CN116120619A - Super macroporous hydrogel and preparation method and application thereof - Google Patents
Super macroporous hydrogel and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 59
- 239000000661 sodium alginate Substances 0.000 claims abstract description 59
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- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 229910000406 trisodium phosphate Inorganic materials 0.000 description 1
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Abstract
The invention discloses a superporous hydrogel and a preparation method and application thereof. The superporous hydrogel is formed by crosslinking sodium alginate and polyvinyl alcohol under the condition that calcium chloride is used as a crosslinking agent. The invention obtains the ultra-large pore sodium alginate hydrogel with high porosity (85 percent) and large pore diameter (100-900 microns) through a simple and rapid preparation method, and simultaneously obtains excellent mechanical properties. The superporous hydrogel can be used for cell culture, and is expected to be applied to the fields of tissue engineering, drug delivery, organoid construction and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a superporous hydrogel and a preparation method and application thereof.
Background
Superporous hydrogels, which are defined as hydrogels with internal pore sizes exceeding 1 μm, are becoming popular for more and more researchers as a novel biomedical material due to their high similarity in structure and composition to many human tissue organs and the like, especially in the fields of tissue regeneration, drug delivery, organoid construction, and the like. In the field of tissue regeneration, by loading cells, medicines or cytokines and the like, the macroporous hydrogel has the characteristics of being highly similar to human tissue and organs in structure and physical characteristics, and can provide mechanical support and biochemical signals for the cells, induce the cells to repair damaged tissues better and reconstruct the structure and functions of the tissues. In the field of drug delivery, the macroporous hydrogel can precisely act on an affected part in a patch form, and the oversized pore diameter is favorable for quick release of cells or drug molecules on the affected part, so that an efficient treatment effect is achieved. In the organoid field, the ultra-large pore structure and the bionic physical property provide a proper environment for proliferation and differentiation of cells, and are ideal scaffold materials for organoid construction.
The methods commonly used for preparing the superporous hydrogels at present are a freeze-drying method, a template method and a pore-forming agent method, however, the superporous hydrogels prepared by the methods have two main disadvantages, namely the complicated preparation process is required, and the excessive preparation process is not beneficial to commercial mass production. In addition, the mechanical property is poor, the ultra-macroporous structure belongs to structural defects in material mechanics, and the ultra-macroporous structure is easier to cause the damage of materials under the action of external force. Therefore, the preparation method of the superporous hydrogel is improved, and the mechanical property of the superporous hydrogel is improved, so that the superporous hydrogel is beneficial to the future wide application in the field of biological medicine.
The invention with publication number CN112725327A discloses a preparation method of a cell immobilization carrier, which comprises the following steps: uniformly mixing a sodium alginate solution and polyvinyl alcohol to obtain a carrier solution; wherein the mass ratio of the sodium alginate to the polyvinyl alcohol is 1:2-10; adding diatomite into the carrier solution, uniformly mixing, gradually extruding the gel mixed solution dropwise into a crosslinking solution in a continuous stirring state for solidification to form coagulated beads, washing the coagulated beads, freezing and crosslinking at the temperature of-25 to-15 ℃, and thawing to obtain the cell immobilization carrier. The sodium alginate solution and the polyvinyl alcohol can be crosslinked into gel under the action of a crosslinking agent.
The invention provides a preparation method of a drug and protein slow-release alginate hybrid gel, which is disclosed in the publication number CN 103040727A. The preparation method comprises the following steps: firstly preparing calcium alginate hydrogel, then soaking the calcium alginate hydrogel into inorganic salt solutions of trisodium phosphate, diammonium hydrogen phosphate, sodium silicate, sodium carbonate, sodium oxalate and the like with certain concentration to swell for a certain time, and then soaking the swelled calcium alginate hydrogel into a medicine and protein water solution containing calcium ions with certain concentration to crosslink for a certain time to obtain the medicine and protein slow-release alginate hybrid gel.
However, there is no report in the prior art of using sodium alginate and polyvinyl alcohol to prepare superporous hydrogels.
Disclosure of Invention
In order to solve the problems in the background art, the invention provides a simple method for preparing the superporous hydrogel by using the polyvinyl alcohol.
