CN109734851B - Temperature-sensitive polymer, synthesis method thereof and temperature-sensitive injectable hydrogel - Google Patents
Temperature-sensitive polymer, synthesis method thereof and temperature-sensitive injectable hydrogel Download PDFInfo
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- C08H1/00—Macromolecular products derived from proteins
Abstract
The invention discloses a temperature-sensitive polymer, a synthesis method thereof and temperature-sensitive injectable hydrogel. When the temperature-sensitive polymer is prepared, GelMA and UPyMA are synthesized; then UPyMA, GelMA and MEOnDispersing three monomers MA into an alkaline aqueous solution with the pH value of 8-11, removing oxygen, adding a redox initiator or a water-soluble initiator at room temperature to perform polymerization reaction until the reaction is complete, purifying the product, and removing the solvent to obtain the temperature-sensitive polymer. The temperature-sensitive polymer can be used for preparing injectable aqueous phase temperature-sensitive polymer sol under the normal pressure environment of not higher than the corresponding low critical phase transition temperature and not lower than the water solidification temperature, and the sol can immediately form gel above the corresponding low critical phase transition temperature. The temperature-sensitive injectable hydrogel has dynamic mechanical properties and good biocompatibility, and can be applied to biomedical fields such as tissue engineering, drug controlled release, cell three-dimensional culture and the like.
Description
Technical Field
The invention belongs to the technical field of polymer physics, polymer chemistry, biomedical materials and tissue engineering, and particularly relates to a method for synthesizing a temperature-sensitive polymer with good biocompatibility and preparing and applying a temperature-sensitive injectable hydrogel corresponding to the temperature-sensitive polymer.
Background
The hydrogel is a gel using an aqueous phase system as a dispersion medium. The molecular composition and structure in the gel network are complex and changeable, and the gel network has good designability; aqueous dispersions can impart good biocompatibility to the gel. Due to the characteristics, the hydrogel can provide a retention or proliferation environment for cells, can also entrap drugs or proteins for controlled slow release, can be biodegraded after the task is completed and discharged out of the body as metabolic waste, and has great application value and potential in the aspect of biomedical use. Therefore, hydrogels containing biocompatible components are often used in the fields of biomedical materials, tissue engineering techniques, and the like.
When the hydrogel is used for tissue engineering in vivo, the implantation method is divided into two types according to the different types of the gel: irreversible chemically crosslinked macroscopic hydrogels mostly require surgical implantation; the reversibly crosslinked macroscopic hydrogel or microgel may be implanted by injection. Compared with the operation, the injection is more moderate and convenient. While chemically crosslinked hydrogels are often potentially toxic. The reversible crosslinking hydrogel is mainly divided into three modes of shear thinning, pH response and temperature response (temperature sensitivity) for realizing injection implantation. Shear thinning and pH response may cause the drug or cells entrapped within the gel to be at a high shear modulus or an inappropriate pH range at or prior to injection, which may compromise the bioactive components entrapped within the gel and thereby affect the therapeutic effect. The temperature-sensitive injectable gel is designed to enable the sol-gel transition point to be in the range from room temperature to body temperature, so that mild sol injection can be realized and gel can be quickly formed at a target implantation position.
Chinese patent 2011103868703 discloses a method for synthesizing a temperature-sensitive polymer capable of being enhanced in situ, which comprises the following steps: in the presence of a hydroxyl-containing initiator and an amine catalyst, the double-bond functional monomer is obtained by homopolymerization, or is obtained by copolymerization of the double-bond functional monomer and one of carbonic ester, lactone and lactide, wherein the temperature of homopolymerization or copolymerization is 50-80 ℃, and the reaction time is 1-4 h. The hydrogel material provided by the invention is in a liquid state at a lower temperature and in a gel state at a physiological temperature, and can conveniently introduce bioactive molecules and biological signals to endow the material with special biological activity. The hydrogel provided by the invention can be gradually degraded in a physiological environment. Similar to the existing temperature-sensitive hydrogel based on the low critical phase transition temperature, the cross-linking structure is formed mainly by hydrophobic aggregation of polymers above the low critical phase transition temperature, and the strength of cross-linking points formed by the hydrophobic aggregation is low, so that unstable gel mechanical properties and a cross-linking structure which is not resistant to swelling are caused, and the application range of the hydrogel is greatly limited.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a temperature-sensitive polymer and a synthesis method thereof.
The invention also aims to provide a temperature-sensitive injectable hydrogel prepared from the temperature-sensitive polymer; the hydrogel has good biocompatibility, and is particularly characterized in that cells are easy to remain, adhere and proliferate in the gel; the hydrogel has practical temperature-sensitive performance, good fluidity at room temperature, is in a sol state easy to inject, and is in a stable gel state at the body temperature of a human body. The gel has good mechanical strength and stability and extremely low cytotoxicity, which means that the temperature-sensitive injectable hydrogel is characterized
The temperature-sensitive polymers are divided into two types, one type is based on the high critical phase transition temperature, aggregation and crosslinking of polymer chain segments occur when the temperature is lower than the phase transition temperature, and dispersion and crosslinking release of the polymer chain segments occur when the temperature is higher than the phase transition temperature; the temperature-sensitive polymer is based on low critical phase transition temperature, and the dispersion and the decrosslinking of the polymer occur when the temperature is lower than the phase transition temperature, and the aggregation and the crosslinking of the polymer occur when the temperature is higher than the phase transition temperature. The temperature-sensitive polymer prepared by the invention belongs to a temperature-sensitive polymer based on low critical phase transition temperature. The general temperature-sensitive hydrogel based on the low critical phase transition temperature has weak mechanical strength and poor stability. The dynamic quadruple hydrogen bond self-assembly reinforcing structure brought by the UPyMA segment remarkably improves the mechanical strength and stability of the gel under the condition of not influencing the fluidity of the sol state. The temperature-sensitive hydrogel only forms a cross-linked network through supermolecule interaction, and no initiator is needed to participate in the formation of the cross-linked network, and no chemical bond is generated, so that the temperature-sensitive hydrogel has extremely low cytotoxicity.
