CN104987473A - Application of high-strength light sensitive hydrogel in action of adjusting cell attachment - Google Patents
Application of high-strength light sensitive hydrogel in action of adjusting cell attachment Download PDFInfo
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Abstract
The invention discloses application of a high-strength light sensitive hydrogel in action of adjusting cell attachment. The high-strength light sensitive hydrogel is formed through free radical polymerization copolymerization of polyethylene glycol methacrylate, 2-vinyl-4,6-diamido-1,3,5-triazine and a monomer containing double bonds of a spiropyrane structure, dissolving the three monomers and an initiator in a solvent, and initiating unsaturated bonds on molecules through the initiator, and preparing the hydrogel through a free radical polymerization reaction. The light sensitive hydrogel disclosed by the invention has higher anti-tensile capacity and compression resistance, and has good biocompatibility, an adsorption capacity of DNA trasfection cells and the action of adjusting cell attachment and deattachment through illumination.
Description
The present patent application is the divisional application of parent application " high-strength light sensitive aquagel and its preparation method and application ", and parent application number is 201310473943.1, and the applying date is on October 11st, 2013.
Technical field
Macromolecule hydrogel technical field of the present invention, more particularly, relate to the preparation of the high intensity hydrogel with desorption cell ability and transgenosis effect, i.e. polyethylene glycol methacrylate-styrene polymer-copolymerization-2-vinyl-4,6-diaminostilbene, 3,5-triazine-copolymerization-containing the preparation of spiro-pyrans monomer copolymer (PEGMA-co-PVDT-co-PSP) hydrogel and the research to its application.
Background technology
Macromolecule hydrogel is that a class has higher water-content and can keep original structure after absorbing water and not by the three-dimensional network polymer dissolved, because its feature class is similar to soft tissue and biocompatibility is good, thus at biomedical engineering field, there is important using value.But, for conventional hydrogels, water molecules in gel is different in size to the diluting effect of macromolecule network, the distribution of cross-linking set randomness and chain link, and the unhomogeneity of cross-linked network causes the mechanical property of hydrogel very weak in plastic process, be difficult to use in the soft tissue bearing high load to substitute, even in moving process, the shake-up of the implements such as tweezers is also easy to make gel damaged, because which limit its application as biomaterial.In recent years, scientific research personnel achieves important progress solving in poor this problem of mechanical property of hydrogel, proposes many neodoxy, method and manufacturing technology of constructing high-strength hydrogel.Such as dual network (DN) gel (J.P.Gong, Y.Katsuyama, T.Kurokawa, Y.Osada, Double-Network Hydrogels with Extremely High Mechanical Strength.Adv.Mater.15 (2003) 1155-1158), the sliding gel (TP) of macromolecular microspheres plural gel (MMC), nano composite material gel (NC), ring, four arms-polyethylene glycol gel and dipole-dipole strengthen gel etc.The technology of these bibliographical informations is mainly devoted to the intensity strengthening hydrogel, as stretching, compression, ductility and energy-to-break etc.Recently, our seminar reports a series of high-strength gel (L.Tang based on diamino triazine hydrogen bond, W.Liu, G.Liu, High-StrengthHydrogels with Integrated Functions ofH ?bonding and Thermoresponsive Surface-MediatedReverse Transfection and Cell Detachment Adv.Mater.22 (2010) 2652-2656).The mechanical strength of its excellence comes from the hydrogen bond of the rigidity supramolecule six-membered ring structure formed between diamino triazine group in gel body; Meanwhile, the diamino triazine of gel surface can form hydrogen bond thus anchoring DNA with the base pair of DNA, can realize the reverse transfection of surface mediation or the transfection of matrix mediation like this.Existing bibliographical information (Z.Cao, W.Liu, D.Liang, G.Guo, J.Zhang, Design of Poly (vinyldiaminotriazine)-Based Nonviral Vectors via Specific Hydrogen Bondingwith Nucleic Acid Base Pairs.Adv.Funct.Mater.17 (2007) 246-252) linear poly-2-vinyl-4,6-diamino, 1,3,5-triazine (PVDT) can form mixture (PVDT/pDNA) with plasmid DNA in cell, realize efficient gene transfection.PVDT/pDNA mixture gel surface can be adsorbed on by the strong hydrogen bonding effect between PVDT and diamino three zin residue of gel surface, effectively transfection efficiency can be improved.
Summary of the invention
Based on above technical background, the object of the present invention is to provide a kind of high-strength light responsive type PEGMA-co-PVDT-co-PSP hydrogel and its preparation method and application.This sensitivity of light hydrogel has higher stretch-proof and resistance to compression, has good biocompatibility.And this hydrogel have adsorption of DNA transfectional cell ability and by the attaching of Illumination adjusting cell and de-attaching behavior.
