CN114957730A - High-reactivity hydrogel microsphere and preparation method and application thereof - Google Patents
High-reactivity hydrogel microsphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a high-reactivity hydrogel microsphere and a preparation method and application thereof, wherein the surface of the high-reactivity hydrogel microsphere contains a large number of double bonds, has good reactivity and is convenient for functional modification; the high-reactivity hydrogel microsphere is prepared by taking a polymer monomer and a macromolecular cross-linking agent as main components and taking W/O micro-droplets as a reaction template; the hydrophilic molecular skeleton of the macromolecular crosslinking agent is copolymerized with a hydrophobic component, so that the macromolecular crosslinking agent has amphiphilicity, and the molecular terminal contains double bonds. The hydrogel microsphere with high reactivity has the advantages of simple preparation, good biocompatibility, high reactivity, easy surface modification and the like, and has wide application prospect in the fields of microcarriers, tissue engineering, drug delivery carriers, cell therapy, bioprinting and the like.
Description
Technical Field
The invention relates to a hydrogel microsphere and a preparation method and application thereof, in particular to a high-reactivity hydrogel microsphere and a preparation method and application thereof.
Background
Cell culture and amplification based on microcarriers have the advantages of high cell yield, simple cell monitoring and harvesting process, high economy and the like, and are widely concerned in the field of biomedicine. Natural polymer materials such as gelatin, collagen, chitosan, alginate, agarose, etc. are sequentially used in the preparation of microcarriers. However, such materials have problems of containing animal-derived components, low modulus, susceptibility to degradation, and difficulty in handling. Therefore, researchers design a series of microsphere carriers applied to cell culture based on synthetic macromolecules (such as polystyrene, polylactic acid, polyurethane, polyhydroxyethyl methacrylate, polyacrylamide and the like), and the carriers have the advantages of easiness in preparation, good biocompatibility, excellent mechanical property and the like. However, there are specific disadvantages:
(1) the surface of the microsphere lacks cell adhesion growth sites, so that cells are difficult to adhere and grow;
(2) the surface of the microsphere is difficult to chemically modify, which relates to a lengthy process flow and has overhigh cost;
(3) toxicity of residual chemical agents during preparation or modification.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a self-assembled hydrogel microsphere with active functional groups enriched on the surface and high reactivity; another objective of the present invention is to provide a method for preparing hydrogel microspheres with high reactivity, which has a simple process; the invention also aims to provide an application of the hydrogel microspheres with high reactivity in a cell culture microcarrier.
The technical scheme is as follows: the preparation method of the high-reactivity hydrogel microsphere comprises the following steps:
(1) dissolving a polymer monomer and a macromolecular cross-linking agent in deionized water or PBS buffer solution to be used as a water phase;
(2) dispersing the solution in an oil phase containing 1-10% by mass of a surfactant to prepare W/O water-in-oil micro-droplets, wherein the diameter of each droplet is 50-5 mm;
(3) after the reaction is finished, the monodisperse high-reactivity hydrogel microspheres are obtained by purification.
Further, the polymer monomer in step (1) includes at least one of acrylamide, isopropylacrylamide, 2-hydroxyethyl methacrylate, N- [ (3- (dimethylamino) propyl ] methacrylamide, poly (ethylene glycol) methacrylate, poly (ethylene glycol) acrylate, a thiol-modified natural polymer, or a double bond-modified natural polymer.
Further, the macromolecular cross-linking agent in the step (1) is obtained by copolymerizing polyethylene glycol polyvinyl monomers and hydrophobic monomers; the molecular weight of the macromolecular cross-linking agent is 5-500 k Da.
Further, the natural polymer is sodium alginate, hyaluronic acid, gelatin, chitosan or cellulose.
Further, the polyethylene glycol polyvinyl monomer is at least one of poly (ethylene glycol) diacrylate, vinyl-terminated multi-arm polyethylene glycol (three-arm polyethylene glycol, four-arm polyethylene glycol, six-arm polyethylene glycol or eight-arm polyethylene glycol).
