WO2020134385A1

WO2020134385A1 - Temperature-sensitive polymer, synthesis method thereof, and temperature-sensitive injectable hydrogel

Info

Publication number: WO2020134385A1
Application number: PCT/CN2019/111529
Authority: WO
Inventors: 任力; 齐大卫; 陈云华; 刘卅
Original assignee: 华南理工大学
Priority date: 2018-12-28
Filing date: 2019-10-16
Publication date: 2020-07-02
Also published as: CN109734851A; CN109734851B

Abstract

Provided are a temperature-sensitive polymer, a synthesis method thereof, and a temperature-sensitive injectable hydrogel. When preparing the temperature-sensitive polymer, first synthesising GelMA and UPyMA; dispersing the three monomers UPyMA, GelMA, and MEOnMA in an alkaline aqueous solution with a pH of 8-11, and removing the oxygen; at room temperature, adding a redox initiator or a water-soluble initiator to make a polymerisation reaction, and continuing until fully reacted; and purifying the product and removing the solvent to obtain a temperature-sensitive polymer. The present temperature-sensitive polymer can formulate an injectable aqueous phase temperature-sensitive polymer sol in a constant pressure environment no higher than a corresponding low critical phase transition temperature and no lower than a water solidification temperature, and the sol can form a gel immediately above the corresponding low critical phase transition temperature. The temperature-sensitive injectable hydrogel has dynamic mechanical properties and good biocompatibility, and can be used in biomedical fields such as tissue engineering, controlled drug release, and three-dimensional cell culture.

Description

Temperature-sensitive polymer, synthesis method thereof and temperature-sensitive injectable hydrogel

Technical field

The invention belongs to the technical field of polymer physics, polymer chemistry, biomedical materials and tissue engineering, in particular to the synthesis of a temperature-sensitive polymer with good biocompatibility and the preparation of a corresponding temperature-sensitive injectable hydrogel and Application method.

Background technique

Hydrogel is a gel with water phase system as dispersion medium. The molecular composition and structure of the gel network are complex and changeable, and have good designability; the aqueous dispersion system can give the gel good biocompatibility. Due to the above characteristics, hydrogels can provide an environment for cells to retain or proliferate, and can also contain drugs or proteins for controlled and sustained release. After the task is completed, they can be biodegraded and discharged as metabolic wastes. Great application value and potential. Therefore, hydrogels containing biocompatible components are often used in biomedical materials or tissue engineering technologies.

When hydrogels are used for in vivo tissue engineering, the implantation methods are divided into two types according to the type of gel: irreversible chemical cross-linked macro hydrogels mostly require surgical implantation; reversible cross-linked macro hydrogels or micro The gel can be implanted by injection. Compared to surgery, injections are gentler and easier. At the same time, chemically crosslinked hydrogels are often potentially toxic. Reversible cross-linked hydrogels are mainly divided into three methods: shear thinning, pH response, and temperature response (temperature sensitive). Shear thinning and pH response may cause the drugs or cells encapsulated in the gel to be in a high shear modulus or inappropriate pH range during or before injection, which may damage the bioactive ingredients encapsulated in the gel Thus affecting the treatment effect. The temperature-sensitive injectable gel is designed so that the sol-gel transition point is in the range of room temperature to body temperature, which can achieve mild sol injection and quickly form a gel at the target implantation site.

Chinese invention patent 2011103868703 discloses a method for synthesizing a temperature-sensitive polymer that can be enhanced in situ, including the following steps: in the presence of a hydroxyl-containing initiator and an amine catalyst, it is obtained by homopolymerization of a double bond functional monomer, or by double The bond functional monomer is obtained by copolymerization with one of carbonate, lactone and lactide, wherein the temperature of the homopolymerization or copolymerization reaction is 50-80°C, and the reaction time is 1-4h. The hydrogel proposed by the present invention is in a liquid form at a lower temperature, and is in a gel form at a physiological temperature. The hydrogel material proposed by the present invention can conveniently introduce biologically active molecules and biological signals, giving the material special Biological activity. The hydrogel proposed by the present invention can gradually degrade under physiological environment. Similar to the existing thermosensitive hydrogels based on low critical phase transition temperature, they mainly rely on the hydrophobic aggregation of polymers above the low critical phase transition temperature to form a crosslinked structure, and the crosslinking point formed by this hydrophobic aggregation has low strength, resulting in unstable The mechanical properties of the gel and the cross-linked structure that are not resistant to swelling greatly limit its scope of application.

Summary of the invention

In order to overcome the shortcomings and shortcomings of the prior art, the object of the present invention is to provide a temperature-sensitive polymer and its synthesis method. The method is simple and easy to perform, with good repeatability, safety and high yield.