Object of the present invention is achieved by following technical proposals:
High-strength light sensitive aquagel, by polyethylene glycol methacrylate-styrene polymer (PEGMA), 2-vinyl-4,6-diaminostilbene, these three kinds of monomers and initiator, linking agent are dissolved in a solvent by 3,5-triazine (VDT) and being formed by radical polymerization copolymerization containing double bond monomer (SP) with spiro-pyrans structure, cause the unsaturated link(age) on their molecules by initiator, prepare hydrogel by Raolical polymerizable.
In technique scheme, described polyethylene glycol methacrylate-styrene polymer is according to the size of polyoxyethylene glycol fragment in molecule, to adjust its number-average molecular weight, select the oligoethylene glycol methacrylic ester (OEGMA) of number-average molecular weight 300-800, due to after initiation radical polymerization, double bond in described polyethylene glycol methacrylate-styrene polymer molecule is initiated and realizes radical polymerization, final formation hydrogel network, therefore specially requirement is not done to the molecular weight of the polyethylene glycol methacrylate-styrene polymer used as initial feed and distribution thereof.
In technique scheme, described with spiro-pyrans structure containing double bond monomer, called after 2-[1-vinylformic acid-3 ', 3 '-dimethyl-6-nitro spiral shell (indoles-2 ', 2 [2 hydrogen-1] chromene) ester] base acrylamide; Be prepared according to following step: by carboxyl spiro-pyrans, (chemical name is 1-(β-propyloic)-3 ', 3 '-dimethyl-6-nitro spiral shell (indoles-2 ', 2 [2 hydrogen-1] chromene)), N-(2-hydroxyethyl) acrylamide and DMAP (DMAP) be dissolved in tetrahydrofuran (THF), drip dicyclohexylcarbodiimide (DCC) wherein, react at least 24 hours at room temperature 20-25 DEG C, preferably 24-30 hours; To be dissolved in after the washing of precipitate obtained in benzene and again filter, gained solution adds in normal hexane, through precipitation obtain purifying with spiro-pyrans structure containing double bond monomer (SP).
The consumption of wherein said carboxyl spiro-pyrans, N-(2-hydroxyethyl) acrylamide, DMAP (DMAP) and dicyclohexylcarbodiimide (DCC) is for wait mole, and namely four mol ratios participating in the material reacted are 1:1:1:1.
Described carboxyl spiro-pyrans is prepared [carboxyl spiro-pyrans reference J.Chen according to following step, F.Zeng, S.Wu, J.Zhao, Q.Chen, Z.Tong, Reversible fluorescence modulation through energy transferwith ABC triblock copolymer micelles as scaffolds.Chem.Commun.43 (2008) 5580 – 5582.]:
2,3,3-trimethylammonium-3H-indoles (0.06mol) and 3-iodopropionic acid (0.06mol) are dissolved in 5ml methyl ethyl ketone, are heated to 100 DEG C of backflows three hours under nitrogen protection.Above-mentioned reaction gained quaternary ammonium salt solution is dissolved in a large amount of distilled water, with 1,2-ethylene dichloride (DCE) repetitive scrubbing solution.Evaporate moisture, obtain iodate l-(β-propyloic)-2,3, the 3-trimethylammonium-3H-indoles of purifying.Above-mentioned iodide and equimolar 5-nitrosalicylaldehyde, piperidines to be dissolved in 5ml methyl ethyl ketone reflux three hours.Reaction gained red tan solution is at room temperature placed and is spent the night, and the yellow crystal particle methyl alcohol repetitive scrubbing of generation is obtained the carboxylic spiro-pyrans of purifying.
Prepare the method for the invention described above hydrogel, carry out according to following step:
By polyethylene glycol methacrylate-styrene polymer (PEGMA), 2-vinyl-4,6-diaminostilbene, 3,5-triazine (VDT), with spiro-pyrans structure containing double bond monomer (SP) and initiator, linking agent dissolving in a solvent, caused the unsaturated link(age) of three kinds of monomers by initiator, being prepared by Raolical polymerizable under the condition of starvation not only can desorption cell but also can the hydrogel of mediated gene transfection.