Further, the hydrophobic monomer is at least one of poly (propylene glycol) monoacrylate, poly (propylene glycol) diacrylate, poly (propylene glycol) monomethacrylate, poly (propylene glycol) dimethacrylate or 3-acrylamidophenylboronic acid.
Further, the mass fraction of the hydrophobic component is 0% -50%.
Further, in the step (1), the concentration of the polymer monomer is 5-20% m/v, and the concentration of the macromolecular cross-linking agent is 0.5-5% m/v.
Further, the oil phase in the step (2) is at least one of simethicone, fluorine oil, stearic acid, paraffin oil, mineral oil, animal oil or vegetable oil; the W/O water-in-oil micro-droplet is obtained by adopting a micro-fluidic method, a membrane emulsification method or a mechanical stirring method.
Furthermore, the surface of the hydrogel microsphere contains double bonds, the interior of the hydrogel microsphere is a three-dimensional network structure formed by crosslinking a polymer monomer and a macromolecular crosslinking agent, and the particle size of the hydrogel microsphere is 50-5 mm.
The hydrogel microspheres can be applied to cell culture microcarriers.
Furthermore, the microsphere can be directly modified by sulfhydryl functionalized biological adhesion peptide and gelatin to obtain a surface suitable for adhesion and growth of adherent cells.
Further, the small molecule peptide is one or more of arginine-glycine-aspartic acid-cysteine, mercaptopropionic acid-arginine-glycine-aspartic acid, glycine-arginine-glycine-aspartic acid-serine-proline-cysteine, and the like, or sulfhydryl RGD containing RGD fragments.
The high-reactivity hydrogel microsphere is prepared by using water drops dispersed in an oil phase as a template, and a macromolecular cross-linking agent can be assembled on a water-oil interface, so that the high-reactivity hydrogel microsphere with a large number of active functional groups (C = C) enriched on the surface is obtained; meanwhile, the small molecular biological adhesion peptide has a sulfydryl functional group, so that when the high-reaction-activity hydrogel microsphere is incubated in a small molecular peptide solution, the double bond on the surface of the microsphere can generate Michael addition reaction with the sulfydryl on the small molecular biological adhesion peptide at room temperature in a neutral environment, and the modification of the small molecular peptide on the surface of the high-reaction-activity microsphere is realized, thereby providing sites for cell adhesion and proliferation.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:
(1) high reaction activity. The hydrophilic molecular skeleton of the macromolecular crosslinking agent is copolymerized with the hydrophobic component, so that the macromolecular crosslinking agent is easy to enrich in an oil-water interface, the double bond content on the surface of the hydrogel microsphere is effectively improved, and subsequent functional modification is facilitated;
(2) the microsphere post-modification conditions are mild and efficient. The high-reactivity hydrogel microspheres obtained by the invention can directly perform Michael addition reaction with sulfhydrylated bioadhesive peptides under physiological conditions, so that cell attachment growth sites are introduced, and the use of organic solvents or toxic reagents is not involved;
(3) good biocompatibility. The high-reactivity hydrogel microsphere provided by the invention is prepared from fully-synthesized high polymers, has definite components, is safe and non-toxic, and is suitable for application of cell culture microcarriers, tissue engineering, drug delivery carriers, biological printing ink and the like.