Another object of the present invention is to use a temperature-sensitive injectable hydrogel prepared by a temperature-sensitive polymer; the hydrogel has good biocompatibility, especially in that cells are easily retained, adhered, and proliferated inside the gel; the hydrogel The glue has practical temperature-sensitive properties, good fluidity at room temperature, is in a sol state that is easy to inject, and is in a stable gel state at body temperature. The mechanical strength and stability of the gel are good, and the low cytotoxicity refers to the characteristics of temperature-sensitive injectable hydrogels.

There are two types of temperature-sensitive polymers. One is a temperature-sensitive polymer based on a high critical phase transition temperature. Polymerization and crosslinking of polymer segments occur below the phase transition temperature, and occur above the phase transition temperature. Segment dispersion and de-crosslinking; one is a temperature-sensitive polymer based on a low critical phase transition temperature. Dispersion and de-crosslinking of the polymer occurs below the phase transition temperature, and occurs when the polymer is above the phase transition temperature. Gather and crosslink. The temperature-sensitive polymer prepared in the present invention belongs to a temperature-sensitive polymer based on a low critical phase transition temperature. General thermosensitive hydrogels based on low critical phase transition temperature have weak mechanical strength and poor stability. The dynamic quaternary hydrogen bond self-assembly enhancement structure brought by the UPyMA fragment of the invention significantly improves the mechanical strength and stability of the gel without affecting the sol-state fluidity. Moreover, the temperature-sensitive hydrogel in the present invention only forms a cross-linked network through supramolecular interaction, and does not require the participation of any initiator nor the formation of chemical bonds in the formation of the cross-linked network, so it has extremely low cytotoxicity.

The purpose of the present invention is achieved by the following technical solutions:

A method for synthesizing temperature-sensitive polymers includes the following steps:

1) GelMA synthesis: dissolve gelatin in water phase buffer system at 40～45℃, add methacrylic acid anhydride, control pH to 7.4～11.0; react for 1～5 hours, terminate the reaction, dissolve the product, purify the product, remove the solvent, The product methacrylic acid acylated gelatin GelMA is obtained;

2) UPyMA synthesis: dissolve aminopyrimidinone in anhydrous polar organic solvent at 150～180℃ to obtain aminopyrimidinone solution; add isocyanate ethyl methacrylate, cool to room temperature, and stir well until white precipitate appears; The solvent is removed to obtain the product UpyMA; the structural formula of the UPyMA is:

3) Polymerization: Disperse the three monomers of UPyMA, GelMA and MEO _n MA with a mass ratio of 1: (1 to 5): (5 to 10) in an alkaline aqueous solution with a pH of 8 to 11 to remove oxygen. At room temperature, a redox initiator or a water-soluble initiator is added to cause a polymerization reaction until the reaction is sufficient; the structural formula of MEO _n MA is:

n is an integer greater than or equal to 2;

4) Purify the product and remove the solvent to obtain a temperature-sensitive polymer.

To further achieve the object of the present invention, preferably, the volume-mass ratio of the methacrylic anhydride to gelatin is 1: 0.5 to 10; the mass unit is g; the volume unit is ml; and the aminopyrimidone and methacrylic acid The molar ratio of ethyl isocyanate is 1:1 to 2.

Preferably, the mass ratio of the aminopyrimidinone to the anhydrous polar solvent is 1:20-30; the methacrylic acid anhydride is added in a dropwise manner, and the dropwise addition speed is not faster than 0.4 ml/min.

Preferably, the termination reaction described in step 1) is to use an aqueous buffer system with a pH of 7.4 to dilute the reaction system 3 to 10 times or adjust the pH of the system to 7.4; the dissolved product is used to terminate the reaction at 40 to 45°C. The water phase buffer system dissolves for 0.5 to 2 hours.

Preferably, the purified product in step 1) is dialysis, ultrafiltration or chromatographic separation to remove solutes with a molecular weight of less than 3000-15000 and water-insoluble products; the solvent removal in step 1) is by freeze-drying or reprecipitation Remove the solvent to obtain dry GelMA; the solvent removal in step 2) is repeated washing the white precipitate with acetone, or the solvent removal in step 2) is achieved by chromatographic separation, and then dried; the step 4) The purified product is a solute with a molecular weight of 3000-15000 separated and purified by dialysis, ultrafiltration or chromatography.

Preferably, the aqueous buffer system in step 1) is Na ₂ CO ₃ —NaHCO ₃ sodium bicarbonate aqueous solution or PBS; the mass ratio of the gelatin to the aqueous buffer system is 1:9 to 1:20; The dissolution time is 1 to 2 hours until a pale yellow clear liquid.

Preferably, the oxidizing agent 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 NaHSO ₃ , Na ₂ One or more of SO ₃ and Na ₂ S ₂ O ₃ ; the organic reducing agent is one or more of alcohol, amine, oxalic acid and glucose; the molar ratio of the oxidant to UPyMA is 2 :3～1:6.