In the inventive solutions, with (low) polyethylene glycol methacrylate-styrene polymer, 2-vinyl-4,6-diaminostilbene, 3,5-triazine, be comonomer containing spiro-pyrans monomer, using polyethyleneglycol diacrylate (PEGDA, Mn=575) as linking agent.Be skeleton structure (the i.e. backbone structure-(CH of peg molecule that " oxygen-carbon-to-carbon-oxygen " singly-bound is connected in the middle of polyethyleneglycol diacrylate molecular chain
2cH
2o)
n), adopt thermal source or light source to make initiator provide free radical, then the double bond being caused oligoethylene glycol methacrylic ester, 2-vinyl-4,6-diamino-1,3,5-triazines by free radical and contained in spiro-pyrans monomer, there is crosslinking copolymerization.In the hydrogel material of final preparation, there is the segment of (low) polyethylene glycol methacrylate-styrene polymer, poly-2-vinyl-4,6-diamino-1,3,5-triazines and poly-spiro-pyrans three kinds of materials.Wherein, form a large amount of hydrogen bond between a large amount of amino on 2-vinyl-4,6-diamino-1,3,5-triazines molecule, greatly improve toughness and the intensity of hydrogel, plasmid DNA can be fixed on the surface of gel simultaneously, give the function of this gel load gene; (low) polyethylene glycol methacrylate-styrene polymer can provide flexible hydrophilic segment, improves the wetting ability of gel; The introducing of spiro-pyrans, hydrogel is made to have photosensitivity, improve the hydrophobicity of gel under visible light, the wetting ability of gel is improved under UV-light, thus make gel surface that the change of reversible hydrophobe occur under ultraviolet/visible light stimulates, be conducive to controlling the attaching of cell and desorption (containing spiro-pyrans structure molecule can under visible ray and UV-light respectively isomery turn to the zwitter-ion structure of closed loop nonionic structure and open loop).Above-mentioned three part synergies, make whole hydrogel material embody high-hydroscopicity, high strength, and good biocompatibility, have dual-use function: Cell detachment and gene transfection simultaneously.
The free radical utilizing initiator to provide causes (low) polyethylene glycol methacrylate-styrene polymer, VDT and SP copolymerization, utilizes linking agent PEGDA to make it be cross-linked.Wherein initiator can select thermal initiator conventional in field of macromolecule polymerization, as Diisopropyl azodicarboxylate (ABIN), benzoyl peroxide (BPO), or light trigger, as 1-[4-(2-hydroxy ethoxy)-phenylene]-2-hydroxyl-2 ', 2 '-dimethyl ethyl ketone (Irgacure 2959), methyl vinyl ketone, st-yrax.If selection thermal initiator, then need first to utilize rare gas element (as nitrogen, argon gas or helium) to get rid of oxygen in reaction system, avoid inhibition; Then according to activity and the consumption of initiator, keep considerable time on kick off temperature reaction system being heated to initiator used, as more than 1h or longer (1-5h), to ensure that initiator can produce abundant free radical, initiation reaction system continues Raolical polymerizable occurs, final preparation hydrogel of the present invention.If selective light initiator, then can select transparent airtight reaction vessel, radical polymerization is caused under the condition of UV-irradiation, because light-initiated efficiency is higher than thermal initiation, need the activity according to selected initiator and consumption adjustment irradiation time, irradiation time can be shorter than the heat-up time of thermal initiation, as 20 minutes or longer (30min-1h).
In the inventive solutions, should according to (low) polyethylene glycol methacrylate-styrene polymer, VDT, SP and the initiator of use, the solvability of linking agent, the solvent that selection can be dissolved above-mentioned four kinds of materials completely or can be dissolved each other completely with above-mentioned four kinds of materials, form homogeneous reaction system, such as dimethyl formamide, N,N-DIMETHYLACETAMIDE, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO) etc.
In preparation scheme, when preparing gel, the mass ratio of VDT and (low) polyethylene glycol methacrylate-styrene polymer is (1-3): 1, the quality of photosensitive monomer SP is the 1%-6% of non-photosensitivity monomer total amount (VDT+ (low) polyethylene glycol methacrylate-styrene polymer), the ratio of linking agent and monomer mass summation is 0.6-0.7:1, and the quality of initiator is the 2%-4% of monomer and linking agent total mass.After the completion of reaction, from reaction vessel, take out multipolymer, after removing monomer, initiator and the solvent not participating in reaction, be immersed in water until reach swelling equilibrium (as soaked 7 days, changing a water every 12h, reaching swelling equilibrium).
A kind of high strength POEGMA-co-PVDT-co-PSP hydrogel provided by the invention is with (low) polyethylene glycol methacrylate-styrene polymer, 2-vinyl-4,6-diaminostilbene, 3,5-triazine and be raw material containing spiro-pyrans monomer, causes crosslinking copolymerization and makes under initiator and linking agent exist.Make hydrogel combine (low) polyethylene glycol methacrylate-styrene polymer, 2-vinyl-4,6-diaminostilbene, 3 simultaneously, the character of 5-triazine and spiro-pyrans, there is very high water-intake rate, very strong stretch-proof and resistance to compression, there is significant sensitivity of light and good biocompatibility.This hydrogel is at room temperature prepared, and preparation method is simple, consumes energy low, and product is easy to long-term and preserves and long-distance transport.As high-strength light sensitive aquagel of the present invention or according to the application of high-strength light sensitive aquagel in the behavior of adjustment cell attachment prepared by preparation method, realize the attaching of cell under visible light illumination, the de-attaching of actual effect cell under UV-irradiation.