Drawings
FIG. 1 is a flow diagram of a chemical preparation of a microfluidic highly reactive hydrogel;
FIG. 2 is a nuclear magnetic hydrogen spectrum of sodium thiolated alginate;
FIG. 3 is a nuclear magnetic hydrogen spectrum of hyperbranched polyethylene glycol diacrylate;
FIG. 4 is a nuclear magnetic hydrogen spectrum of phenylboronic acid modified hyperbranched polypropylene glycol diacrylate;
FIG. 5 is a fluorescence map and a fluorescence intensity map of the reaction of microspheres containing no hydrophobic component (A) and containing a hydrophobic component (B) and thiolated rhodamine prepared based on the W/O template method in example 5;
FIG. 6 is a graph of the cytotoxicity test data of co-incubation of hMSC with the high reactivity hydrogel microspheres of modified bioadhesive peptide of example 3;
FIG. 7 is a diagram showing the co-culture of the non-modified bioadhesive peptide-modified highly reactive hydrogel microspheres (left) and the modified bioadhesive peptide-modified highly reactive hydrogel microspheres with L-929 in example 1 (right);
FIG. 8 is a schematic diagram of co-culture of the RGD-modified high-reactivity hydrogel microcarrier and L-929 and a live-dead staining pattern in example 1.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
(1) Preparation of hydrogel microspheres
Dissolving a surfactant (FE-surf) in fluorine oil to prepare a continuous phase A1 (oil phase) with the mass fraction of 2%; dissolving 20mg of sodium sulfhydrylated alginate (SH-SA) and 3mg of TCEP in 1mL of deionized water to prepare 2% (w/v) which is marked as a disperse phase B1; 100mg of hyperbranched polyethylene glycol diacrylate (HB-PEGDA) was dissolved in deionized water to prepare 10% (w/v), which was designated as dispersed phase B2. Connecting the prepared solution with a microfluidic chip through a pipeline; and adjusting the flow rate of the oil phase A1 to be 16 mu L/min, the flow rate of the water phase B1 to be 1 mu L/min and the flow rate of the water phase B2 to be 1 mu L/min, preparing monodisperse hydrogel micro-droplets, and washing out the micro-droplets after 20min solidification, wherein the particle size distribution is 140-160 mu m.
(2) Preparation of bioadhesive peptide modified hydrogel microcarrier
Dissolving 500 mu G of tetrapeptide (R-G-D-C) containing RGD fragments in 1ml PBS to obtain RGD aqueous solution, dissolving 1G of the hydrogel microspheres in the RGD aqueous solution, adjusting the pH value to 7 by using 1mol/L NaOH solution, and incubating at normal temperature for 15 min; centrifuging to obtain the RGD modified hydrogel microcarrier.
(3) Co-culture of L-929 with hydrogel microcarriers
6 cell suspensions of 100. mu.L were prepared in a 96-well plate, about 5000L-929 cells per well, 1000 hydrogel microspheres not modified with RGD were added to 3 of the wells and RGD-modified hydrogel microspheres were added to the other 3 of the wells, and the plates were cultured in an incubator for 24 hours (at 37 ℃ C., 5% CO) 2 Under the conditions of (a).
Example 2
(1) Preparation of hydrogel microspheres
Dissolving a surfactant (FE-surf) in fluorine oil to prepare a continuous phase A1 (oil phase) with the mass fraction of 2%; dissolving 20mg of thiolated hyaluronic acid (SH-HA) and 3mg of TCEP in 1mL of deionized water to prepare 2% (w/v) which is marked as a disperse phase B1; 100mg HB-PEGDA was dissolved in deionized water to make up 10% (w/v) as dispersed phase B2. Connecting the prepared solution with a microfluidic chip through a pipeline; and adjusting the flow rate of the oil phase A1 to be 24 mu L/min, the flow rate of the water phase B1 to be 2 mu L/min and the flow rate of the water phase B2 to be 2 mu L/min, preparing monodisperse hydrogel micro-droplets, and washing out the microspheres after 20min solidification, wherein the particle size distribution is 140-170 mu m.
(2) Preparation of RGD-modified hydrogel microcarrier
Dissolving 500 mu G of heptapeptide (G-R-G-D-S-P-C) containing RGD fragment in 1ml PBS to obtain RGD aqueous solution, dissolving 1G of the hydrogel microspheres in the RGD aqueous solution, adjusting the pH value to 7 by using 1mol/L NaOH solution, and incubating for 15min at normal temperature; centrifuging to obtain the RGD modified hydrogel microcarrier.
(3) Co-culture of HEK-293T and hydrogel microcarrier
100 μ L of cell suspension was prepared in a 96-well plate, about 5000 HEK-293T cells per well, and 1000 RGD-modified hydrogel microcarriers were added to the well, and the plates were cultured in an incubator for 24 hours (at 37 ℃, 5% CO) 2 Under the conditions of (a).
