CN103224679A - Chitosan polyacrylamide hydrogel base material and preparation method thereof - Google Patents
Chitosan polyacrylamide hydrogel base material and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000000017 hydrogel Substances 0.000 title abstract description 11
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims abstract description 23
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Abstract
The invention discloses a chitosan polyacrylamide hydrogel base material and a preparation method thereof. The chitosan polyacrylamide hydrogel base material is composed of an acrylamide solution, a methylene diacrylamide solution, H2O, an ammonium persulfate solution, tetramethyl ethylenediamine and a chitosan solution. The preparation method of the chitosan polyacrylamide hydrogel base material comprises a step of mixing and stirring, a step of transferring to a glass clamping plate for reacting, and a step of immersion. The chitosan polyacrylamide hydrogel base material has a simple technology and allows hydrogels having different hardnesses to be obtained, and the obtained hydrogels have the advantages of non-toxicity, easy adherence growth of cells, reduction of the reaching complexity of the cytomechanics, benefiting for the researches on the mechanical properties of the cells, and reduction of the researching cost.
Description
Technical field
The invention belongs to the hydrogel technical field, specifically, relate to chitosan polyacrylamide hydrophilic gel base material and preparation method thereof.
Background technology
Cell can be experienced the stimulation of the factors such as physics, chemistry and biology that come from the outside as the fundamental unit of organism 26S Proteasome Structure and Function, and response is made in stimulation.The substrate hardness of cell growth is an important mechanics study parameter, also is the focus of current cyto-mechanics area research.Engler etc. studies show that stem cell is very responsive to tissue elasticity, can break up (specialization) and produce corresponding phenotype with the variation of the elasticity indexes in the tissue and to a certain pedigree.The soft matrix that is similar to cerebral tissue has " neural to " inducing function, and the hard slightly matrix of muscle tissue has " flesh to " inducing function, and the hardness of cartilage has " bone to " inducing function.
The investigator carries out the influence of cell culture studies different soft and hard substrate pair cell elasticity and cell physiological with polyacrylamide hydrophilic gel for the variable hardness base material, yet cell need show to serve as a contrast material such as collagen, ln, fibronectin, gelatin, poly-lysine and the polypeptide etc. that are beneficial to the cell attaching at the polyacrylamide hydrophilic gel substrate grown.Tony Yeung etc. has studied the motion of variation such as morphocytology, cytoskeleton and plain expressions of integration and cell on the polyacrylamide hydrophilic gel of the different soft and hard degree of table lining fibronectin or collagen of inoblast, endotheliocyte and neutrophil leucocyte and has tended to.Above-mentioned research adopts the substrate of different table lining material cell growth to show lining according to different research purposes and content usually.The main focus of cyto-mechanics research field is the influence of the change pair cell of mechanics parameter at present, and with these extracellular matrix proteins to substrate surface show the lining after, will consider the influence of the material pair cell showing to serve as a contrast, but so but increased the complicacy of research and can not go to study the influence of this mechanics factor pair cell of substrate hardness separately, as elasticity and the cytoskeleton influence of research collagen protein such as Denitsa Docheva and fibronectin to prostate cancer cell, the cell elasticity that discovery is grown in table former albumen of line with rubber and fibronectin substrate can change, because these extracellular matrix proteins and epicyte protein are integrated plain interaction of molecules, thereby the reorganization and the depolymerization running balance that cause microfilament protein in the cell change, and microfilament protein is to keep the elastic important albumen of cell in the cell.So when with polyacrylamide hydrophilic gel substrate research different soft and hard degree pair cell elasticity effect, the protein molecular of table lining and the soft or hard of substrate all can influence cell elasticity.
And chitosan is subjected to extensive concern with advantageous properties such as its biological functionality, blood compatibility, security and biocidal properties in fields such as medicine, biomedical engineerings.As differing materials and chitosan are made film, allow these two kinds of material original functions obtain coordinating; With the various microcarriers of Preparation of Chitosan; The preparation tissue engineering bracket material.Positively charged the adherent of cell that help of chitosan, we make film and inoculating cell thereon with chitosan, find that the cell attachment time shortens on chitosan film, have the obvious development potentiality.