A method for preparing superporous hydrogel, comprising the following steps:
(1) Preparing sodium alginate solution, polyvinyl alcohol solution and calcium chloride solution respectively;
(2) Mixing sodium alginate solution and polyvinyl alcohol solution to obtain polyvinyl alcohol/sodium alginate mixed solution;
(3) Adding a cross-linking agent calcium chloride solution into the polyvinyl alcohol/sodium alginate mixed solution, cross-linking to obtain the polyvinyl alcohol and sodium alginate composite super-macroporous hydrogel,
wherein the concentration of the sodium alginate solution is 4wt%, the concentration of the polyvinyl alcohol solution is 10wt%, and the sodium alginate solution and the polyvinyl alcohol solution are mixed according to the volume ratio of 1:1.
Preferably, in the step (1), the concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol/sodium alginate mixed solution is 3:2.
Preferably, in the step (3), bubbles in the mixed solution of polyvinyl alcohol and sodium alginate are removed before the calcium chloride solution is added.
Preferably, in step (3), the crosslinking process is left to stand for at least 12 hours.
Preferably, in the step (3), the resulting superporous hydrogel is immersed in ultrapure water to remove free calcium ions after the completion of the crosslinking.
Preferably, the pore size distribution in the superporous hydrogel is 100-900 μm.
The invention also provides the superporous hydrogel prepared by the preparation method.
The invention also provides application of the superporous hydrogel in cell culture, wherein the superporous hydrogel is soaked in a culture medium when in application, and then cells are injected into holes in the superporous hydrogel for cell culture.
The invention also provides a cell culture method, which uses the superporous hydrogel to soak the superporous hydrogel in a culture medium, and then the cells are injected into the pores inside the superporous hydrogel for cell culture.
The invention selects sodium alginate as a framework material of gel, provides necessary mechanical properties for the material, and adds polyvinyl alcohol into the gel to induce phase separation, so as to form a high-density phase formed by aggregation of sodium alginate and a low-density phase formed by polyvinyl alcohol. The high-density phase formed by closely gathering the crosslinked sodium alginate is a framework structure, and the low-density phase formed by the free polyvinyl alcohol and containing a large amount of water is a hole structure.
The invention obtains the ultra-large pore sodium alginate hydrogel with high porosity (85 percent) and large pore diameter (100-900 microns) through a simple and rapid preparation method, and simultaneously obtains excellent mechanical properties. The superporous hydrogel can be used for cell culture, and is expected to be applied to the fields of tissue engineering, drug delivery, organoid construction and the like.
When the superporous hydrogel is prepared, the concentration and the proportion of the specific sodium alginate and the specific polyvinyl alcohol are required, otherwise, the pore structure with large pore diameter inside the hydrogel cannot be effectively obtained during crosslinking.
Drawings
FIG. 1 is a flow chart of the preparation of superporous hydrogels in example 1 and example 2.
FIG. 2 is a cross-sectional view of the superporous hydrogel in example 1 and the structure of the superporous structure observed under a microscope.
FIG. 3 shows the hydrogel prepared by the different polyvinyl alcohol and sodium alginate ratios in example 1 and the mechanism observed under a microscope.
Fig. 4 shows a pore size distribution obtained by statistically analyzing the microscopic image in example 1.
FIG. 5 is the results of the compression performance test performed on the superporous hydrogels in example 2.
FIG. 6 shows the growth of cells in the superporous hydrogels under the microscope in example 3, on a scale of 200. Mu.m.
Detailed Description
Example 1
(1) Preparing sodium Alginate (Alginate) solution: 8g of sodium alginate powder was dissolved in 192g of ultrapure water at room temperature to obtain a sodium alginate solution of 4 wt%.
(2) Preparing a polyvinyl alcohol (PVA) solution: 20g of PVA powder was dissolved in hot water (180 g) at 90℃with continuous stirring to give a 10wt% PVA solution.
(3) Preparing a polyvinyl alcohol/sodium alginate mixed solution: blending the sodium alginate solution and the PVA solution in the steps (1) and (2) according to the volume ratio of 1:1, and continuously stirring at room temperature until the mixture is in a uniform steam drum-free state.
(4) Preparing a control group solution: the sodium alginate and PVA solution in the steps (1) and (2) are respectively blended according to the volume ratio of 5:1 and 1:5 to be used as the proportion of a control group.
(5) Preparing a calcium chloride solution: 11.1g of calcium chloride particles were weighed, dissolved in 200ml of ultrapure water, and treated by ultrasonic treatment to obtain a 0.5M calcium chloride solution.