Accompanying drawing explanation
Fig. 1 is the nuclear magnetic spectrogram of carboxyl spiro-pyrans monomer in deuterated DMSO.
Fig. 2 is the nuclear magnetic spectrogram of spiro-pyrans monomer in deuterated DMSO that double bond is modified.
Fig. 3 is the subject hydrogel photo through visible ray and UV-irradiation.
Fig. 4 is the microphotograph that L929 cell attaches on inventive gel, and wherein (A) is the microphotograph that under visible ray, L929 cell attaches on inventive gel; (B) be after UV-irradiation 15min cell at the microphotograph of gel surface desorption; (C) be the microphotograph that cell removes rear gel surface residual cell.
Fig. 5 is the microphotograph of subject hydrogel surface after partial irradiation UV-light 15min.
Embodiment
Further illustrate technical scheme of the present invention below in conjunction with specific embodiment, use medicine from sigma company, grade is analytical pure, and oligoethylene glycol methacrylic ester number-average molecular weight is 475, and polyethyleneglycol diacrylate number-average molecular weight is 575.
First, the carboxylic spiro-pyrans of preparation is recorded according to document
2,3,3-trimethylammonium-3H-indoles (0.06mol) and 3-iodopropionic acid (0.06mol) are dissolved in 5ml methyl ethyl ketone, are heated to 100 DEG C of backflows three hours under nitrogen protection.Above-mentioned reaction gained quaternary ammonium salt solution is dissolved in a large amount of distilled water, with 1,2-ethylene dichloride (DCE) repetitive scrubbing solution.Evaporate moisture, obtain iodate l-(β-propyloic)-2,3, the 3-trimethylammonium-3H-indoles of purifying.Above-mentioned iodide and equimolar 5-nitrosalicylaldehyde, piperidines to be dissolved in 5ml methyl ethyl ketone reflux three hours.Reaction gained red tan solution is at room temperature placed and is spent the night, and the yellow crystal particle methyl alcohol repetitive scrubbing of generation is obtained the carboxylic spiro-pyrans of purifying.
Secondly, the spiro-pyrans monomer with double bond structure is prepared
Above-mentioned for 0.05mol carboxyl spiro-pyrans and 0.05molN-(2-hydroxyethyl) acrylamide, 0.05mol DMAP (DMAP) are dissolved in 15ml tetrahydrofuran (THF), dropwise add 0.05mol dicyclohexylcarbodiimide (DCC), room temperature reaction 24 hours.Surplus solution is poured into washing in a large amount of distilled water and obtain red-purple precipitation.This precipitation is dissolved in benzene and also again filters.Gained solution adds in a large amount of normal hexane, obtains the spiro-pyrans monomer (SP) containing double bond of purifying through precipitation.
Nucleus magnetic resonance (German Bruker Advance 400MHz HIGH RESOLUTION SUPERCONDUCTING nuclear magnetic resonance analyser) is used to analyze the product of above-mentioned preparation, result as shown in figure 1 and 2, [J.Chen in Fig. 1 and carboxyl spiro-pyrans reference, F.Zeng, S.Wu, J.Zhao, Q.Chen, Z.Tong, Reversible fluorescence modulation through energy transferwith ABC triblock copolymer micelles as scaffolds.Chem.Commun.43 (2008) 5580 – 5582.] nuclear magnetic spectrogram that provides is consistent; In Fig. 2, after double bond is modified, the characteristic peak (δ 12.2) of carboxyl-COOH disappears, carbon-carbon double bond-CH=CH
2characteristic peak (δ 5.6,6.1,6.2) occur, prove that this small molecules is successfully modified upper double bond.
Utilize the spiro-pyrans monomer (SP) with double bond structure of above-mentioned preparation, oligoethylene glycol methacrylic ester (OEGMA) and 2-vinyl-4,6-diaminostilbene, 3,5-triazine (VDT) prepares hydrogel, use POVSPx-y-z to represent, x-y-z represents the mass ratio of OEGMA/VDT/SP.
Embodiment 1:
By monomeric oligomeric glycolmethacrylate (6mg), 2-vinyl-4,6-diaminostilbene, 3,5-triazine (6mg), SP (0.6mg) and linking agent polyethyleneglycol diacrylate (8.4mg) join in 1.5ml centrifuge tube, after the DMSO dissolved monomer of 210 μ l and linking agent, add light trigger Irgacure 2959 (0.5mg).Will containing monomer, the solvent of linking agent and initiator injects closed mold, and mould irradiates 30min in ultra-violet curing case, to cause radical polymerization.Open mould subsequently and take out gel, repeatedly rinse for several times with deionized water and soak 7 days, changing above-mentioned deionized water every 12h.