The shortcoming of prior art: carry out the influence of cell culture studies different soft and hard substrate pair cell elasticity and cell physiological with polyacrylamide hydrophilic gel for the variable hardness base material, yet cell need show to serve as a contrast material such as collagen, ln, fibronectin, gelatin, poly-lysine and the polypeptide etc. that are beneficial to the cell attaching at the polyacrylamide hydrophilic gel substrate grown, influence factor is too much, research is complicated, influence that can not single research substrate hardness pair cell.
Summary of the invention
For solving above technical problem, the object of the present invention is to provide a kind ofly can obtain chitosan different hardness, that add and help cell attachment growth, the complicacy that does not increase research and difficulty and chitosan polyacrylamide hydrophilic gel base material with low cost, that can study the influence of substrate hardness pair cell separately and preparation method thereof.
The present invention seeks to realize like this: a kind of chitosan polyacrylamide hydrophilic gel base material, its key is to be made up of the component of following volume parts:
Acrylamide soln: 1.25 parts;
Methylene diacrylamide solution: 0.5～3 part;
H
2O:0.75～3.25 part;
0.03 part of ammonium persulfate solution;
0.02 part of Tetramethyl Ethylene Diamine;
0.5～1 part of chitosan solution.
The mass concentration of above-mentioned chitosan solution is 1%, and its compound method is: the 0.5g chitosan is joined in the 50mL1% acetum, and 37 ℃ are stirred to dissolving, leave standstill 24h after, obtain mass concentration and be 1% chitosan solution.
The mass concentration of above-mentioned methylene diacrylamide solution is 1%; The mass concentration of described acrylamide soln is 30%.
Above-mentioned methylene diacrylamide solution and H
2The volume parts sum of O is 3.75 parts.
A kind of preparation method of described chitosan polyacrylamide hydrophilic gel base material, its key is to be made up of following steps:
(1) mixes stirring: after mixing by the described volume parts adding of claim 1 acrylamide soln, methylene diacrylamide solution, ammonium persulfate solution, Tetramethyl Ethylene Diamine, chitosan solution, join in the centrifuge tube, on whirl mixer, stir 5～10min;
(2) move into the reaction of glass clamping plate: the solution that will mix after stirring with liquid-transfering gun pours in the glass clamping plate of 10cm*6cm, presss from both sides steady clamping plate and coats anti-leakage, at room temperature fully reaction;
(3) soak: then the gel of making is put into medical saline and two anti-mixing solutions, the long-pending portion rate of described medical saline and double antibody is 100:1, soak 48h, every 12h changes soak solution one time, obtains chitosan polyacrylamide hydrophilic gel base material at last.
The mass concentration of above-mentioned chitosan solution is 1%, and its compound method is: the 0.5g chitosan is joined in the 50mL1% acetum, and 37 ℃ are stirred to dissolving, leave standstill 24h after, obtain mass concentration and be 1% chitosan solution.
The mass concentration of above-mentioned methylene diacrylamide solution is 1%; The mass concentration of described acrylamide soln is 30%.
Above-mentioned methylene diacrylamide solution and H
2The volume parts sum of O is 3.75 parts.
1, measures chitosan polyacrylamide hydrophilic gel swelling property
The chitosan polyacrylamide hydrophilic gel is got small pieces to be put into the 35mm culture dish and to be built in 37 ℃ of baking ovens and to dry the record of weighing.In culture dish, add PBS solution, under the room temperature, unnecessary PBS solution in every 20min exhaustion culture dish, the record of weighing no longer changes until weight so repeatedly, then looks hydrogel and reaches capacity, and the result is as depicted in figs. 1 and 2.
The result shows: the chitosan polyacrylamide hydrophilic gel of different ingredients preparation is behind 120min, the gel suction reaches capacity, water regain no longer includes considerable change, this can illustrate, with gel through after 24h hatches, reach capacity fully, can get rid of the influence of the stretching pulling force pair cell that the substrate water-swelling brings thus, thereby be easy to the growth of cell.