(6) Preparation of cellular hydrogels in centrifuge tubes: firstly, taking 2ml of the mixed solution in the step (3), adding the mixed solution into a 5ml centrifuge tube, removing bubbles generated in the adding process of the solution through the centrifuge, slowly adding the calcium chloride solution in the step (5) into the centrifuge tube, and filling the residual space in the centrifuge tube with the calcium chloride solution.
(7) Control gels were prepared in centrifuge tubes: taking 2ml of the sodium alginate solution in the step (1) and the control group solution in the step (4), adding the solution into a 5ml centrifuge tube, removing bubbles generated in the adding process of the solution through a centrifuge, slowly adding the calcium chloride solution in the step (5) into the centrifuge tube, and filling the residual space in the centrifuge tube with the calcium chloride solution.
(8) Standing and crosslinking: and (3) standing the centrifuge tube samples obtained in the step (6) and the step (7) for 12 hours at room temperature, and fully crosslinking the calcium chloride solution and the sodium alginate to obtain the polyvinyl alcohol and sodium alginate compounded superporous hydrogel and the control group gel.
(9) Removing free calcium ions: taking out the porous hydrogel and the control group gel in the step (8), soaking in ultrapure water for 24 hours, and changing water once in the middle for 12 hours to obtain a final hydrogel sample. Finally, the gel is obtained as a super-macroporous hydrogel (abbreviated as 1-1) with the mixing ratio of polyvinyl alcohol/sodium alginate of 1:1, and the gel of the control group comprises a composite gel with the mixing ratio of 5:1 (abbreviated as 5-1) and 1:5 (abbreviated as 1-5), and a hydrogel of pure sodium alginate (abbreviated as Alg).
(10) Pore size distribution: firstly, cutting the ultra-large pore gel and the control group gel in the step (9) into slices with the thickness of about 1mm through a sample cutting machine, placing the slices in a central area of a section of an observation sample under an optical microscope, and carrying out statistical analysis on a pore structure in a microscopic photo by utilizing software after photographing.
(11) Porosity (P) test: cutting the ultra-macroporous hydrogel (1-1) in the step (9) into a thickness of 3mm, and then weighing the mass (m) 1 ) Then placing the cut sample on a paper towel, squeezing gel by hand, removing water from the sample, repeatedly squeezing for three times, and weighing the sample mass (m 2 ) The porosity can be calculated by two weighed mass changes:
P=(m 1 -m 2 )/m 1 ×100%
(12) Moisture content test (Q): slicing the superporous hydrogel in the step (9), and weighing to obtain the mass m 1 Then placing in an oven at 80 ℃ for 24 hours, weighing after the sample is completely dried to obtain m 2 The water content of the superporous hydrogel can be calculated through the mass change of the sample before and after drying:
Q=(m 1 -m 2 )/m 1 ×100%。
analysis of results:
as shown in FIG. 1, after polyvinyl alcohol and sodium alginate solution are mixed according to the volume ratio of 1:1, the superporous hydrogel prepared in a centrifuge tube is in a white bullet shape, the middle part of the superporous hydrogel is cut by a sample cutting machine to obtain a wafer sample shown in FIG. 2, the outer layer of the gel is in a white compact structure as seen in a section view, and the inside of the gel is in a loose cavity structure. Accordingly, as shown in FIG. 3, when the volume ratio of polyvinyl alcohol to sodium alginate is 5:1 and 1:5, or when the hydrogel is prepared by using pure sodium alginate solution, the ultra-large pore structure cannot be obtained. When the central area of the superporous hydrogel wafer sample is observed under an optical microscope, the pore diameter of the central area can be found to be distributed in a gradient way within the range of 100-900 microns. The pore size of the superporous hydrogel prepared by mixing polyvinyl alcohol and sodium alginate solution according to the volume ratio of 1:1 is statistically analyzed by using analysis software of an optical microscope, and further, as shown in fig. 4, sample 1 and sample 2 are repeated twice, and it can be seen that the pore size of most macropores is distributed in the range of 100-500 micrometers, the pore size of more than 500 micrometers is intensively distributed in the middle area, and the phenomenon that the pore sizes are distributed from large to small in a gradient manner in the interior is related to the manner that calcium ions penetrate from outside to inside for crosslinking.
In addition, in order to measure the porosity of the superporous hydrogel, a sample cutting machine is used for cutting the superporous hydrogel to a thickness of about 5mm, free water in holes is drained through a repeated extrusion mode, and the porosity of the superporous hydrogel is calculated to be about 85% with the mass change before and after water is drained, so that the superporous hydrogel belongs to a higher level in the reported superporous hydrogel.