A temperature-sensitive polymer obtained by the above synthesis method; the temperature-sensitive polymer has a low critical phase transition temperature in an aqueous phase solvent.

A temperature-sensitive injectable hydrogel prepared by using the temperature-sensitive polymer: 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; Or the substance of the auxiliary treatment is evenly mixed into the temperature-sensitive polymer aqueous phase dispersion system to obtain the bioactive substance sol encapsulation system; the bioactive substance sol encapsulation system is heated in situ to above the low critical phase transition temperature of the sol to form bioactivity Substance gel encapsulation system; or form a sol below the low critical phase transition temperature and above the solidification temperature of the bioactive substance sol encapsulation system, and then move into the sol into the injection tool and inject to the temperature above the low critical phase transition temperature Formed in situ to form a gel.

Preferably, the cells are autologous stem cells, corneal endothelial cells, corneal epithelial cells, or chondrocytes; or the cells are ethically derived allogeneic cells for treatment or research;

The substances for adjuvant therapy are antitumor drugs, differentiation promoting drugs, antibiotic drugs.

The MEO _n MA of the present invention is a monomer having a double bond and containing a polyethylene glycol repeating unit.

The LCST initiating the sol-gel transition of the present invention is inversely related to the polymer concentration, the lowest is about 21°C, the corresponding solid content is about 8wt%-20wt%, the highest is about 29°C, and the corresponding solid content is about 4-7wt%.

In the present invention, the temperature-sensitive polymer is dispersed in the aqueous phase system with a solid content of 5wt% to 20wt%, and the solid content should be such that the obtained polymer aqueous dispersion system is at a room temperature under normal pressure and room temperature, Sol state to obtain a temperature-sensitive polymer sol; the temperature-sensitive polymer sol has a low critical phase transition temperature, which can be gelled above this temperature, this temperature-sensitive polymer aqueous system has dynamics such as temperature sensitivity and thixotropy Mechanical properties and good biocompatibility can be used as biomedical materials.

Compared with the prior art, the present invention has the following advantages:

1) UPyMA fragments in polymers can significantly enhance the weaker hydrophobic aggregate cross-linked structure above the low critical phase transition temperature and improve the mechanical stability of the gel, while below the low critical phase transition temperature, due to the collapse of the hydrophobic structure , And can be well dispersed in the water phase environment, without affecting the fluidity and injectability of the sol, thereby improving the overall application potential of the temperature-sensitive hydrogel.

2) The polymer synthesis and gel preparation of the present invention are simple, safe, low cost, and the raw materials are cheap and easily available. The simple realization of the mixing and injection of finished products is extremely easy to operate, and it has great significance for clinical application.

3) As a potential injection implant, the present invention can be gradually degraded in a physiological environment. The degradation products are non-toxic biomolecules such as amino acids and cytosine, and have good biocompatibility. Compared with most synthetic polymer implants, the possibility of rejection is extremely low, and the trouble of surgical removal is also avoided.

BRIEF DESCRIPTION

Figure 1 is the NMR spectrum of UPyMA.

Figure 2 is the nuclear magnetic spectrum of GelMA and temperature-sensitive polymer Gel-MEO-UPy.

Figure 3 is an amplitude scan of 10wt% Gel-MEO-UPy hydrogel at 37°C.

Figure 4 is a frequency scan of 10wt% Gel-MEO-UPy hydrogel at 37°C.

Figure 5 shows the thixotropy of 10wt% Gel-MEO-UPy hydrogel at 37°C.

Figure 6 is a temperature scan of a 10 wt% Gel-MEO-UPy hydrogel.

Fig. 7A is a photograph of the shear viscosity curve of the sol at 20°C and the injection of 10wt% Gel-MEO-UPy sol into 37°C water to form a gel.

7B is a photograph of a gel shape after injection of 10wt% Gel-MEO-UPy sol into a mold at 37°C at 20°C.

FIG. 8A is a histogram of Gel-MEO-UPy polymer cytotoxicity test;

FIG. 8B is a bar graph of the CCK-8 experiment to quantitatively characterize the proliferation of cells in the gel.