2, measure polyacrylamide hydrophilic gel and chitosan polyacrylamide hydrophilic gel Young's modulus
Rheometer is one of common instrument in the rheology experiment, rotational rheometer (AR200ex0) belongs to stress control class rheometer, its principle of work: be used in the multiple rheological properties such as visco-elasticity that the shear flow that produces in the rotary movement is come the rapid determination material.Prepare the polyacrylamide hydrophilic gel and the chitosan polyacrylamide hydrophilic gel of different ingredients before measuring respectively by table 1, put it into soaked overnight in the PBS solution then, be cut into diameter and be about the 2cm disk, last machine is measured, the parameter setting: 25 ℃ of temperature, shearing stress are 1%, oscillation frequency 0.2Hz.The gained data are carried out test of significance with Spss software, and the result as shown in Figure 3 and Figure 4.
The polyacrylamide hydrophilic gel of table 1 different ingredients and chitosan polyacrylamide hydrophilic gel
The result shows: in the Young's modulus that rotational rheometer measures the chitosan polyacrylamide hydrophilic gel and the polyacrylamide hydrophilic gel of different ingredients preparation: 0.5-and 1.5-, 0.5+ and observed value has significant difference (p＜0.05) between the 1.5+, and 0.5-and 0.5+, 1.5-and observed value difference not obvious (p＞0.05) between the 1.5+, illustrate by changing the amount of linking agent, just can change the hardness of chitosan polyacrylamide hydrophilic gel, and the gel hardness after the interpolation chitosan is little with the gel hardness difference of not adding chitosan, and then explanation, the gel that adds chitosan does not change the hardness property of original gel, thereby does not influence cell elasticity.
3, mtt assay test material toxicity
3.1 the material vat liquor extracts
The ratio of the quality of polyacrylamide hydrophilic gel or chitosan polyacrylamide hydrophilic gel and lixiviate medium volume is the extraction that 0.2g/ml carries out vat liquor, and the lixiviate medium is the modified form RPMI-1640 substratum of 10% foetal calf serum (Gibco), at 37 ℃, 5%CO
2Incubator in hatch 72h after, the 0.22um filter filters, it is 25%, 50% and 100% vat liquor that vat liquor is diluted to concentration respectively, carries out the MTT experiment then.
3.2MTT experiment
To be in the HepG2 cell of logarithmic growth, be digested to cell suspension after, cell concn is adjusted to 7 * 10
4Individual/ml, with 96 orifice plate MTT experiment, every hole adds 0.1mL.At 37 ℃, 5%CO
2Incubator in abandon substratum after cultivating 24h, add different concns vat liquor 0.1mL/ hole, do not add vat liquor as negative control group, every kind of concentration vat liquor is cooked 6 multiple holes, and cultivates 24h respectively, behind 48h and the 72h, add MTT liquid 20 μ L/ holes, add DMSO100 μ L/ hole behind the 4h, vibration 10min, measure the absorbancy OD value in each hole, result such as table 2, table 3 and shown in Figure 5 with microplate reader wavelength 490nm.
The different incubation time MTT of table 2 detect and respectively organize HepG2 cell absorbance
The different incubation times of table 3 are respectively organized HepG2 cell degree of propagation and toxicity grading relatively
Carry out the rank scoring according to national security standard pair cell toxicity test: 0 grade of relative propagation degree 〉=100%; 1 grade of relative propagation degree 75%～99%; 2 grades of relative propagation degree 50%～74%; 3 grades of relative propagation degree 25%～49%; 4 grades of relative propagation degree 1%～24%; 5 grades of relative propagation degree are 0.The evaluation material rate.
Show as table 2 and Fig. 5 and to draw: from 24h, 48h and 72h absorbance measuring result as can be seen, compare with the absorbance of negative control group that the absorbancy of cell is to increase along with the increase of time in the different vat liquors.Statistical study obtains: difference nonsignificance (p＞0.05) between each group of negative control group and vat liquor, and illustrate that the toxic action of vat liquor is little or do not have, illustrate that then the toxic action of chitosan polyacrylamide hydrophilic gel pair cell is little or do not have.