Example 2
(1) Preparing sodium Alginate (Alginate) solution: 8g of sodium alginate powder was dissolved in 192g of ultrapure water at room temperature to obtain a sodium alginate solution of 4 wt%.
(2) Preparing a polyvinyl alcohol (PVA) solution: 20g of PVA powder was dissolved in hot water (180 g) at 90℃with continuous stirring to give a 10wt% PVA solution.
(3) Preparing a polyvinyl alcohol/sodium alginate mixed solution: blending the sodium alginate solution and the PVA solution in the steps (1) and (2) according to the volume ratio of 1:1, and continuously stirring at room temperature until the mixture is in a uniform steam drum-free state.
(4) Preparing a calcium chloride solution: 11.1g of calcium chloride particles were weighed, dissolved in 200ml of ultrapure water, and treated by ultrasonic treatment to obtain a 0.5M calcium chloride solution.
(5) Preparation of cellular hydrogels in centrifuge tubes: firstly, taking 2ml of the mixed solution in the step (3), adding the mixed solution into a 5ml centrifuge tube, removing bubbles generated in the adding process of the solution through a centrifuge, slowly adding the calcium chloride solution in the step (4) into the centrifuge tube, and filling the residual space in the centrifuge tube with the calcium chloride solution.
(6) Standing and crosslinking: and (3) standing the centrifuge tube sample obtained in the step (5) for 12 hours at room temperature, and fully crosslinking the calcium chloride solution and sodium alginate to obtain the polyvinyl alcohol and sodium alginate compounded superporous hydrogel.
(7) Removing free calcium ions: taking out the superporous hydrogel in the step (6), soaking in ultrapure water for 24 hours, and changing water once in the middle for 12 hours to obtain a final macroporous hydrogel sample.
Analysis of results:
the superporous hydrogel prepared from the centrifuge tube is cut into cylindrical samples with the height of 9mm and the height of 16mm by a sample cutting machine, the compression performance test is carried out at room temperature, the compression rate is set to 5mm/min, the test result is shown in figure 5 (each of the height detection is repeated and is respectively used as a sample 1 and a sample 2), the breaking stress of the sample with the height of 16mm can reach more than 6MPa, the breaking stress of the sample with the height of 9mm can also reach approximately 1MPa, in addition, the compression modulus of the sample can be calculated by the ratio of the stress at the 1% compression strain, and the compression modulus of the superporous hydrogel is about 0.3MPa as shown in the right graph in figure 5 and is equivalent to the superporous hydrogel reported at present.
Example 3
(1) Preparing sodium Alginate (Alginate) solution: 8g of sodium alginate powder was dissolved in 192g of ultrapure water at room temperature to obtain a sodium alginate solution of 4 wt%.
(2) Preparing a polyvinyl alcohol (PVA) solution: 20g of PVA powder was dissolved in hot water (180 g) at 90℃with continuous stirring to give a 10wt% PVA solution.
(3) Preparing a polyvinyl alcohol/sodium alginate mixed solution: blending the sodium alginate solution and the PVA solution in the steps (1) and (2) according to the volume ratio of 1:1, and continuously stirring at room temperature until the mixture is in a uniform steam drum-free state.
(4) Preparing a calcium chloride solution: 11.1g of calcium chloride particles were weighed, dissolved in 200ml of ultrapure water, and treated by ultrasonic treatment to obtain a 0.5M calcium chloride solution.
(5) Preparation of cellular hydrogels in centrifuge tubes: firstly, taking 2ml of the mixed solution in the step (3), adding the mixed solution into a 5ml centrifuge tube, removing bubbles generated in the adding process of the solution through a centrifuge, slowly adding the calcium chloride solution in the step (4) into the centrifuge tube, and filling the residual space in the centrifuge tube with the calcium chloride solution.
(6) Standing and crosslinking: and (3) standing the centrifuge tube sample obtained in the step (5) for 12 hours at room temperature, and fully crosslinking the calcium chloride solution and sodium alginate to obtain the polyvinyl alcohol and sodium alginate compounded superporous hydrogel.
(7) Removing free calcium ions: taking out the superporous hydrogel in the step (6), soaking in ultrapure water for 24 hours, and changing water once in the middle for 12 hours to obtain a final superporous hydrogel sample.