The purpose of the invention is realized by the following technical scheme:
a method for synthesizing a temperature-sensitive polymer comprises the following steps:
1) GelMA Synthesis: dissolving gelatin in a water phase buffer system at 40-45 ℃, adding methacrylic anhydride, and controlling the pH to be 7.4-11.0; reacting for 1-5 hours, stopping the reaction, dissolving the product, purifying the product, and removing the solvent to obtain a product of methacrylic acid acylated gelatin GelMA;
2) synthesis of UPyMA: dissolving aminopyrimidinone in an anhydrous polar organic solvent at 150-180 ℃ to obtain an aminopyrimidinone solution; adding ethyl methacrylate, cooling to room temperature, and fully stirring until white precipitate appears; removing the solvent to obtain a product UpyMA; the structural formula of the UPyMA is as follows:
3) polymerization reaction: mixing the components in a mass ratio of 1: (1-5): (5-10) UPyMA, GelMA and MEOnDispersing three monomers MA into an alkaline aqueous solution with the pH value of 8-11, removing oxygen, adding a redox initiator or a water-soluble initiator at room temperature to perform polymerization reaction until the reaction is sufficient; the MEOnThe structural formula of MA is:
4) purifying the product, and removing the solvent to obtain the temperature-sensitive polymer.
To further achieve the object of the present invention, preferably, the volume mass ratio of the methacrylic anhydride to the gelatin is 1: 0.5 to 10; the mass unit is g; the volume unit is ml; the molar ratio of the aminopyrimidinone to the ethyl methacrylate is 1: 1 to 2.
Preferably, the mass ratio of the aminopyrimidinone to the anhydrous polar solvent is 1: 20-30; the methacrylic anhydride is added in a dropwise manner, and the dropwise adding speed is not faster than 0.4 ml/min.
Preferably, the termination reaction in the step 1) is to dilute the reaction system by 3-10 times by using a water phase buffer system with the pH of 7.4 or to adjust the pH of the system to 7.4; the dissolution product is dissolved for 0.5-2 hours at 40-45 ℃ by using a water phase buffer system for terminating the reaction.
Preferably, the purified product in the step 1) is obtained by removing solutes with molecular weight less than 3000-15000 and water-insoluble products through dialysis, ultrafiltration or chromatographic separation; the solvent removal in the step 1) is to remove the solvent by freeze-drying or reprecipitation to obtain dry GelMA; the removal of the solvent in step 2) is carried out by repeatedly washing the white precipitate with acetone, or the removal of the solvent in step 2) is carried out by chromatographic separation and then dried; and 4) separating and purifying the solute with the molecular weight of 3000-15000 by dialysis, ultrafiltration or chromatography.
Preferably, the aqueous phase buffer system in the step 1) is Na2CO3-NaHCO3Aqueous sodium bicarbonate or PBS; the mass ratio of the gelatin to the water phase buffer system is 1: 9-1: 20; the dissolving time is 1-2 hours until the solution is a light yellow clear liquid.
Preferably, the oxidant in the redox initiator is hydrogen peroxide, persulfate, hydroperoxide; the reducing agent is an inorganic reducing agent or an organic reducing agent; the inorganic reducing agent is NaHSO3、Na2SO3And Na2S2O3One or more of; the organic reducing agent is one or more of alcohol, amine, oxalic acid and glucose; the molar ratio of the oxidant to the UPyMA is 2: 3-1: 6.
A temperature-sensitive polymer obtained by the synthesis method; the temperature-sensitive polymer has low critical phase transition temperature in a water phase solvent.
The temperature-sensitive injectable hydrogel prepared by applying the temperature-sensitive polymer comprises the following components in percentage by weight: dispersing the temperature-sensitive polymer in an aqueous phase system at a solid content of 5-20 wt% to form a temperature-sensitive polymer aqueous phase dispersion system; uniformly mixing cells or substances for adjuvant therapy into the temperature-sensitive polymer aqueous phase dispersion system to obtain a bioactive substance sol encapsulation system; heating the bioactive substance sol encapsulation system in situ to a temperature above the low critical phase transition temperature of the sol to form a bioactive substance gel encapsulation system; or firstly forming sol below the low critical phase transition temperature and above the solidification temperature of the bioactive substance sol encapsulation system, and then transferring the sol into an injection tool to inject a part with the temperature higher than the low critical phase transition temperature to form gel in situ.
Preferably, the cells are autologous stem cells, corneal endothelial cells, corneal epithelial cells or chondrocytes; or the cells are ethically derived cells of xenogenic origin for use in therapy or research;
the auxiliary therapeutic substances are antitumor drugs, differentiation promoting drugs and antibiotic drugs.