Draw as shown in Table 3: by the HepG2 cell relatively degree of propagation find out: degree of propagation increases in time and increases relatively, carries out rank scoring according to national security standard pair cell toxicity test: mostly be 0 grade and 1 grade.It is little or do not have to further specify the toxic action of vat liquor, illustrates that then the toxic action of chitosan polyacrylamide hydrophilic gel pair cell is little or does not have.
4, cell cultures
On aseptic worktable, cut chitosan polyacrylamide hydrophilic gel and polyacrylamide hydrophilic gel, with tweezers the gel thin slice is put into culture dish and flatten, sterilization PBS solution flushing 3～5 times, 2min/ time according to the culture dish size.After modified form RPMI-1640 substratum is hatched 24h, abandon substratum.Cultivate the HepG2 cell with containing 10% foetal calf serum modified form RPMI-1640 substratum on gel, take pictures behind the 24h and carry out morphological observation, the result as shown in Figure 6 and Figure 7.
The result shows: HepG2 cell fully normal adherent growth on chitosan/polyacrylamide hydrophilic gel, and the cell quantity of adherent growth is less on polyacrylamide hydrophilic gel, and circular cell not adherent or death is more.
5, atomic force microscope impression pattern is measured HepG2 cell Young's modulus in the substrate of different soft and hard degree
The HepG2 cell inoculation that will be in exponential phase of growth to the 35mm culture dish and hardness be on the chitosan polyacrylamide hydrophilic gel of 3500Pa, after 48 hours, measure with atomic force microscope pair cell Young's modulus, 10 force curves of each cell measurement are measured 27 cells respectively on two kinds of different hardness.With JPK Image Processing software measurement, the gained data are carried out Gauss curve fitting with Origin pro7.0 software again and are obtained the cell Young's modulus, result such as Fig. 8 and shown in Figure 9.
Found that: the HepG2 cell Young's modulus of growing in culture dish is 1644.3Pa, is that the Young's modulus of cell is 472Pa on the chitosan polyacrylamide hydrophilic gel of 3500Pa in hardness.The elasticity of HepG2 cell is on the 3500Pa 3.5 times approximately on the culture dish, this studies show that with Engler etc. stem cell is similar to the highstrung conclusion of tissue elasticity, be that substrate hardness is influential to the cell elasticity of HepG2 cell, the elasticity that is cell can change along with the variation of substrate hardness, utilize the chitosan polyacrylamide hydrophilic gel to carry out cyto-mechanics research, effect is obvious.
Beneficial effect:
The present invention is a kind of chitosan polyacrylamide hydrophilic gel base material and preparation method thereof, it is simple to have technology, can obtain the hydrogel of different hardness, and the hydrogel that obtains has free of toxic effects, is easy to the cell attachment growth, simplified the complicacy of research cyto-mechanics, help the pair cell mechanical property and study, and the price of chitosan is much lower than the albumen of table lining, reduced the cost of research.
Description of drawings
Fig. 1 is the water regain of the chitosan polyacrylamide hydrophilic gel that adds 0.5 part of linking agent graphic representation, wherein 0.5+ over time " amount of expression 1% methylene diacrylamide solution is 0.5ml and added the 0.5ml1% chitosan solution;
Fig. 2 is the water regain of the chitosan polyacrylamide hydrophilic gel that adds 1.5 parts of linking agents graphic representation, wherein 1.5+ over time " amount of expression 1% methylene diacrylamide solution is 1.5ml and added the 0.5ml1% chitosan solution;
Fig. 3 is the amount and the gel hardness graph of relation of methylene diacrylamide;
Fig. 4 is the elasticity comparison diagram of different ingredients gel, and wherein X-coordinate is the amount of linking agent in the gelling system (methylene diacrylamide), adds the chitosan solution of 0.5ml1% in "+" expression gel.The chitosan solution that does not add 0.5ml1% in "-" expression gel;
Fig. 5 is that the different concns vat liquor is at the column diagram of different time to the HepG2 impact cell;
Fig. 6 is the growing state figure of HepG2 cell behind 24h on the chitosan polyacrylamide hydrophilic gel;
Fig. 7 is HepG2 cell growing state figure behind 24h on the polyacrylamide hydrophilic gel;
HepG2 Young's modulus distribution plan on (A) in Tissue Culture Dish that Fig. 8 obtains for Gauss curve fitting, wherein curve is the Gauss curve fitting curve;
Fig. 9 goes up the Young's modulus distribution plan for the HepG2 that Gauss curve fitting obtains in chitosan/polyacrylamide hydrophilic gel (B) of 3500Pa, and wherein curve is the Gauss curve fitting curve.