(8) Preparing a cell complete medium: 10% fetal bovine serum and 1% penicillin-streptomycin solution were added to high-sugar DMEM medium.
(9) KPC mouse pancreatic cancer cell culture: culturing KPC mouse pancreatic cancer cells by using the complete culture medium prepared in the step (8), and placing at 37 ℃ and 5% CO 2 The cells were cultured in an incubator, medium was changed every day, and passaging was performed every 3d using trypsin.
(10) Immersing the gel in a culture medium: cutting the superporous hydrogel sample obtained in the step (7) into a cylinder with the height of 9mm, performing high-pressure sterilization treatment, placing the cylinder into a sterile 48-well plate, then soaking the cylinder into the cell culture medium prepared in the step (8), and soaking the cylinder for 12 hours to reach an equilibrium state.
(11) Cells were cultured in ultra-macroporous gel: digesting the KPC cells cultured in step (9) with trypsin, adding fresh medium, and re-suspending the cells to 2×10 4 Each of the superporous hydrogels was then cultured by injecting 1mL of the cell suspension into the interior of each superporous hydrogel using a pipette. Since the pore structure of the superporous hydrogel is interconnected with the outside, cells inside the injected pore structure can continuously obtain nutrients from the medium. The medium replacement is also consistent with the traditional cell culture method, and no special treatment is needed.
(12) Cell growth was observed: the growth state of the cells was observed under an optical microscope after the cells were cultured in the superporous hydrogel for a certain period of time.
Analysis of results:
as shown in FIG. 6, after KPC cells were cultured in macroporous gel for 24 hours, the cells grown in the superporous hydrogel showed round, semitransparent particles, about 20-30 microns in size, which were much smaller than those grown in the bottom of the pore plate, and after 48 hours of culture, the cells proliferated inside the macroporous gel to form distinct cytoballs, unlike the cells grown in the bottom of the pore plate by general adherence. These results show that the macroporous gel environment can effectively support the growth of KPC cells and induce the formation of cytoballs, and compared with cells cultured in common pore plates, the cells cultured in macroporous gel are more similar in cell state in real tissue organs, and are expected to be applied to the fields of animal cancer model establishment, organoid construction and other tissue engineering.
Claims (9)
1. The preparation method of the superporous hydrogel is characterized by comprising the following steps:
(1) Preparing sodium alginate solution, polyvinyl alcohol solution and calcium chloride solution respectively;
(2) Mixing sodium alginate solution and polyvinyl alcohol solution to obtain polyvinyl alcohol/sodium alginate mixed solution;
(3) Adding a cross-linking agent calcium chloride solution into the polyvinyl alcohol/sodium alginate mixed solution, cross-linking to obtain the polyvinyl alcohol and sodium alginate composite super-macroporous hydrogel,
wherein the concentration of the sodium alginate solution is 4wt%, the concentration of the polyvinyl alcohol solution is 10wt%, and the sodium alginate solution and the polyvinyl alcohol solution are mixed according to the volume ratio of 1: 1.
2. The method for preparing superporous hydrogel according to claim 1, wherein in step (1), the concentration of the calcium chloride solution is 0.5M, and the volume ratio of the calcium chloride solution to the polyvinyl alcohol/sodium alginate mixed solution is 3:2.
3. the method for preparing superporous hydrogel according to claim 1, wherein in step (3), bubbles in the mixed solution of polyvinyl alcohol and sodium alginate are removed before adding the calcium chloride solution.
4. The method for preparing superporous hydrogels as claimed in claim 1, wherein in the step (3), the crosslinking process is standing for at least 12 hours.
5. The method for preparing superporous hydrogels as claimed in claim 1, wherein in the step (3), the obtained superporous hydrogels are immersed in ultrapure water to remove free calcium ions after the crosslinking is completed.
6. The method for preparing superporous hydrogels as claimed in claim 1, wherein the pore size distribution in said superporous hydrogels is 100-900. Mu.rm.
7. The superporous hydrogel made by the method of claim 1-6.
8. The use of the superporous hydrogels of claim 7 in cell culture, wherein said superporous hydrogels are immersed in a culture medium and then cells are injected into the pores inside said superporous hydrogels for cell culture.
9. A cell culture method, characterized in that the superporous hydrogel of claim 7 is used, the superporous hydrogel is immersed in a culture medium, and then cells are injected into the pores inside the superporous hydrogel for cell culture.
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