Gel surface cell cultures and desorption: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, add 200 μ lDMEM substratum displacement medical alcohols.Being added to by L929 cell suspension in the culture plate of gel to cultivate makes it attach in gel surface and sprawl for 24 hours, irradiating 15min under culture plate being placed in 365nm UV-light makes gel spiro-pyrans isomery turn to zwitter-ion structure again, gel surface becomes more hydrophilic, and cell comes off from gel.Measuring by mtt assay the desorption rate recording cell from the cell of desorption gel and the cell remained on gel is: 86.5%.
The gene transfection of gel surface mediation: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, adds 200 μ l DMEM substratum displacement medical alcohols.Gel immerse DMEM substratum 24 as a child after, the PVDT/pDNA mixture (mass ratio 0.3125:1) containing 1 μ g plasmid DNA of compound 4h is in advance joined in the orifice plate of gel.Adsorb after 24 hours, the COS-7 cell suspension being cultured to exponential phase of growth is joined in the gel having adsorbed DNA, cultivate after 24 hours, change nutrient solution and cultivate 24 hours.Be placed on 365nm UV-light under irradiate 15min and make Cell detachment, lysing cell, the uciferase activity measured wherein is: 1.85 × 10
6.
Embodiment 2:
By monomeric oligomeric glycolmethacrylate (6mg), 2-vinyl-4,6-diaminostilbene, 3,5-triazine (12mg), SP (0.9mg) and linking agent polyethyleneglycol diacrylate (12.6mg) join in 1.5ml centrifuge tube, after the DMSO dissolved monomer of 315 μ l and linking agent, add light trigger Irgacure 2959 (0.7mg).Will containing monomer, the solvent of linking agent and initiator injects closed mold, and mould irradiates 30min in ultra-violet curing case, to cause radical polymerization.Open mould subsequently and take out gel, repeatedly rinse for several times with deionized water and soak 7 days, changing above-mentioned deionized water every 12h.
Gel surface cell cultures and desorption: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, add 200 μ lDMEM substratum displacement medical alcohols.Being added to by L929 cell suspension in the culture plate of gel to cultivate makes it attach in gel surface and sprawl for 24 hours, irradiating 15min under culture plate being placed in 365nm UV-light makes gel spiro-pyrans isomery turn to zwitter-ion structure again, gel surface becomes more hydrophilic, and cell comes off from gel.Measuring by mtt assay the desorption rate recording cell from the cell of desorption gel and the cell remained on gel is: 84.1%.
The gene transfection of gel surface mediation: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, adds 200 μ l DMEM substratum displacement medical alcohols.Gel immerse DMEM substratum 24 as a child after, the PVDT/pDNA mixture (mass ratio 0.3125:1) containing 1 μ g plasmid DNA of compound 4h is in advance joined in the orifice plate of gel.Adsorb after 24 hours, the COS-7 cell suspension being cultured to exponential phase of growth is joined in the gel having adsorbed DNA, cultivate after 24 hours, change nutrient solution and cultivate 24 hours.Be placed on 365nm UV-light under irradiate 15min and make Cell detachment, lysing cell, the uciferase activity measured wherein is: 3.89 × 10
6.
Embodiment 3:
By monomeric oligomeric glycolmethacrylate (6mg), 2-vinyl-4,6-diaminostilbene, 3,5-triazine (18mg), SP (1.2mg) and linking agent polyethyleneglycol diacrylate (16.8mg) join in 1.5ml centrifuge tube, after the DMSO dissolved monomer of 420 μ l and linking agent, add light trigger Irgacure 2959 (0.9mg).Will containing monomer, the solvent of linking agent and initiator injects closed mold, and mould irradiates 30min in ultra-violet curing case, to cause radical polymerization.Open mould subsequently and take out gel, repeatedly rinse for several times with deionized water and soak 7 days, changing above-mentioned deionized water every 12h.
Gel surface cell cultures and desorption: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, add 200 μ lDMEM substratum displacement medical alcohols.Being added to by L929 cell suspension in the culture plate of gel to cultivate makes it attach in gel surface and sprawl for 24 hours, irradiating 15min under culture plate being placed in 365nm UV-light makes gel spiro-pyrans isomery turn to zwitter-ion structure again, gel surface becomes more hydrophilic, and cell comes off from gel.Measuring by mtt assay the desorption rate recording cell from the cell of desorption gel and the cell remained on gel is: 82%.
