. | Alginate based hydrogel as a potential biopolymeric carrier for drug delivery and cell delivery systems: present status and applications | |
| Choi et al. | Biodegradable porous beads and their potential applications in regenerative medicine | |
| CN102070895B (en) | Core-shell microcapsule and preparation method thereof | |
| CN102172498B (en) | Three-dimensional porous chitosan/gelatine microsphere, preparation method thereof and application in liver cell culture | |
| JP6573716B2 (en) | Method for producing hollow porous microspheres | |
| Datta et al. | Importance of alginate bioink for 3D bioprinting in tissue engineering and regenerative medicine | |
| CN101249077A (en) | Preparation of degradable pollutant polyalcohol stephanoporate microballoons and uses thereof | |
| CN102319449B (en) | Poly(lactic-co-glycolic acid)-based growth factor gradient release microsphere stent as well as preparation method and application thereof | |
| CN102397585A (en) | Fiber bracket containing growth factors and preparation method thereof | |
| Porta et al. | Biomaterials and supercritical fluid technologies: which perspectives to fabricate artificial extracellular matrix? | |
| CN111704739A (en) | Porous microsphere particles for three-dimensional cell culture and preparation method thereof | |
| CN101134784A (en) | Agarose and hyaluronic acid grafts and preparation method and uses thereof | |
| Lu et al. | Preparation of chitosan microcarriers by high voltage electrostatic field and freeze drying | |
| WO2012099482A2 (en) | Device, method and system for preparing microcapsules | |
| CN112494728A (en) | Microsphere-based stent and preparation method and application thereof | |
| CN113368304B (en) | Method for preparing multifunctional sodium alginate scaffold embedded with drug-loaded microspheres by using in-situ emulsification-based 3D printing technology | |
| EP3468631B1 (en) | Multicellular lay-up process | |
| CN115475284B (en) | Microsphere with three-dimensional porous structure and preparation method thereof | |
| Mucha et al. | Novel technique of polymer composite preparation for bone implants | |
| Marison et al. | Liquid Core Caspules for Applications in Biotechnology | |
| CN111909417A (en) | Preparation method of polylactic acid film with uniform pore on surface | |
| Kulkarni et al. | Cell immobilization strategies for tissue engineering: Recent trends and future perspectives |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210813 |