MEO of the inventionnMA is a monomer with a double bond and comprising polyethylene glycol repeating units.
The L CST for inducing sol-gel transition in the present invention is inversely related to the polymer concentration, with a minimum of about 21 ℃ corresponding to a solids content of about 8 wt% to 20 wt%, and a maximum of about 29 ℃ corresponding to a solids content of about 4 wt% to 7 wt%.
The temperature-sensitive polymer is dispersed in an aqueous phase system at a solid content of 5-20 wt%, wherein the solid content is such that the obtained polymer aqueous dispersion system is in a sol state under the condition of standing at normal pressure and room temperature, so as to obtain temperature-sensitive polymer sol; the temperature-sensitive polymer sol has low critical phase transition temperature, can be gelled when the temperature is higher than the critical phase transition temperature, has dynamic mechanical properties such as temperature sensitivity, thixotropy and the like and good biocompatibility, and can be used as a biomedical material.
Compared with the prior art, the invention has the following advantages:
1) the UPyMA segment in the polymer can obviously enhance the originally weak hydrophobic aggregation cross-linked structure above the low critical phase transition temperature, and improve the mechanical stability of the gel, and can be well dispersed in the water phase environment due to the collapse of the hydrophobic structure below the low critical phase transition temperature, so that the fluidity and the injectability of the sol are not influenced, and the application potential of the temperature-sensitive hydrogel is integrally improved.
2) The polymer synthesis and gel preparation of the invention are simple, safe, low in cost and cheap and easily available in raw materials. The application and implementation mode of the finished product, namely simple mixing and injection, is extremely easy to operate, and has great significance for wide clinical application.
3) The invention can be degraded gradually in physiological environment as a potential injection implant, and degradation products are nontoxic biomolecules such as amino acid, cytosine and the like, and have good biocompatibility. Compared with most of artificially synthesized polymer implants, the possibility of rejection reaction is reduced to be extremely low, and the trouble of taking out the implant by an operation is avoided.
Drawings
FIG. 1 is a nuclear magnetic spectrum of UPyMA.
FIG. 2 is a nuclear magnetic spectrum of GelMA and a temperature sensitive polymer Gel-MEO-UPy.
FIG. 3 is an amplitude scan of a 10 wt% Gel-MEO-UPy hydrogel at 37 ℃.
FIG. 4 is a frequency sweep at 37 ℃ of a 10 wt% Gel-MEO-UPy hydrogel.
FIG. 5 is the thixotropy of a 10 wt% Gel-MEO-UPy hydrogel at 37 ℃.
FIG. 6 is a temperature scan of a 10 wt% Gel-MEO-UPy hydrogel.
FIG. 7A is a photograph of a Gel-forming Gel by injecting a 10 wt% Gel-MEO-UPy sol into water at 37 ℃ and a shear viscosity curve of the sol at 20 ℃.
FIG. 7B is a photograph of a Gel shape after injection of 10 wt% Gel-MEO-UPy sol at 20 ℃ into a 37 ℃ mold.
FIG. 8A is a bar graph of cytotoxicity assays for Gel-MEO-UPy polymers;
FIG. 8B is a bar graph of the CCK-8 assay quantitatively characterizing cell proliferation within the gel.
FIG. 9 is a confocal laser photograph of a 10 wt% Gel-MEO-UPy sol coated mesenchymal stem cells injected into a 37 ℃ mold, gelled, cultured in three dimensions and subjected to a live-dead staining experiment.
Detailed Description
The present invention will be further described with reference to the following examples for better understanding of the present invention, but the embodiments of the present invention are not limited thereto.
Example 1
1. Synthesis of temperature-sensitive polymers
(1) GelMA (methacrylic acylated gelatin) synthesis using gelatin and methacrylic anhydride:
① putting a 4cm round-bottom flask with a polytetrafluoroethylene magnetic rotor with two tips at two ends, adding 6g of gelatin solid and 60ml of 1 × PBS, sealing the flask, stirring at a constant speed of 300rpm, and dissolving in water bath at 40 ℃ for 1 hour until the substances in the flask become light yellow clear liquid;
② heating the gelatin solution obtained in step ① to 50 ℃, keeping the stirring at 300rpm, and then slowly injecting and dripping 12ml of methacrylic anhydride into the gelatin solution, wherein the dripping speed is 0.2 ml/min;
③ is fully reacted for 4 hours in a water bath at 50 ℃ and at the stirring speed of 300rpm after the dropwise addition is finished;
④ the reaction was stopped, the product was dissolved, the reaction system was diluted with 1 × PBS by 10 volumes, and then dissolved in a water bath at 40 ℃ for 1 hour;
⑤ purifying the product, dialyzing the liquid obtained in step ④ with 5 liters of ultrapure water, changing water once every 12 hours by using a dialysis bag with the molecular weight cutoff of 8000-14400, dialyzing for 7 days, taking out the liquid after dialysis after 7 days, centrifuging at the rotating speed of 9000rpm, and taking the supernatant;