The gene transfection of gel surface mediation: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, adds 200 μ l DMEM substratum displacement medical alcohols.Gel immerse DMEM substratum 24 as a child after, the PVDT/pDNA mixture (mass ratio 0.3125:1) containing 1 μ g plasmid DNA of compound 4h is in advance joined in the orifice plate of gel.Adsorb after 24 hours, the COS-7 cell suspension being cultured to exponential phase of growth is joined in the gel having adsorbed DNA, cultivate after 24 hours, change nutrient solution and cultivate 24 hours.Be placed on 365nm UV-light under irradiate 15min and make Cell detachment, lysing cell, the uciferase activity measured wherein is: 6.40 × 10
6.
Embodiment 4:
By monomeric oligomeric glycolmethacrylate (6mg), 2-vinyl-4,6-diaminostilbene, 3,5-triazine (12mg), SP (0.18mg) and linking agent polyethyleneglycol diacrylate (12.7mg) join in 1.5ml centrifuge tube, after the DMSO dissolved monomer of 309 μ l and linking agent, add light trigger Irgacure 2959 (1.24mg).Will containing monomer, the solvent of linking agent and initiator injects closed mold, and mould irradiates 30min in ultra-violet curing case, to cause radical polymerization.Open mould subsequently and take out gel, repeatedly rinse for several times with deionized water and soak 7 days, changing above-mentioned deionized water every 12h.
Gel surface cell cultures and desorption: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, add 200 μ lDMEM substratum displacement medical alcohols.Being added to by L929 cell suspension in the culture plate of gel to cultivate makes it attach in gel surface and sprawl for 24 hours, irradiating 15min under culture plate being placed in 365nm UV-light makes gel spiro-pyrans isomery turn to zwitter-ion structure again, gel surface becomes more hydrophilic, and cell comes off from gel.Measuring by mtt assay the desorption rate recording cell from the cell of desorption gel and the cell remained on gel is: 85%.
The gene transfection of gel surface mediation: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, adds 200 μ l DMEM substratum displacement medical alcohols.Gel immerse DMEM substratum 24 as a child after, the PVDT/pDNA mixture (mass ratio 0.3125:1) containing 1 μ g plasmid DNA of compound 4h is in advance joined in the orifice plate of gel.Adsorb after 24 hours, the COS-7 cell suspension being cultured to exponential phase of growth is joined in the gel having adsorbed DNA, cultivate after 24 hours, change nutrient solution and cultivate 24 hours.Be placed on 365nm UV-light under irradiate 15min and make Cell detachment, lysing cell, the uciferase activity measured wherein is: 4.10 × 10
6.
Embodiment 5:
By monomeric oligomeric glycolmethacrylate (6mg), 2-vinyl-4,6-diaminostilbene, 3,5-triazine (18mg), SP (1.44mg) and linking agent polyethyleneglycol diacrylate (15.26mg) join in 1.5ml centrifuge tube, after the DMSO dissolved monomer of 407 μ l and linking agent, add light trigger Irgacure 2959 (1.22mg).Will containing monomer, the solvent of linking agent and initiator injects closed mold, and mould irradiates 30min in ultra-violet curing case, to cause radical polymerization.Open mould subsequently and take out gel, repeatedly rinse for several times with deionized water and soak 7 days, changing above-mentioned deionized water every 12h.
Gel surface cell cultures and desorption: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, add 200 μ lDMEM substratum displacement medical alcohols.Being added to by L929 cell suspension in the culture plate of gel to cultivate makes it attach in gel surface and sprawl for 24 hours, irradiating 15min under culture plate being placed in 365nm UV-light makes gel spiro-pyrans isomery turn to zwitter-ion structure again, gel surface becomes more hydrophilic, and cell comes off from gel.Measuring by mtt assay the desorption rate recording cell from the cell of desorption gel and the cell remained on gel is: 80%.
The gene transfection of gel surface mediation: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, adds 200 μ l DMEM substratum displacement medical alcohols.Gel immerse DMEM substratum 24 as a child after, the PVDT/pDNA mixture (mass ratio 0.3125:1) containing 1 μ g plasmid DNA of compound 4h is in advance joined in the orifice plate of gel.Adsorb after 24 hours, the COS-7 cell suspension being cultured to exponential phase of growth is joined in the gel having adsorbed DNA, cultivate after 24 hours, change nutrient solution and cultivate 24 hours.Be placed on 365nm UV-light under irradiate 15min and make Cell detachment, lysing cell, the uciferase activity measured wherein is: 6.10 × 10
6.
Comparative example:
Monomeric oligomeric glycolmethacrylate (12mg) and linking agent polyethyleneglycol diacrylate (8mg) are joined in 1.5ml centrifuge tube, after the DMSO dissolved monomer of 200 μ l and linking agent, add light trigger Irgacure 2959 (0.4mg).Will containing monomer, the solvent of linking agent and initiator injects closed mold, and mould irradiates 30min in ultra-violet curing case, to cause radical polymerization.Open mould subsequently and take out gel, repeatedly rinse for several times with deionized water and soak 7 days, changing above-mentioned deionized water every 12h.