Fig. 9 is a laser confocal photograph taken by 10wt% Gel-MEO-UPy sol-coated bone marrow mesenchymal stem cells injected into a mold at 37°C to form a gel for three-dimensional culture and live-dying staining experiments.

detailed description

In order to better understand the present invention, the present invention will be further described below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Example 1

1. Synthesis of temperature-sensitive polymers

(1) Synthesis of GelMA (methacrylic acid acylated gelatin) using gelatin and methacrylic anhydride:

① Put 6g gelatin solid and 60ml 1×PBS in a 250ml round bottom flask with a 4cm two-pointed Teflon magnetic rotor, close the container, stir at 300rpm at a constant speed, dissolve for 1 hour in a 40°C water bath until the content in the container is light Yellow clear liquid;

②Temperature raising step①The obtained gelatin solution was brought to 50°C, kept stirring at 300 rpm, and then 12 ml of methacrylic acid anhydride was slowly added dropwise to it, with a drop rate of 0.2 ml/min;

③ After the dropwise addition, fully react in a 50°C water bath at 300 rpm stirring rate for 4 hours;

④ Stop the reaction, dissolve the product, use 1×PBS to dilute the reaction system with 10 times the volume, and then dissolve in a 40°C water bath for 1 hour;

⑤ Purify the product, 5 liters of ultrapure water dialysis step ④ The obtained liquid, use a dialysis bag with a molecular weight cut-off of 8000 ~ 14400, change the water every 12 hours, dialyze for 7 days, take out the liquid after dialysis after 7 days, at a speed of 9000rpm Centrifuge and take supernatant;

⑥ Freeze-drying step ⑤ The supernatant obtained, to obtain a dry white solid GelMA;

(2) Synthesis of UPyMA (2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-one)-ureido) using aminopyrimidinone and isocyanatoethyl methacrylate ) Ethyl methacrylate);

1) Put 2g of semiuracil and 50ml of anhydrous dimethyl sulfoxide into a dry and sealed 150ml round bottom flask with two tips of 4cm polytetrafluoroethylene magnetic rotor, stir at 300rpm and heat to 170°C in an oil bath, then continue Stir until the semicarbacil is completely dissolved;

2) Remove the heating and immediately add 2.75 g of isocyanatoethyl methacrylate to the aminopyrimidone solution obtained in step 1) to form a reaction system;

3) Use a water bath to cool the reaction system to react, and at the same time fully stir at 500rpm, white precipitate gradually appears in the system;

4) Repeat the washing step 3) using acetone to leave a white solid, and then vacuum dry to remove the remaining acetone and dimethyl sulfoxide to obtain the product as white powder UPyMA;

The supramolecular monomer UPyMA prepared in this example was dissolved in deuterated chloroform and subjected to ¹ H-NMR nuclear magnetic test. The results are shown in FIG. 1. Observing the ¹ H-NMR spectrum displayed by UPyMA in deuterated chloroform, we can see the following chemical shifts and corresponding H atoms in different chemical environments: 12.97 (u ₁ , s, 1H, NH), 11.95 (u ₂ , s , 1H, NH), 10.46 (u ₃ , s, 1H, NH), 6.20 and 5.56 (a, c, s, 2H, C=CH ₂ ), 5.64 (b, s, 1H, aromatic ring H), 4.26 (d, s, 2H, OCH ₂ ), 3.59 (e, m, 2H, NCH ₂ ), 2.38 (f, s, 3H, ArCH ₃ ), 1.92 (g, s, 3H, -CH ₃ ), proof The UPyMA with the following structure was obtained. UPyMA builds a stable self-assembled quaternary hydrogen bond in the hydrophobic cross-linked region formed by the temperature-sensitive polymer, thereby enhancing the hydrophobic cross-linking strength and improving the mechanical strength and stability of the temperature-sensitive hydrogel.

(3) Purchase 2-methyl-2-acrylic acid 2-(2-methoxyethoxy) ethyl ester as MEO ₂ MA;

(4) Add 250g of GelMA and 100ml of deionized water to a 250ml round-bottom flask with a 4cm two-pointed Teflon magnetic rotor, heat in a 40°C water bath, and stir at 300rpm. After 1 hour, GelMA is completely dissolved;

Add 2ml of 1M NaOH aqueous solution, add 0.1g of UPyMA and keep stirring. After 10 minutes, UPyMA is completely dissolved, and then the water bath is cooled to 20°C;

Add 1ml MEO ₂ MA, 0.03g potassium persulfate, stir until dissolved;

To seal the system, insert a long needle passing nitrogen below the liquid level of the system, and insert a short needle at the mouth of the bottle, stirring at 300 rpm under a 20°C water bath for 1 hour to remove oxygen from the system, then pull out all needles and seal the container ;

Add 1ml of deionized water to the 5ml sample bottle, and then drop 50μl of tetramethylethylenediamine. After mixing, use a nitrogen-filled needle to extend below the liquid surface for 5 minutes to remove oxygen. Then immediately aspirate with a syringe and inject The reaction system starts to react;

After 10 hours of reaction, the reaction solution was dialyzed with 5L of ultrapure water, the molecular weight cut off of the dialysis bag was 8000-14400, the water was changed every 6 hours, and dialysis was performed for 3 days. Then, the liquid in the dialysis bag was taken out and lyophilized, finally obtaining a white dry solid Temperature-sensitive polymer Gel-MEO-UPy (hydrogen bond self-assembly low critical phase transition temperature-sensitive hybrid polymer).