Embodiment
Embodiment 1:
A kind of chitosan polyacrylamide hydrophilic gel base material, form by the component of following volume parts:
30%(w/v) acrylamide soln: 1.25 parts;
1%(w/v) methylene diacrylamide solution: 0.5 part;
H
2O:3.25 part;
0.03 part of ammonium persulfate solution;
0.02 part of Tetramethyl Ethylene Diamine;
1%(w/v) chitosan solution is 0.5 part.
The preparation 1%(w/v) chitosan solution: the 0.5g chitosan is joined in the 50mL1% acetum, and 37 ℃ are stirred to dissolving, leave standstill 24h after, obtain 1%(w/v) chitosan solution.
A kind of preparation method of chitosan polyacrylamide hydrophilic gel base material, be made up of following steps:
(1) mix to stir: by above-mentioned volume parts adding 30%(w/v) acrylamide soln, 1%(w/v) methylene diacrylamide solution, ammonium persulfate solution, Tetramethyl Ethylene Diamine, 1%(w/v) after chitosan solution mixes, join in the centrifuge tube, on whirl mixer, stir 5～10min;
(2) move into the reaction of glass clamping plate: the solution that will mix after stirring with liquid-transfering gun pours in the glass clamping plate of 10cm*6cm, presss from both sides steady clamping plate and coats anti-leakage, at room temperature fully reaction;
(3) soak: then the gel of making is put into medical saline and two anti-mixing solutions, described medical saline and two anti-volume parts are than being 100:1, soak 48h, every 12h changes soak solution one time, obtains chitosan polyacrylamide hydrophilic gel base material at last.
Embodiment 2:
A kind of chitosan polyacrylamide hydrophilic gel base material, form by the component of following volume parts:
30%(w/v) acrylamide soln: 1.25 parts;
1%(w/v) methylene diacrylamide solution: 3 parts;
H
2O:0.75 part;
0.03 part of ammonium persulfate solution;
0.02 part of Tetramethyl Ethylene Diamine;
1%(w/v) chitosan solution is 0.5 part.
The preparation 1%(w/v) chitosan solution: the 0.5g chitosan is joined in the 50mL1% acetum, and 37 ℃ are stirred to dissolving, leave standstill 24h after, obtain 1%(w/v) chitosan solution.
A kind of preparation method of chitosan polyacrylamide hydrophilic gel base material, be made up of following steps:
(1) mix to stir: by above-mentioned volume parts adding 30%(w/v) acrylamide soln, 1%(w/v) methylene diacrylamide solution, ammonium persulfate solution, Tetramethyl Ethylene Diamine, 1%(w/v) after chitosan solution mixes, join in the centrifuge tube, on whirl mixer, stir 5～10min;
(2) move into the reaction of glass clamping plate: the solution that will mix after stirring with liquid-transfering gun pours in the glass clamping plate of 10cm*6cm, presss from both sides steady clamping plate and coats anti-leakage, at room temperature fully reaction;
(3) soak: then the gel of making is put into medical saline and two anti-mixing solutions, described medical saline and two anti-volume parts are than being 100:1, soak 48h, every 12h changes soak solution one time, obtains chitosan polyacrylamide hydrophilic gel base material at last.
Embodiment 3:
A kind of chitosan polyacrylamide hydrophilic gel base material, form by the component of following volume parts:
30%(w/v) acrylamide soln: 1.25 parts;
1%(w/v) methylene diacrylamide solution: 1.5 parts;
H
2O:2.25 part;
0.03 part of ammonium persulfate solution;
0.02 part of Tetramethyl Ethylene Diamine;
1%(w/v) chitosan solution is 0.5 part.
The preparation 1%(w/v) chitosan solution: the 0.5g chitosan is joined in the 50mL1% acetum, and 37 ℃ are stirred to dissolving, leave standstill 24h after, obtain 1%(w/v) chitosan solution.