Example 3
(1) Preparation of hydrogel microspheres
Dissolving a surfactant (FE-surf) in the fluorine oil to prepare a continuous phase A1 (oil phase) with the mass fraction of 2.5%; dissolving 20mg of sodium thiolated alginate and 3mg of TCEP in 1mL of deionized water to prepare 2% (w/v) which is marked as a dispersion phase B1; 100mg HB-PEGDA was dissolved in deionized water to make up 10% (w/v) as dispersed phase B2. Connecting the prepared solution with a microfluidic chip through a pipeline; and adjusting the flow rate of the oil phase A1 to be 16 mu L/min, the flow rate of the water phase B1 to be 1 mu L/min and the flow rate of the water phase B2 to be 1 mu L/min, preparing monodisperse hydrogel micro-droplets, and washing out the microspheres after 20min solidification, wherein the particle size distribution is 120-140 mu m.
(2) Preparation of RGD-modified hydrogel microcarrier
Dissolving 500 mu G of heptapeptide (G-R-G-D-S-P-C) containing RGD fragment in 1ml PBS to obtain RGD aqueous solution, dissolving 1G of the hydrogel microspheres in the RGD aqueous solution, adjusting the pH value to 7 by using 1mol/L NaOH solution, and incubating for 15min at normal temperature; centrifuging to obtain the RGD modified hydrogel microcarrier.
(3) Co-culture of hMSC and hydrogel microcarrier
100 μ L of cell suspension was prepared in a 96-well plate, about 5000 hMSC cells per well, 1000 RGD-modified hydrogel microcarriers were added to the well, and the plates were cultured in an incubator for 24 hours (at 37 ℃, 5% CO) 2 Under the conditions of (a).
Example 4
(1) Preparation of hydrogel microspheres
Dissolving a surfactant (FE-surf) in the fluorine oil to prepare a continuous phase A1 (oil phase) with the mass fraction of 2.5%; dissolving 20mg of thiolated hyaluronic acid (SH-HA) and 3mg of TCEP in 1mL of deionized water to prepare 2% (w/v) which is marked as a disperse phase B1; 100mg HB-PEGDA was dissolved in deionized water to make up 10% (w/v) as dispersed phase B2. Connecting the prepared solution with a microfluidic chip through a pipeline; and adjusting the flow rate of the oil phase A1 to 36 mu L/min, the flow rate of the water phase B1 to 4 mu L/min and the flow rate of the water phase B2 to 4 mu L/min, preparing monodisperse hydrogel micro-droplets, and washing out the microspheres after 20min solidification, wherein the particle size distribution is 130-160 mu m.
(2) Preparation of hydrogel microcarrier modified by thiolated gelatin (Gel-SH)
Dissolving 2mg of Gel-SH in 1ml PBS to obtain a Gel-SH aqueous solution, dissolving 1g of the hydrogel microspheres in the Gel-SH aqueous solution, adjusting the pH value to 7 by using 1mol/L NaOH solution, and incubating for 15min at normal temperature; centrifuging to obtain the Gel-SH modified hydrogel microcarrier.
(3) Co-culture of Hacat and hydrogel microcarrier
100 μ L of cell suspension was prepared in a 96-well plate, about 5000 Hacat cells per well, and 1000 Gel-SH modified hydrogel microcarriers were added to the well, and the plates were cultured in an incubator for 24 hours (at 37 ℃, 5% CO) 2 Under the conditions of (a).
Example 5
(1) Preparation of hydrogel microspheres
Hydrogel microspheres (denoted a) without hydrophobic component: dissolving a surfactant (FE-surf) in the fluorine oil to prepare a continuous phase A1 (oil phase) with the mass fraction of 2.5%; dissolving 20mg of sodium thiolated alginate and 3mg of TCEP in 1mL of deionized water to prepare 2% (w/v) which is marked as a dispersion phase B1; 100mg HB-PEGDA was dissolved in deionized water to make up 10% (w/v) as dispersed phase B2. Connecting the prepared solution with a microfluidic chip through a pipeline; and adjusting the flow rate of the oil phase A1 to be 24 mu L/min, the flow rate of the water phase B1 to be 2 mu L/min and the flow rate of the water phase B2 to be 2 mu L/min, preparing monodisperse hydrogel micro-droplets, and washing out the microspheres after 20min solidification, wherein the particle size distribution is 140-160 mu m.