The ¹ H-NMR nuclear magnetic test was performed on the macromonomer GelMA and the temperature-sensitive polymer Gel-MEO-UPy prepared in this example, and the results are shown in FIG. 2. Observing the ¹ H-NMR spectrum displayed by GelMA in DMSO-d ₆ , the peaks a and b at 5.6 ppm and 5.3 ppm chemical shifts prove the successful grafting of double bonds on gelatin molecules, and the peak at 3.1 ppm chemical shifts Represents a hydrogen atom attached to the methylene group of unreacted primary ammonia. GelMA is a molecular monomer based on gelatin and has similar biocompatibility and bioactivity as gelatin. The specific performance is: low immunogenicity, can support cell adhesion, and can be degraded by matrix metalloproteinase and other enzymes. Observe the ¹ H-NMR spectrum displayed by Gel-MEO-UPy in DMSO-d _6. First, the peaks e, f, and g at 3.4 ppm, 3.9 ppm, and 3.2 ppm prove the existence of the MEO ₂ MA fragment, 5.755 ppm chemical The peak h at the displacement represents the hydrogen atom on the aromatic ring of UPyMA, proving the existence of the UPyMA fragment. Gel-MEO-UPy is a temperature-sensitive polymer with a low critical phase transition temperature lower than the body temperature of the human body and good biocompatibility, which can be used to prepare good biocompatibility, bioactivity, biodegradability Injectable self-healing temperature-sensitive supramolecular hydrogel.

2. Preparation and application of temperature-sensitive injectable hydrogel

(1) The temperature-sensitive polymer Gel-MEO-UPy is immersed in a pH value of 7.4 at 20 ℃ with a solid content of 10wt%, the composition is NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂ HPO ₄ 10mmol/L , KH ₂ PO ₄ 2mmol/L phosphate buffer solution, after swelling is complete, slowly stir until the dispersion is uniform, to obtain a polymer sol;

(2) The bone marrow mesenchymal stem cells are evenly mixed into the temperature-sensitive polymer sol; the cell concentration is 10 ⁶ cells/mL, which is the concentration of 1 million cells per ml of sol. The highest concentration does not exceed 10 ⁷ cells/ml.

(3) Move the temperature-sensitive polymer sol obtained in step (2) into an injection tool (medical syringe) at room temperature and inject into a cell complete medium (89% high glucose medium, 10% fetus) at a temperature of 37°C Bovine serum, 1% penicillin-streptomycin double antibody solution) formed a gel, and three-dimensional culture of stem cells was performed. Change fresh complete medium at 37°C every day. As the number of days increases, stem cells proliferate steadily in the gel. On the third day, the number of cells expanded by about 10 times, on the fifth day, the number of cells expanded by about 15 times, and on the seventh day, the number of cells expanded by about 30 times. Depending on the needs, the gel was lowered to room temperature on different days at 1000 rpm Centrifuge for 5 min to obtain expanded stem cells.

The temperature-sensitive hydrogel prepared in this example was subjected to an oscillating rheological test. The results are shown in Figures 3-6. The solid data points are the storage modulus of the polymer system, and the hollow 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 a gel state; when the storage modulus is less than the loss modulus, the polymer system is in a sol state. Figure 3 shows the amplitude sweep of the 10wt% Gel-MEO-UPy hydrogel at 37°C with an angular frequency of 10 rad/s. As the amplitude changes from 0.01% to 40%, the storage modulus is always about the loss modulus and stored There is basically no obvious change in modulus and loss modulus, so the hydrogel exhibits linear viscoelasticity within this range. This amplitude range is a linear viscoelastic region. When the amplitude exceeds 1000%, the gel breaks down and becomes a sol Compared with most LCST-based thermosensitive hydrogels, Gel-MEO-UPy hydrogels have a longer linear viscoelastic zone and greater strain to failure, indicating that Gel-MEO-UPy hydrogels are stable Linear viscoelasticity. Fig. 4 is the frequency sweep of 10wt% Gel-MEO-UPy hydrogel at 37℃, the amplitude is 1%, the lower the frequency, the longer the time zone, from the frequency of 0.1rad/s representing the longer time zone to the representative At a frequency of 100 rad/s in the short time zone, the storage modulus is always greater than the loss modulus, and the storage modulus does not decrease significantly, indicating that Gel-MEO-UPy hydrogels have good mechanical stability in the long and short time zones. Figure 5 shows the thixotropy of 10wt% Gel-MEO-UPy hydrogel at 37℃, the angular frequency is constant at 10rad/s, the amplitude is periodically switched between 1% and 2000%, within the range of 1% amplitude, storage The modulus is always greater than the loss modulus, and there is no significant change between the two. It is in a stable gel state. When the storage modulus is 2000%, the loss modulus is greater than the storage modulus, indicating that the gel structure is destroyed by large amplitude oscillations. When recovering from 2000% to 1%, the storage modulus immediately returns to a state greater than the loss modulus, and with time, the storage modulus increases steadily, and the difference between the storage modulus and the loss modulus also gradually increases It shows that Gel-MEO-UPy hydrogel can self-heal in the face of structural damage. The self-healing property is a kind of superior mechanical stability that is beneficial to the application of hydrogel in complex dynamic mechanical environment. Figure 6 is a temperature scan of a 10wt% Gel-MEO-UPy hydrogel with an angular frequency of 10 rad/s, an amplitude of 1%, a heating rate of 1°C/min, and a loss modulus in the range of temperatures below 21°C Greater than the storage modulus, indicating that the polymer system is in a sol state. In the region where the temperature is greater than 21°C, the storage modulus is greater than the loss modulus. At the same time, the difference between the storage modulus, the storage modulus and the loss modulus varies with the temperature. The increase is increasing and the polymer system is in an increasing gel state, indicating that the Gel-MEO-UPy hydrogel has a temperature-sensitive sol-gel transition point based on a low critical phase transition temperature. This property can be used to achieve temperature Minco injection application.