⑥ lyophilizing the supernatant from step ⑤ to obtain a dried white solid GelMA;
(2) synthesizing UPyMA (2- (3- (6-methyl-4-oxo-1, 4-dihydropyrimidin-2-one) -ureido) ethyl methacrylate) by using aminopyrimidinone and isocyano ethyl methacrylate;
1) adding 2g of semicarbazide pyrimidine and 50ml of anhydrous dimethyl sulfoxide into a dry and sealed 150ml round-bottom flask provided with a polytetrafluoroethylene magnetic rotor with two sharp ends of 4cm, stirring at 300rpm, heating in an oil bath to 170 ℃, and then continuing stirring until the semicarbazide pyrimidine is completely dissolved;
2) removing the heating, and immediately slowly injecting and adding 2.75g of ethyl isocyanate methacrylate into the aminopyrimidinone solution obtained in the step 1) to form a reaction system;
3) cooling the reaction system by using a water bath to react, and simultaneously fully stirring at a rotating speed of 500rpm to gradually generate white precipitates in the system;
4) repeatedly washing the suspension obtained in the step 3) by using acetone to leave a white solid, and then, carrying out vacuum drying to remove the residual acetone and dimethyl sulfoxide to obtain a white powdery UPyMA product;
the supramolecular monomer UPyMA prepared in this example was dissolved in deuterated chloroform1H-NMR nuclear magnetic tests, the results are shown in FIG. 1. Observation of the display of UPyMA in deuterated chloroform1H-NMR spectrum, the following chemical shifts and their corresponding H atoms of different chemical environments can be seen: 12.97 (u)1，s，1H，NH)，11.95(u2，s，1H，NH)，10.46(u3S, 1H, NH), 6.20 and 5.56(a, C, s, 2H, C ═ CH)2) 5.64(b, s, 1H, H on aromatic ring), 4.26(d, s, 2H, OCH)2)，3.59(e，m，2H，NCH2)，2.38(f，s，3H，ArCH3)，1.92(g，s，3H，-CH3) It was confirmed that UPyMA of the following structure was obtained. The stable self-assembly quadruple hydrogen bonds are constructed in a hydrophobic cross-linking area formed by the temperature-sensitive polymer by the UPyMA, so that the hydrophobic cross-linking strength is enhanced, and the mechanical strength and the stability of the temperature-sensitive hydrogel are improved.
(3) Commercially available 2-methyl-2-propenoic acid 2- (2-methoxyethoxy) ethyl ester is purchased as MEO2MA；
(4) Adding 0.5g of GelMA and 100ml of deionized water into a 250ml round-bottom flask with a 4cm magnetic rotor with two sharp ends, heating in a water bath at 40 ℃, stirring at 300rpm, and completely dissolving GelMA after 1 hour;
adding 2ml of 1M NaOH aqueous solution, adding 0.1g of UPyMA, keeping stirring, completely dissolving the UPyMA after 10 minutes, and then cooling to 20 ℃ in a water bath;
1ml of MEO was added2MA, 0.03g of potassium persulfate, stirred until dissolved;
sealing the system, inserting a long needle which is introduced with nitrogen below the liquid level of the system, inserting a short needle at the bottle mouth, stirring in water bath at 20 ℃ at 300rpm for 1 hour to remove oxygen in the system, and then pulling out all needle heads and sealing the container;
adding 1ml of deionized water into a 5ml sample bottle, then dripping 50 mul of tetramethylethylenediamine, uniformly mixing, extending a needle with nitrogen below the liquid level for 5 minutes to remove oxygen, then immediately sucking out by using an injector and injecting into a reaction system to start reaction;
and after reacting for 10 hours, dialyzing the reaction solution by using 5L ultrapure water, changing water once every 6 hours by using a dialysis bag with the cut-off molecular weight of 8000-14400, dialyzing for 3 days, taking out the liquid in the dialysis bag, and freeze-drying to finally obtain a white dry solid temperature-sensitive polymer Gel-MEO-UPy (hydrogen bond self-assembly low-critical phase transition temperature-sensitive hybrid polymer).
The macromonomer GelMA and the temperature sensitive polymer Gel-MEO-UPy prepared in this example were subjected to1H-NMR nuclear magnetic tests, the results are shown in FIG. 2. Observation of GelMA in DMSO-d6As shown in1H-NMR spectrum, peaks a, b at 5.6ppm and 5.3ppm chemical shifts, which represent the hydrogen atom on the methylene group attached to the unreacted primary amine, demonstrate successful grafting of the double bond on the gelatin molecule. GelMA is a molecular monomer based on gelatin, and has biocompatibility and bioactivity similar to that of gelatin. The concrete expression is as follows: low immunogenicity, support cell adhesion, and be degraded by various enzymes such as matrix metalloproteinases. Observation of Gel-MEO-UPy in DMSO-d6As shown in1H-NMR spectrum, first, peaks e, f, g at 3.4ppm, 3.9ppm, 3.2ppm confirmed MEO2The presence of the MA fragment, peak h at 5.755ppm chemical shift, represents a hydrogen atom on the UPyMA aromatic ring, evidencing the presence of the UPyMA fragment. Gel-MEO-UPy is a temperature-sensitive polymer, has low critical phase transition temperature lower than the body temperature of a human body and good biocompatibility, and can be used for preparing injectable self-healing temperature-sensitive supramolecular hydrogel with good biocompatibility, bioactivity and biodegradability.