Gel surface cell cultures and desorption: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, add 200 μ lDMEM substratum displacement medical alcohols.Being added to by L929 cell suspension in the culture plate of gel to cultivate makes it attach in gel surface and sprawl for 24 hours, irradiating 15min under culture plate being placed in 365nm UV-light makes gel spiro-pyrans isomery turn to zwitter-ion structure again, gel surface becomes more hydrophilic, and cell comes off from gel.Measuring by mtt assay the desorption rate recording cell from the cell of desorption gel and the cell remained on gel is: 4.7%.
The gene transfection of gel surface mediation: immersed in medical alcohol by the gel obtained and put into 96 orifice plates after sterilizing 4h, adds 200 μ l DMEM substratum displacement medical alcohols.Gel immerse DMEM substratum 24 as a child after, the PVDT/pDNA mixture (mass ratio 0.3125:1) containing 1 μ g plasmid DNA of compound 4h is in advance joined in the orifice plate of gel.Adsorb after 24 hours, the COS-7 cell suspension being cultured to exponential phase of growth is joined in the gel having adsorbed DNA, cultivate after 24 hours, change nutrient solution and cultivate 24 hours.Lysing cell afterwards, the uciferase activity measured wherein is: 5.82 × 10
5.
For respectively organizing hydrogel above, preparing sheet or column sample by same steps, using WGW microcomputer controlled electronic universal tester (Jinan Instrument Ltd. in epoch in epoch) to carry out Mechanics Performance Testing.The sample wherein carrying out tensile property test is of a size of the sheet gel of 20mm × 5mm × 0.4mm.The sample size of carrying out compression performance test is the cylinder of diameter 4mm, high 6mm.Elongation test speed is 100mm/min, and compression speed is 10mm/min.The various performance parameters of the hydrogel sample that table 1 obtains for above-described embodiment.Visible its has higher equilibrium water absorption, and good mechanical property.
The physical function parameter of table 1.POEGMA-co-PVDT-co-PSP hydrogel
--
arefer to that this gel is too weak to such an extent as to its intensity cannot be measured
Subject hydrogel is irradiated, as shown in Figure 3, to realize the metamorphosis of subject hydrogel with visible ray and UV-irradiation.Hydrogel of the present invention is utilized to carry out cell experiment as follows:
Be laid in 96 orifice plates, after PBS swelling equilibrium, by L929 cell suspension with 1 × 10 after hydrogel sterilization
4the density in/hole is inoculated into gel surface, cell is placed in 37 DEG C, 5%CO
2, cultivate 24 hours under 100% humidity condition, make cell attachment in gel surface.Gel is placed in 365nm ultraviolet source (Spectronics company of the U.S., XL-1000,2mW/cm
2) under irradiate 15min, make cell from gel surface desorption.Before and after UV-irradiation, the cell state of gel surface all uses OLYMPUS CKX41 (Japanese Olympus company) microscope to take pictures observation.
The desorption rate mtt assay of cell is tested.MTT chemistry 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt by name.Process is as follows: gel surface culturing cell is after 24 hours, 15min is irradiated under UV-light, the cell suspension of desorption is made in the medium with liquid-transfering gun piping and druming, again by complete for the substratum in every hole sucking-off, be added dropwise in new culture plate respectively, in the new culture plate at the cell place of the culture plate of the gel with residual attaching cell and desorption being got off, every hole adds the MTT solution that the fresh DMEM medium of the serum-free of 180 μ l and 20 μ l concentration are 5mg/ml respectively, continues cultivation 4 hours.Siphon away the substratum in every hole, add the lysigenous purple crystal of DMSO of 200 μ l, test the absorbancy of DMSO solution in every hole.Desorption rate is defined as: absorbancy/(absorbancy+remnants of desorption cell attach the absorbancy of cell) × 100% of desorption cell.
The cell free method in local: with the light shield covering part region that tinfoil is made in UV-irradiation process, part cell can be made to be retained in gel surface.
From accompanying drawing 4, can find out in (A), under visible ray, cell can uniform spreading grow in gel surface, in good condition, after UV-irradiation, cell bounces back gradually in gel surface, just retract into spherical after 15min, from gel surface desorption, as shown in (B).After leniently blowing and beating with liquid-transfering gun, cell departs from from surface, and form cell suspension, gel surface has a small amount of cell rests, as shown in (C).The reason of this phenomenon is caused to be that photosensitive composition SP in hydrogel becomes hydrophobic closed-loop structure under visible light, gel surface is made to present more hydrophobic state, be conducive to cell adhesion growth, and under uv irradiating, the rapid isomery of SP turns to hydrophilic open loop structure, make gel surface become more hydrophilic, be unfavorable for cell adhesion, cell from surface gradually desorption get off.