The temperature-sensitive hydrogel prepared in this example was subjected to injection gelation performance verification, and the results are shown in FIG. 7. Fig. 7A shows the change curve of the viscosity of the sol with different polymer concentrations at 20℃ obtained by the rheometer test when the logarithmic change rate increases with the 10wt% Gel-MEO-UPy sol mixed with tetramethylrhodamine The photo of the dye after injection into 37℃ water to form a gel. The viscosity of the curve decreases with the increase of the shear rate, indicating that the sol has shear thinning properties. 1～10 1/s is for intramuscular, subcutaneous or intravenous injection. The shear rate at this time, the viscosity of the sol is not higher than 0.2Pa.s, which is roughly equivalent to the viscosity of edible olive oil; in FIG. 7A, the sol at room temperature forms a linear shape immediately after being injected into 37°C water through a syringe The gel, the viscosity curve of FIG. 7A and the injection photograph together illustrate the good injectability of the sol state. Fig. 7B shows that 10wt% Gel-MEO-UPy sol at 20°C is injected into different shapes of molds at a temperature of 37°C to form a gel conforming to the shape of the mold; it shows that Gel-MEO-UPy hydrogel has good temperature sensitivity The injection performance also shows that the injectable hydrogel has good plasticity, which is of great significance for its injection into the body to fully fill tissue defects of different shapes.

Using the CCK-8 kit, the temperature-sensitive polymer Gel-MEO-UPy prepared in this example was subjected to a cytotoxicity test. The results are shown in FIG. 8A. The three-dimensional temperature-sensitive hydrogel encapsulating bone marrow mesenchymal stem cells prepared in the application section of this example The qualitative and quantitative characterization of cell proliferation was carried out using live death staining kit and CCK-8 kit. The results are shown in Figure 9 and Figure 8B.

Figure 8A is the cytotoxicity of Gel-MEO-UPy polymer by measuring the complete medium (89% high glucose medium, 10% fetal bovine serum, 1% penicillin-streptomyces) with different Gel-MEO-UPy polymer concentrations The cell viability of bone marrow mesenchymal stem cells after 48 hours of incubation is obtained. The cell viability is in the range of 1 to 5 mg/mL polymer concentration, which increases with the increase of polymer concentration. At a very low concentration of 1 to 5 mg/mL, it can promote cell proliferation without cytotoxicity. It shows that Gel-MEO-UPy polymer can promote cell proliferation and reflects its good biocompatibility.

Fig. 9 shows that 10wt% Gel-MEO-UPy sol-coated bone marrow mesenchymal stem cells are injected into a mold at 37°C to form a gel for three-dimensional culture and live-dying staining experiments to qualitatively characterize the proliferation and distribution of cells. Light-colored areas represent live cells and light-colored cells. The number of colored areas increased significantly with the increase of days, indicating that the cells were steadily proliferating with the increase of days. The lower right corner of Figure 9 is a further enlargement of the photo of the live and dead staining, showing the three-dimensional distribution of the community of living cells in the gel.

Figure 8B is a quantitative characterization of the proliferation of cells in the gel by the CCK-8 experiment. The absorbance is proportional to the number of cells, and the increase in absorbance with the increase in culture days indicates a corresponding increase in the amount of cells.

The stable proliferation and extremely high survival rate of the cells in FIGS. 9 and 8B illustrate the ability of Gel-MEO-UPy hydrogel to culture cells in three dimensions, indicating that it is a hydrogel with excellent biocompatibility.