2. Preparation and application of temperature-sensitive injectable hydrogel
(1) Immersing temperature-sensitive polymer Gel-MEO-UPy at 20 deg.C with 10 wt% solid content in pH of 7.4, wherein the component is NaCl 137 mmol/L2.7 mmol/L, Na2HPO410mmol/L，KH2PO4In 2 mmol/L phosphate buffer solution, after swelling completely, slowly stirring until dispersing uniformly to obtain polymer sol;
(2) uniformly mixing the mesenchymal stem cells into the temperature-sensitive polymer sol; cell concentration 106cells/m L, i.e., a concentration of 100 ten thousand cells per ml of sol, the highest concentration not exceeding 107cells/ml。
(3) And (3) transferring the temperature-sensitive polymer sol obtained in the step (2) into an injection tool (medical injector) at room temperature, injecting the temperature-sensitive polymer sol into a cell complete culture medium (89% high-sugar culture medium, 10% fetal calf serum and 1% penicillin-streptomycin double-antibody solution) at the temperature of 37 ℃ to form gel, and performing three-dimensional culture on stem cells. Fresh complete medium at 37 ℃ was replaced daily. With increasing days, stem cells proliferated stably within the gel. The cell number was expanded about 10-fold on the third day, about 15-fold on the fifth day, and about 30-fold on the seventh day, and the gel was lowered to room temperature on different days and centrifuged at 1000rpm for 5min to obtain expanded stem cells, as required.
The temperature-sensitive hydrogel prepared in this example is subjected to oscillatory rheological testing, and the results are shown in FIGS. 3-6, solid data points are the storage modulus of the polymer system, open data points are the loss modulus of the polymer system, when the storage modulus is greater than the loss modulus, the polymer system is in the Gel state, when the storage modulus is less than the loss modulus, the polymer system is in the sol state, FIG. 3 is an amplitude sweep of a 10 wt% Gel-MEO-UPy hydrogel at 37 ℃, the angular frequency is 10rad/s, the storage modulus always remains at about the loss modulus as the amplitude changes from 0.01% to 40%, and the storage modulus and the loss modulus do not substantially change significantly, so that the hydrogel exhibits linear viscoelasticity in this range, which is a linear viscoelastic region, when the amplitude exceeds 1000%, the Gel fails to become a sol state, compared to most temperature-sensitive hydrogels based on L CST, Gel-MEO-UPy has a longer linear viscoelastic region, greater failure is illustrated by a longer linear viscoelastic hydrogel region, and greater than a shorter dynamic change in the Gel storage modulus at a temperature sweep temperature range, the temperature-1 ℃ and the Gel stability is greater than the temperature-1% when the Gel-1 ℃ and the Gel phase storage modulus is greater than the temperature-1% and the temperature-1% of the Gel stability of the Gel phase change is greater.
The temperature-sensitive hydrogel prepared in this example was verified for gel forming performance by injection, and the results are shown in fig. 7. FIG. 7A is a graph of viscosity change of sol of different polymer concentrations at 20 ℃ according to a logarithmic change increase of shear rate obtained by rheometer test and a photograph of sol formed by injecting the sol of 10 wt% Gel-MEO-UPy mixed with tetramethylrhodamine dye into water at 37 ℃ to perform Gel formation, wherein the decrease of viscosity according to the increase of shear rate in the curve indicates that the sol has shear thinning property, 1-101/s is the shear rate in intramuscular injection, subcutaneous injection or intravenous injection, and the viscosity of the sol is not higher than 0.2Pa.s and is approximately equal to the viscosity of edible olive oil; in fig. 7A, a linear gel is formed immediately after the sol at room temperature is injected into water at 37 c by a syringe, and the viscosity curve of fig. 7A and the injection photograph together illustrate the good injectability of the sol phase. FIG. 7B is a Gel conforming to the shape of a mold after injecting 10 wt% Gel-MEO-UPy sol at 20 ℃ into molds of different shapes at 37 ℃; the Gel-MEO-UPy hydrogel has good temperature-sensitive injectable performance, and also has good plasticity, and has important significance for fully filling tissue defects of different shapes when being injected into a body.
The result of cytotoxicity test of the temperature-sensitive polymer Gel-MEO-UPy prepared in this example by using the CCK-8 kit is shown in FIG. 8A, and the result of qualitative and quantitative characterization of cell proliferation of the temperature-sensitive hydrogel three-dimensionally encapsulating the mesenchymal stem cells prepared in this example by using the live-dead staining kit and the CCK-8 kit is shown in FIGS. 9 and 8B.
FIG. 8A shows cytotoxicity of Gel-MEO-UPy polymer, which is obtained by measuring cell viability of mesenchymal stem cells incubated for 48 hours by complete media (89% high sugar media, 10% fetal calf serum, 1% penicillin-streptomycin double antibody solution) with different concentrations of Gel-MEO-UPy polymer, wherein the cell viability is in the range of polymer concentration of 1-5 mg/m L, and increases with the increase of polymer concentration, which indicates that the polymer has the effect of promoting cell proliferation at the extremely low concentration of 1-5 mg/m L, and has no cytotoxicity, and indicates that the Gel-MEO-UPy polymer has the effect of promoting cell proliferation and embodies good biocompatibility.
FIG. 9 is 10 wt% Gel-MEO-UPy sol coated bone marrow mesenchymal stem cells injected into a 37 ℃ mold, then the Gel is formed to carry out three-dimensional culture and a live-dead staining experiment is carried out to qualitatively characterize the proliferation and distribution of the cells, light-colored areas represent live cells, the number of the light-colored areas is obviously increased along with the increase of days, the stable proliferation of the cells is illustrated along with the increase of days, and the lower right corner of FIG. 9 is further amplification of a live-dead staining picture to show the three-dimensional distribution of the swarms of the live cells in the Gel.