Preparation of hydrogel containing hydrophobic component (noted as B): dissolving a surfactant (FE-surf) in the fluorine oil to prepare a continuous phase A1 (oil phase) with the mass fraction of 5%; dissolving 20mg of sodium thiolated alginate and 3mg of TCEP in 1mL of deionized water to prepare 2% (w/v) which is marked as a dispersion phase B1; 100mg of phenylboronic acid modified hyperbranched polypropylene glycol diacrylate (HB-PEGDA-APBA) is dissolved in deionized water to prepare 10% (w/v), which is marked as dispersed phase B2. Connecting the prepared solution with a microfluidic chip through a pipeline; and adjusting the flow rate of the oil phase A1 to 24 mu L/min, the flow rate of the water phase B1 to 2 mu L/min and the flow rate of the water phase B2 to 2 mu L/min, preparing monodisperse hydrogel micro-droplets, and washing out the micro-droplets after 20min solidification, wherein the particle size distribution is 140-160 mu m.
(2) Preparation of SH-PEG-Rhodamine aqueous solution
2mg of SH-PEG-Rhodamine was dissolved in 1mL of PBS and the pH was adjusted to 7.4 (three parallel aqueous solutions a were prepared) 1 、a 2 ) 100mg of each of the three hydrogels (A, B) was placed in an aqueous solution a 1 、a 2 And incubating for 20 min. After the incubation, excess SH-PEG-Rhodamine solution was washed away with PBS and observed with a fluorescence microscope.
FIG. 2 is a nuclear magnetic hydrogen spectrum of sodium hydrosulphonate, which proves the successful modification of sulfydryl on the structure of sodium alginate.
FIG. 3 is a nuclear magnetic hydrogen spectrum of hyperbranched polyethylene glycol diacrylate, which shows that the hyperbranched polyethylene glycol diacrylate has carbon-carbon double bonds.
FIG. 4 is nuclear magnetic hydrogen spectrum of phenylboronic acid modified hyperbranched polypropylene glycol diacrylate, which proves the successful modification of the hydrophobic component phenylboronic acid.
FIG. 5 is a fluorescence map and fluorescence intensity map (9.5 for A and 39.8 for B) of microspheres containing no hydrophobic component (A) and hydrophobic component (B) prepared by example 5 based on the W/O template method, which react with thiolated rhodamine, and shows that the hydrogel microspheres containing the hydrophobic component have more double bonds on the surface and higher activity than the microspheres containing no hydrophobic component.
FIG. 6 is a graph of data of cytotoxicity experiments in which hMSC of example 3 was co-incubated with high-reactivity hydrogel microspheres modified with bioadhesive peptides, and it can be seen that 1000 microspheres were not toxic to cells, and the cell activity decreased with the increase of the number of microspheres, but still exhibited more than 75% of cell activity, where the decrease of cell activity was more than the increase of the number of microspheres, which inhibited the expansion of cells. Therefore, the high-reactivity hydrogel microsphere is low in toxicity and good in biocompatibility.
FIG. 7 is a co-culture of the unmodified bioadhesive peptide-modified hydrogel microspheres and the modified bioadhesive peptide-modified hydrogel microspheres with L-929 in example 1, demonstrating that the modified bioadhesive peptide microspheres are more favorable for cell adhesion.
Fig. 8 is a schematic diagram of co-culture and a live-dead staining diagram of the RGD-modified hydrogel microspheres and L-929 in example 1, and it can be seen from the diagrams that cells are obviously adhered to the microcarriers, and almost all the cells adhered to the microcarriers are green (living cells), which proves that the bioadhesive peptide-modified hydrogel microspheres have low toxicity and good biocompatibility, and are beneficial to adhesion and proliferation of cells.