The Gel-MEO-UPy hydrogel of the present invention can still provide a stable gel environment to support the three-dimensional growth of cells on the seventh day of three-dimensional culture in a hydrophilic environment, and the Pluronic hydrogel collapses within a day .

It can be seen from FIGS. 3-9 that the hydrogel obtained by the present invention requires good biocompatibility, especially in that cells are easy to retain, adhere and proliferate inside the gel; the hydrogel has practical temperature-sensitive properties, It has good fluidity at room temperature, is in a sol state that is easy to inject, and is in a stable gel state at body temperature.

Example 2

1. Synthesis of temperature-sensitive polymers

(1) Synthesis of GelMA (methacrylic acid acylated gelatin) using gelatin and methacrylic anhydride

① Put 10g gelatin solid and 100ml of 0.3M (0.09M Na ₂ CO ₃ , 0.21M NaHCO ₃ ) Na ₂ CO ₃ -NaHCO ₃ sodium bicarbonate in a 250ml round bottom flask with a 4cm two-pointed Teflon magnetic rotor Aqueous solution, closed container, stirring at 300 rpm at a constant speed, and dissolved in a 40°C water bath for 1 hour until the contents of the container are light yellow clear liquid, and the pH value is adjusted to 9.0 using 5M NaOH aqueous solution;

②Temperature raising step①The obtained gelatin solution was brought to 50°C, kept stirring at 300 rpm, and then 1 ml of methacrylic acid anhydride was slowly added dropwise to it, with a drop rate of 0.2 ml/min;

③ After the dropwise addition, the reaction was fully conducted in a 50°C water bath at a stirring rate of 300 rpm for 2 hours;

④ Stop the reaction, dissolve the product, use 5M HCl aqueous solution to adjust the pH to 7.4, and then dissolve in a 40°C water bath for 1 hour;

⑤ Purify the product, 5 liters of ultrapure water dialysis step ④ The obtained liquid, use a dialysis bag with a molecular weight cut-off of 8000 ~ 14400, change the water every 12 hours, dialyze for 7 days, after 7 days, take out the dialyzed liquid, filter, remove liquid;

⑥ The lyophilization step ⑤ the obtained clear liquid to obtain a dry white solid GelMA;

The degree of substitution of acid anhydride on gelatin is slightly increased, which mainly improves the reaction speed and reduces the amount of acid anhydride. It shows that the increase of buffer alkalinity increases the reactivity of amino groups and hydroxyl groups on gelatin, and accelerates the reaction speed.

(2) Synthesis of UPyMA using aminopyrimidinone and isocyanatoethyl methacrylate;

1) In a dry and sealed 150ml round bottom flask with two 4cm Teflon magnetic rotors, add 4g semiuracil and 100ml anhydrous dimethyl sulfoxide, stir at 300rpm and heat to 170°C in an oil bath, then continue Stir until the semicarbacil is completely dissolved;

2) Remove the heating and immediately add 4.5g of ethyl methacrylate isocyanate to the aminopyrimidone solution obtained in step 1) to form a reaction system;

3) Use an ice bath to lower the temperature of the reaction system to react, and at the same time fully stir at 500rpm, white precipitate gradually appears in the system;

(4) Add 0.4g GelMA and 100ml deionized water to a 250ml round bottom flask with a 4cm two-pointed Teflon magnetic rotor, heated in a 40°C water bath, and stir at 300rpm. After 1 hour, GelMA is completely dissolved; add 2ml 1M NaOH Add 0.2g UPyMA to the aqueous solution and keep stirring. After 10 minutes, UPyMA is completely dissolved, and then the water bath is cooled to 20°C; add 1ml MEO ₂ MA, 0.02g potassium persulfate, and stir until dissolved;

Add 1ml of deionized water into a 5ml sample bottle, then drop 100μl of tetramethylethylenediamine, mix it with a nitrogen-filled needle and protrude below the liquid surface for 5 minutes to remove oxygen, then immediately aspirate with a syringe and inject The reaction system starts to react;

After 10 hours of reaction, the reaction solution was dialyzed with 5L of ultrapure water, the molecular weight cut off of the dialysis bag was 8000-14400, the water was changed every 6 hours, and dialysis was performed for 3 days. Then, the liquid in the dialysis bag was taken out and lyophilized, finally obtaining a white dry solid Temperature-sensitive polymer Gel-MEO-UPy.