FIG. 8B is a CCK-8 assay quantitatively characterizing cell proliferation in a gel, with absorbance proportional to cell number and the increase in absorbance with increasing days of culture indicating a corresponding increase in cell number.
The stable proliferation and high survival rate of the cells in FIGS. 9 and 8B illustrate the good three-dimensional cell culturing ability of the Gel-MEO-UPy hydrogel, indicating that it is a hydrogel with excellent biocompatibility.
The Gel-MEO-UPy hydrogel can still provide stable Gel environment to support the three-dimensional growth of cells when being subjected to three-dimensional culture in a hydrophilic environment on the seventh day, and the pluronic hydrogel collapses within one day.
As can be seen from fig. 3-9, the hydrogel obtained by the present invention requires good biocompatibility, especially in that cells are easy to remain, adhere and proliferate inside the gel; the hydrogel has practical temperature-sensitive performance, good fluidity at room temperature, is in a sol state easy to inject, and is in a stable gel state at the body temperature of a human body.
Example 2
1. Synthesis of temperature-sensitive polymers
(1) GelMA (methacrylic acylated gelatin) Synthesis Using gelatin and methacrylic anhydride
① A250 ml round bottom flask with a 4cm magnetic rotor of PTFE with two tips was charged with 10g of gelatin solids and 100ml0.3M (0.09M Na)2CO3，0.21M NaHCO3)Na2CO3-NaHCO3Sodium bicarbonate water solution, a closed container and uniform stirring at 300rpm, dissolving for 1 hour in water bath at 40 ℃ until the substance in the container is light yellow clear liquid, and adjusting the pH value to 9.0 by using 5M NaOH water solution;
② heating the gelatin solution obtained in step ① to 50 ℃, keeping the stirring at 300rpm, and then slowly injecting and dripping 1ml of methacrylic anhydride into the gelatin solution, wherein the dripping speed is 0.2 ml/min;
③ is added in water bath at 50 ℃ and stirred at 300rpm for full reaction for 2 hours;
④ the reaction was stopped, the product was dissolved, the pH was adjusted to 7.4 using 5M aqueous HCl and then dissolved in a water bath at 40 ℃ for 1 hour;
⑤ purifying the product, dialyzing the liquid obtained in step ④ with 5 liters of ultrapure water, changing water once every 12 hours by using a dialysis bag with the molecular weight cutoff of 8000-14400, dialyzing for 7 days, taking out the liquid after dialysis after 7 days, filtering, and taking out clear liquid;
⑥ lyophilizing the supernatant from step ⑤ to obtain a dried white solid GelMA;
the substitution degree of the anhydride on the gelatin is slightly improved, the reaction speed is mainly improved, and the consumption of the anhydride is reduced. The improvement of the alkalinity of the buffer solution increases the reactivity of the amino and the hydroxyl on the gelatin and accelerates the reaction speed.
(2) Synthesizing UPyMA by using aminopyrimidinone and isocyano ethyl methacrylate;
1) adding 4g of semicarbazide pyrimidine and 100ml of anhydrous dimethyl sulfoxide into a dry and sealed 150ml round-bottom flask provided with a polytetrafluoroethylene magnetic rotor with two sharp ends of 4cm, stirring at 300rpm, heating in an oil bath to 170 ℃, and then continuing stirring until the semicarbazide pyrimidine is completely dissolved;
2) removing the heating, and immediately slowly injecting and adding 4.5g of ethyl isocyanate methacrylate into the aminopyrimidinone solution obtained in the step 1) to form a reaction system;
3) cooling the reaction system by using an ice bath to react, and simultaneously fully stirring at the rotating speed of 500rpm to gradually generate white precipitates in the system;
4) repeatedly washing the suspension obtained in the step 3) by using acetone to leave a white solid, and then, carrying out vacuum drying to remove the residual acetone and dimethyl sulfoxide to obtain a white powdery UPyMA product;
(3) commercially available 2-methyl-2-propenoic acid 2- (2-methoxyethoxy) ethyl ester is purchased as MEO2MA；
(4) Adding 0.4g GelMA and 100ml deionized water into a 250ml round-bottom flask with a 4cm magnetic rotor with two tips, heating in a water bath at 40 ℃, stirring at 300rpm, and completely dissolving GelMA after 1 hour; adding 2ml of 1M NaOH aqueous solution, adding 0.2g of UPyMA, keeping stirring, completely dissolving the UPyMA after 10 minutes, and then cooling to 20 ℃ in a water bath; adding 1ml MEO2MA, 0.02g of potassium persulfate, stirred until dissolved;
sealing the system, inserting a long needle which is introduced with nitrogen below the liquid level of the system, inserting a short needle at the bottle mouth, stirring in water bath at 20 ℃ at 300rpm for 1 hour to remove oxygen in the system, and then pulling out all needle heads and sealing the container;
adding 1ml of deionized water into a 5ml sample bottle, then dripping 100 mul of tetramethylethylenediamine, uniformly mixing, extending a needle with nitrogen below the liquid level for 5 minutes to remove oxygen, then immediately sucking out by using an injector and injecting into a reaction system to start reaction;
and after 10 hours of reaction, dialyzing the reaction solution by using 5L ultrapure water, changing water every 6 hours in a dialysis bag with the cut-off molecular weight of 8000-14400, dialyzing for 3 days, taking out the liquid in the dialysis bag, and freeze-drying to finally obtain a white dry solid temperature-sensitive polymer Gel-MEO-UPy.