Claims (10)
1. A preparation method of hydrogel microspheres with high reactivity is characterized by comprising the following steps: the method comprises the following steps:
(1) dissolving a polymer monomer and a macromolecular cross-linking agent in deionized water or PBS buffer solution to be used as a water phase;
(2) dispersing the solution in an oil phase containing 1-10% by mass of a surfactant to prepare W/O water-in-oil micro-droplets, wherein the diameter of each droplet is 50-5 mm;
(3) after the reaction is finished, the monodisperse high-reactivity hydrogel microspheres are obtained by purification.
2. The high-reactivity hydrogel microsphere and the preparation method thereof according to claim 1, wherein the high-reactivity hydrogel microsphere comprises: the polymer monomer in the step (1) comprises at least one of acrylamide, isopropyl acrylamide, 2-hydroxyethyl methacrylate, N- [ (3- (dimethylamino) propyl ] methacrylamide, poly (ethylene glycol) methacrylate, poly (ethylene glycol) acrylate, sulfydryl modified natural polymer or double bond modified natural polymer.
3. The high-reactivity hydrogel microsphere and the preparation method thereof according to claim 1, wherein the high-reactivity hydrogel microsphere comprises: the macromolecular cross-linking agent in the step (1) is obtained by copolymerizing polyethylene glycol polyvinyl monomers and hydrophobic monomers; the molecular weight of the macromolecular cross-linking agent is 5-500 k Da.
4. The high-reactivity hydrogel microsphere and the preparation method thereof according to claim 2, wherein the high-reactivity hydrogel microsphere comprises: the natural polymer is sodium alginate, hyaluronic acid, gelatin, chitosan or cellulose.
5. The high-reactivity hydrogel microsphere and the preparation method thereof according to claim 3, wherein the high-reactivity hydrogel microsphere comprises: the polyethylene glycol polyvinyl monomer is at least one of poly (ethylene glycol) diacrylate and vinyl-terminated multi-arm polyethylene glycol (three-arm polyethylene glycol, four-arm polyethylene glycol, six-arm polyethylene glycol or eight-arm polyethylene glycol).
6. The high-reactivity hydrogel microsphere and the preparation method thereof according to claim 3, wherein the high-reactivity hydrogel microsphere comprises: the hydrophobic monomer is at least one of poly (propylene glycol) monoacrylate, poly (propylene glycol) diacrylate, poly (propylene glycol) monomethacrylate, poly (propylene glycol) dimethacrylate or 3-acrylamidophenylboronic acid.
7. The high-reactivity hydrogel microsphere and the preparation method thereof according to claim 1, wherein the high-reactivity hydrogel microsphere comprises: in the step (1), the concentration of the polymer monomer is 5-20% m/v, and the concentration of the macromolecular cross-linking agent is 0.5-5% m/v.
8. The high-reactivity hydrogel microsphere and the preparation method thereof according to claim 1, wherein the high-reactivity hydrogel microsphere comprises: in the step (2), the oil phase is at least one of simethicone, fluorine oil, stearic acid, paraffin oil, mineral oil, animal oil or vegetable oil; the W/O water-in-oil micro-droplet is obtained by adopting a micro-fluidic method, a membrane emulsification method or a mechanical stirring method.
9. A hydrogel microsphere produced by the production method according to any one of claims 1 to 8, wherein: the hydrogel microsphere has double bonds on the surface, a three-dimensional network structure formed by crosslinking a polymer monomer and a macromolecular crosslinking agent is arranged inside the hydrogel microsphere, and the particle size is 50 micrometers-5 mm.
10. Use of the hydrogel microspheres of claim 9 in a cell culture microcarrier.
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CN115558133A (en) * | 2022-10-20 | 2023-01-03 | 南京工业大学 | Temperature response type particle gel and preparation method thereof |
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CN115505160B (en) * | 2022-09-01 | 2024-07-09 | 南京工业大学 | Preparation method of hydrogel microsphere carrier, product obtained by preparation method and application of hydrogel microsphere carrier |
CN115558133A (en) * | 2022-10-20 | 2023-01-03 | 南京工业大学 | Temperature response type particle gel and preparation method thereof |
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