As seen from Figure 5, after gel section being hidden, irradiating ultraviolet light can reach the effect of local cells desorption.Blowing and beating whole gel surface with liquid-transfering gun after ultraviolet partial irradiation, undosed part cell still keeps original state sprawling adhesion, and substantially comes off through the part cell of uv irradiating.This changes because the irradiated site of gel there occurs wetting ability, and hide position and still keep former hydrophobic state.
The known high-strength light sensitive aquagel prepared as high-strength light sensitive aquagel of the present invention or foundation preparation method is regulating the application in cell attachment behavior, realize the attaching of cell under visible light illumination, the de-attaching of actual effect cell under UV-irradiation.
Above to invention has been exemplary description; should be noted that; when not departing from core of the present invention, any simple distortion, amendment or other those skilled in the art can not spend the equivalent replacement of creative work all to fall into protection scope of the present invention.
Claims (5)
1. high-strength light sensitive aquagel is regulating the application in cell attachment behavior, it is characterized in that, realize the attaching of cell under visible light illumination, the de-attaching of cell is realized under UV-irradiation, described high-strength light sensitive aquagel is by polyethylene glycol methacrylate-styrene polymer, 2-vinyl-4, 6-diaminostilbene, 3, 5-triazine and being formed by radical polymerization copolymerization containing double bond monomer with spiro-pyrans structure, by this three kinds of monomers and initiator, linking agent dissolves in a solvent, the unsaturated link(age) on their molecules is caused by initiator, hydrogel is prepared by Raolical polymerizable, described is 2-[1-vinylformic acid-3 ', 3 '-dimethyl-6-nitro spiral shell (indoles-2 ', 2 [2 hydrogen-1] chromene) ester] base acrylamide with spiro-pyrans structure containing double bond monomer, described linking agent is polyethyleneglycol diacrylate Mn=575, and described polyethylene glycol methacrylate-styrene polymer, according to the size of polyoxyethylene glycol fragment in molecule, to adjust its number-average molecular weight, selects the oligoethylene glycol methacrylic ester of number-average molecular weight 300-800,
When preparing gel, 2-vinyl-4, 6-diaminostilbene, 3, the mass ratio of 5-triazine and polyethylene glycol methacrylate-styrene polymer is (1-3): 1, the quality containing double bond monomer with spiro-pyrans structure is non-photosensitivity monomer 2-vinyl-4, 6-diaminostilbene, 3, the 1%-6% of 5-triazine and polyethylene glycol methacrylate-styrene polymer total amount, linking agent and monomer polyethylene glycol methacrylate-styrene polymer, 2-vinyl-4, 6-diaminostilbene, 3, 5-triazine and the ratio containing double bond monomer quality summation with spiro-pyrans structure are 0.6-0.7:1, the quality of initiator is polyethylene glycol methacrylate-styrene polymer, 2-vinyl-4, 6-diaminostilbene, 3, 5-triazine, with the 2%-4% containing double bond monomer and linking agent total mass of spiro-pyrans structure, described solvent is dimethyl formamide, N,N-DIMETHYLACETAMIDE, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO).
2. high-strength light sensitive aquagel according to claim 1 is regulating the application in cell attachment behavior, it is characterized in that, described initiator selects thermal initiator conventional in field of macromolecule polymerization or light trigger.
3. high-strength light sensitive aquagel according to claim 1 is regulating the application in cell attachment behavior, it is characterized in that, thermal initiator conventional in described field of macromolecule polymerization is Diisopropyl azodicarboxylate, benzoyl peroxide, described light trigger be 1-[4-(2-hydroxy ethoxy)-phenylene]-2-hydroxyl-2 ', 2 '-dimethyl ethyl ketone, methyl vinyl ketone, st-yrax.
4. high-strength light sensitive aquagel according to claim 1 is regulating the application in cell attachment behavior, it is characterized in that, when preparing gel, selects thermal initiator, then need first to utilize the oxygen in rare gas element eliminating reaction system, avoids inhibition; Then according to activity and the consumption of initiator, keep 1-5h on kick off temperature reaction system being heated to initiator used, to ensure that initiator can produce abundant free radical, initiation reaction system continues Raolical polymerizable occurs.
5. high-strength light sensitive aquagel according to claim 1 is regulating the application in cell attachment behavior, it is characterized in that, when preparing gel, selective light initiator, then select transparent airtight reaction vessel, under the condition of UV-irradiation, cause radical polymerization, according to activity and the consumption adjustment irradiation time of selected initiator, because light-initiated efficiency is higher than thermal initiation, irradiation time can be shorter than the heat-up time of thermal initiation, and described irradiation time is 30min-1h.
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