2. Preparation and application of temperature-sensitive injectable hydrogel
Immersing the temperature-sensitive polymer Gel-MEO-UPy in a complete culture medium at 20 ℃ with a solid content of 12 wt%, swelling completely and dispersing uniformly to obtain Gel-MEO-UPy complete culture medium sol, and adding 10% of the Gel-MEO-UPy complete culture medium sol6Mixing cells/m L with chondrocytes, blowing, stirring, adding 5% CO at 37 deg.C2The culture box is used for culturing, the gel is taken out after four days of culture, the gel is immersed and incubated for 1 hour by using a type II collagenase solution of 200U/m L, and then the gel is incubatedThe extracellular matrix can be used as an implantation bracket for cooperative stem cells or chondrocytes and can also be used as an in vitro research model of cartilage tissues.
Example 3
1. Synthesis of temperature-sensitive polymers
(1) GelMA Synthesis Using gelatin and methacrylic acid anhydride
① A250 ml round bottom flask with a 4cm magnetic rotor of PTFE with two tips was charged with 10g of gelatin solid and 100ml of 0.2M (0.03M Na)2CO3，0.17M NaHCO3)Na2CO3-NaHCO3Sodium bicarbonate water solution, a closed container and uniform stirring at 300rpm, dissolving for 1 hour in water bath at 40 ℃ until the substance in the container is light yellow clear liquid, and adjusting the pH value to 9.0 by using 5M NaOH water solution;
② heating the gelatin solution obtained in step (1) ① to 50 ℃, keeping the stirring at 300rpm, and then slowly injecting and dripping 1ml of methacrylic anhydride into the gelatin solution, wherein the dripping speed is 0.2 ml/min;
③ is added in water bath at 50 ℃ and stirred at 300rpm for full reaction for 2 hours (1) ② to obtain a system;
④ the reaction was stopped, the product was dissolved, the pH was adjusted to 7.4 using 6M aqueous HCl and then dissolved in a water bath at 40 ℃ for 1 hour;
⑤ purifying the product, dialyzing the liquid obtained in step (1) ④ with 5 liters of ultrapure water, changing water once every 12 hours by using a dialysis bag with the molecular weight cutoff of 8000-14400, dialyzing for 7 days, taking out the liquid after dialysis after 7 days, and filtering to obtain clear liquid;
⑥ lyophilizing the supernatant of (1) ⑤ to obtain dried white solid GelMA;
(2) synthesizing UPyMA by using aminopyrimidinone and isocyano ethyl methacrylate;
① A dry, sealed 150ml round bottom flask with a 4cm magnetic polytetrafluoroethylene rotor with two tips at both ends was charged with 4g semicarbazide pyrimidine and 100ml anhydrous dimethyl sulfoxide, stirred at 300rpm and heated in an oil bath to 170 ℃, followed by continued stirring until the semicarbazide pyrimidine was completely dissolved;
② the heating was removed, and 4.5g of ethyl isocyanate methacrylate was immediately added by slow injection to the aminopyrimidinone solution obtained in (2) ① to form a reaction system;
③ cooling the reaction system by using ice bath to react, and stirring fully at 500rpm to obtain white precipitate;
④ washing the suspension obtained in step (2) ③ repeatedly with acetone to leave a white solid, followed by vacuum drying to remove the remaining acetone and dimethyl sulfoxide to obtain the product UPyMA in the form of white powder;
(3) commercially available 2-methyl-2-propenoic acid 2- (2-methoxyethoxy) ethyl ester is purchased as MEO2MA；
(4) ① putting a 250ml round bottom flask with a 4cm magnetic rotor with two tips of polytetrafluoroethylene, adding 0.5g GelMA and 100ml deionized water, heating in water bath at 40 ℃, stirring at 300rpm, and completely dissolving GelMA after 1 hour;
② adding 2ml of 1M NaOH aqueous solution into the system obtained in (4) ①, adding 0.1g of UPyMA, keeping stirring, completely dissolving the UPyMA after 10 minutes, and then cooling to 20 ℃ in a water bath;
③ Add 2ml MEO to the system2MA, 0.04g of potassium persulfate, stirred until dissolved;
④ sealing the system, inserting long needle filled with nitrogen gas below the liquid level of the system, inserting short needle at the bottle mouth, stirring at 300rpm in water bath at 20 deg.C for 1 hr to remove oxygen in the system, pulling out all needles and sealing the container;
⑤ adding 1ml deionized water into a 5ml sample bottle, then dripping 200 μ l tetramethylethylenediamine, mixing uniformly, extending into the liquid level below the liquid level for 5 minutes by a needle with nitrogen to remove oxygen, then immediately sucking out by a syringe and injecting into the reaction system to start reaction;
⑥ reacting for 10 hours, dialyzing the reaction solution with 5L ultrapure water, changing water every 6 hours for 3 days with the cut-off molecular weight of a dialysis bag being 8000-14400, taking out the liquid in the dialysis bag, and freeze-drying to obtain the white dry solid temperature-sensitive polymer Gel-MEO-UPy.