A kind of preparation method of chitosan polyacrylamide hydrophilic gel base material, be made up of following steps:
(1) mix to stir: by above-mentioned volume parts adding 30%(w/v) acrylamide soln, 1%(w/v) methylene diacrylamide solution, ammonium persulfate solution, Tetramethyl Ethylene Diamine, 1%(w/v) after chitosan solution mixes, join in the centrifuge tube, on whirl mixer, stir 5～10min;
(2) move into the reaction of glass clamping plate: the solution that will mix after stirring with liquid-transfering gun pours in the glass clamping plate of 10cm*6cm, presss from both sides steady clamping plate and coats anti-leakage, at room temperature fully reaction;
(3) soak: then the gel of making is put into medical saline and two anti-mixing solutions, described medical saline and two anti-volume parts are than being 100:1, soak 48h, every 12h changes soak solution one time, obtains chitosan polyacrylamide hydrophilic gel base material at last.
Claims (8)
1. chitosan polyacrylamide hydrophilic gel base material is characterized in that being made up of the component of following volume parts:
Acrylamide soln: 1.25 parts;
Methylene diacrylamide solution: 0.5～3 part;
H
2O:0.75～3.25 part;
0.03 part of ammonium persulfate solution;
0.02 part of Tetramethyl Ethylene Diamine;
0.5～1 part of chitosan solution.
2. according to the described chitosan polyacrylamide hydrophilic gel of claim 1 base material, it is characterized in that: the mass concentration of described chitosan solution is 1%, its compound method is: it is in 1% the acetum that the 0.5g chitosan is joined the 50mL volumetric concentration, 37 ℃ are stirred to dissolving, after leaving standstill 24h, obtain mass concentration and be 1% chitosan solution.
3. according to the described chitosan polyacrylamide hydrophilic gel of claim 2 base material, it is characterized in that: the mass concentration of described methylene diacrylamide solution is 1%; The mass concentration of described acrylamide soln is 30%.
4. according to the described chitosan polyacrylamide hydrophilic gel of claim 3 base material, it is characterized in that: described methylene diacrylamide solution and H
2The volume parts sum of O is 3.75 parts.
5. the preparation method of the described chitosan polyacrylamide hydrophilic gel of claim 1 base material is characterized in that being made up of following steps:
(1) mixes stirring: after mixing by the described volume parts adding of claim 1 acrylamide soln, methylene diacrylamide solution, ammonium persulfate solution, Tetramethyl Ethylene Diamine, chitosan solution, join in the centrifuge tube, on whirl mixer, stir 5～10min;
(2) move into the reaction of glass clamping plate: the solution that will mix after stirring with liquid-transfering gun pours in the glass clamping plate of 10cm*6cm, presss from both sides steady clamping plate and coats anti-leakage, at room temperature fully reaction;
(3) soak: then the gel of making is put into medical saline and two anti-mixing solutions, the long-pending portion rate of described medical saline and double antibody is 100:1, soak 48h, every 12h changes soak solution one time, obtains chitosan polyacrylamide hydrophilic gel base material at last.
6. according to the preparation method of the described chitosan polyacrylamide hydrophilic gel of claim 5 base material, it is characterized in that: the mass concentration of described chitosan solution is 1%, its compound method is: the 0.5g chitosan is joined in the 50mL1% acetum, 37 ℃ are stirred to dissolving, after leaving standstill 24h, obtain mass concentration and be 1% chitosan solution.
7. according to the preparation method of the described chitosan polyacrylamide hydrophilic gel of claim 6 base material, it is characterized in that: the mass concentration of described methylene diacrylamide solution is 1%; The mass concentration of described acrylamide soln is 30%.
8. according to the preparation method of the described chitosan polyacrylamide hydrophilic gel of claim 7 base material, it is characterized in that: described methylene diacrylamide solution and H
2The volume parts sum of O is 3.75 parts.
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| CN112250805A (en) * | 2020-10-27 | 2021-01-22 | 中国人民解放军空军特色医学中心 | Preparation method of artificial nervous tissue engineering material, material and application thereof |
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