2. Preparation and application of temperature-sensitive injectable hydrogel
(1) Dispersing a temperature-sensitive polymer Gel-MEO-UPy in a complete culture medium at a solid content of 15 wt% to obtain a polymer sol;
(2) tumor cells were treated with 106The cell/m L is mixed into the temperature sensitive polymer sol;
(3) transferring the sol into an injection tool (medical injector), injecting the sol into any part in the body of an experimental nude mouse, manufacturing a tumor model, and obtaining a mature tumor model within seven days. The method has the advantages that the traditional method for manufacturing the tumor model can only inject the cancer cell suspension into the part with sufficient blood circulation, otherwise the model is easy to establish failure, for example, the tumor model is difficult to form by the cancer cell suspension injected subcutaneously at the back. However, the microenvironment established by the Gel-MEO-UPy hydrogel and having good biocompatibility and bioactivity provides comfortable survival conditions for cancer cells, and conditions are created for establishing a successful tumor model at any part.
The embodiments of the present invention are not limited to the above embodiments, and other changes, modifications, substitutions, combinations and simplifications which are made under the spirit and principle of the present invention should be equivalents and all fall within the protection scope of the present invention.
Claims (9)
1. A method for synthesizing a temperature-sensitive polymer is characterized by comprising the following steps:
1) GelMA Synthesis: dissolving gelatin in a water phase buffer system at 40-45 ℃, adding methacrylic anhydride, and controlling the pH to be 7.4-11.0; reacting for 1-5 hours, stopping the reaction, dissolving the product, purifying the product, and removing the solvent to obtain a product of methacrylic acid acylated gelatin GelMA;
2) synthesis of UPyMA: dissolving 2-amino-6-methyl-4 (1H) -pyrimidone in an anhydrous polar organic solvent at 150-180 ℃ to obtain a 2-amino-6-methyl-4 (1H) -pyrimidone solution; adding isocyano ethyl methacrylate, cooling to room temperature, and fully stirring until white precipitate appears; removing the solvent to obtain a product UpyMA; the structural formula of the UPyMA is as follows:
3) polymerization reaction: mixing the components in a mass ratio of 1: (1-5): (5-10) UPyMA, GelMA and MEOnDispersing three monomers MA into an alkaline aqueous solution with the pH value of 8-11, removing oxygen, adding a redox initiator or a water-soluble initiator at room temperature to perform polymerization reaction until the reaction is sufficient; the MEOnThe structural formula of MA is:
4) purifying the product, and removing the solvent to obtain the temperature-sensitive polymer.
2. The method for synthesizing the temperature-sensitive polymer according to claim 1, wherein the volume mass ratio of the methacrylic anhydride to the gelatin is 1: 0.5 to 10; the mass unit is g; the volume unit is ml; the molar ratio of the 2-amino-6-methyl-4 (1H) -pyrimidone to the isocyano ethyl methacrylate is 1: 1 to 2.
3. The method for synthesizing the temperature-sensitive polymer according to claim 1, wherein the mass ratio of the 2-amino-6-methyl-4 (1H) -pyrimidone to the anhydrous polar solvent is 1: 20-30; the methacrylic anhydride is added in a dropwise manner, and the dropwise adding speed is not faster than 0.4 ml/min.
4. The method for synthesizing the temperature-sensitive polymer according to claim 1, wherein the terminating reaction in step 1) is to dilute the reaction system by 3-10 times using a water phase buffer system with a pH of 7.4 or to adjust the pH of the system to 7.4; the dissolution product is dissolved for 0.5-2 hours at 40-45 ℃ by using a water phase buffer system for terminating the reaction.
5. The method for synthesizing the temperature-sensitive polymer according to claim 1, wherein the purified product of step 1) is obtained by removing solutes with molecular weight less than 3000 and water-insoluble products by dialysis, ultrafiltration or chromatographic separation; the solvent removal in the step 1) is to remove the solvent by freeze-drying or reprecipitation to obtain dry GelMA; the removal of the solvent in step 2) is carried out by repeatedly washing the white precipitate with acetone, or the removal of the solvent in step 2) is carried out by chromatographic separation and then dried; and 4) separating and purifying the solute with the molecular weight of 3000-15000 by dialysis, ultrafiltration or chromatography.
6. The method for synthesizing temperature-sensitive polymer according to claim 1, wherein the aqueous buffer system in step 1) is Na2CO3-NaHCO3Aqueous solution or PBS; the mass ratio of the gelatin to the water phase buffer system is 1: 9-1: 20; the dissolving time is 1-2 hours until the solution is a light yellow clear liquid.
7. The method for synthesizing the temperature-sensitive polymer according to claim 1, wherein the oxidant in the redox initiator is hydrogen peroxide, persulfate or hydroperoxide; the reducing agent is an inorganic reducing agent or an organic reducing agent; the inorganic reducing agent is NaHSO3、Na2SO3And Na2S2O3One or more of; the organic reducing agent is one or more of alcohol, amine, oxalic acid and glucose; the molar ratio of the oxidant to the UPyMA is 2: 3-1: 6.
8. A temperature-sensitive polymer obtained by the synthesis method according to any one of claims 1 to 7.
9. The temperature-sensitive injectable hydrogel prepared by applying the temperature-sensitive polymer according to claim 8, wherein the temperature-sensitive injectable hydrogel is prepared by dispersing the temperature-sensitive polymer in a water phase system at a solid content of 5-20 wt% to form a temperature-sensitive polymer water phase dispersion system, namely the temperature-sensitive injectable hydrogel.
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