2. Preparation and application of temperature-sensitive injectable hydrogel

The temperature-sensitive polymer Gel-MEO-UPy was immersed in the complete medium at 20°C at a solid content of 12wt% at room temperature. After the swelling was completed and dispersed uniformly, the Gel-MEO-UPy complete medium sol was obtained. Chondrocytes were mixed at a concentration of ⁶ cells/mL, mixed by pipetting, and then placed in an incubator at 37°C and 5% CO ₂ for incubation. After four days of culture, they were taken out and incubated with 200U/mL type II collagenase solution in the gel. After 1 hour, incubate with a 500 mg/L trypsin solution for 1 min, then cool to room temperature, then aspirate the sol and rinse with PBS to obtain chondrocyte extracellular matrix. The extracellular matrix can be used as a scaffold for cooperating with stem cells or chondrocytes, and can also be used as an in vitro research model for cartilage tissue. This extracellular matrix preparation method benefits from Gel-MEO-UPy's sensitive dynamic sol-gel transition and good biocompatibility, bioactivity, easy operation, and fast matrix growth rate (traditional methods generally require at least one month).

Example 3

1. Synthesis of temperature-sensitive polymers

(1) Synthesis of GelMA using gelatin and methacrylic anhydride

① Put 10g gelatin solid and 100ml of 0.2M (0.03M Na ₂ CO ₃ , 0.17M NaHCO ₃ ) Na ₂ CO ₃ -NaHCO ₃ sodium bicarbonate in a 250ml round bottom flask with a 4cm two-pointed Teflon magnetic rotor Aqueous solution, sealed container, stirring at 300 rpm at a constant speed, and dissolved in a 40°C water bath for 1 hour until the content in the container is a pale yellow clear liquid. Use 5M NaOH aqueous solution to adjust the pH to 9.0;

②Temperature raising step (1)① The obtained gelatin solution was brought to 50°C, kept stirring at 300 rpm, and then 1 ml of methacrylic anhydride was slowly added dropwise to it, and the drop rate was 0.2 ml/min;

③ After the dropwise addition, the system was fully reacted for 2 hours in a water bath at 50°C and a stirring rate of 300 rpm (1) ②The obtained system;

④ Stop the reaction, dissolve the product, use 6M HCl aqueous solution to adjust the pH to 7.4, and then dissolve in a 40°C water bath for 1 hour;

⑤ Purify the product, dialyze with 5 liters of ultrapure water (1) ④ The obtained liquid, use a dialysis bag with a molecular weight cut-off of 8000-14400, change the water every 12 hours, dialyze for 7 days, take out the liquid after 7 days and filter, Take the clear solution;

⑥ Freeze-drying (1) ⑤ The resulting clear liquid to obtain a dry white solid GelMA

①Add 4g semiuracil and 100ml anhydrous dimethyl sulfoxide into a dry and sealed 150ml round bottom flask with two tips of 4cm polytetrafluoroethylene magnetic rotor, stir at 300rpm and heat to 170°C in an oil bath, then continue to stir Until the semicarbacil is completely dissolved;

②Remove the heating and immediately inject 4.5g ethyl methacrylate isocyanate into the aminopyrimidone solution obtained in (2)① to form a reaction system;

③ Use an ice bath to cool the reaction system to react, and at the same time fully stir at 500rpm, white precipitate gradually appears in the system;

④ Repeated washing with acetone (2) ③ The resulting suspension left a white solid, followed by vacuum drying to remove the remaining acetone and dimethyl sulfoxide to obtain the product as a white powder UPyMA;

(4) ① Put 0.5g GelMA and 100ml deionized water in a 250ml round bottom flask with a 4cm two-pointed Teflon magnetic rotor, heated at 40℃ water bath, and stir at 300rpm. After 1 hour, GelMA is completely dissolved;

②Add 2ml of 1M NaOH aqueous solution to the system obtained in (4)①, add 0.1g of UPyMA and keep stirring. After 10 minutes, UPyMA is completely dissolved, and then the water bath is cooled to 20℃;

③Add 2ml MEO ₂ MA, 0.04g potassium persulfate to the system, stir until dissolved;

④Seal the system, insert a long needle with nitrogen gas below the liquid level of the system, and insert a short needle at the mouth of the bottle, stirring at 300 rpm under a 20°C water bath for 1 hour to remove oxygen from the system, then pull out all the needles and seal container;

⑤Add 1ml of deionized water to the 5ml sample bottle, and then drop 200μl of tetramethylethylenediamine, mix it with a nitrogen-filled needle and extend it under the liquid surface for 5 minutes to remove oxygen, then immediately aspirate and inject with a syringe Enter the reaction system to start the reaction;

⑥After 10 hours of reaction, dialyze the reaction solution with 5L ultrapure water. The molecular weight of the dialysis bag is 8000～14400. Change the water every 6 hours and dialyze for 3 days. Then take out the liquid in the dialysis bag and freeze-dry it. Solid temperature-sensitive polymer Gel-MEO-UPy.

2. Preparation and application of temperature-sensitive injectable hydrogel

(1) Disperse the temperature-sensitive polymer Gel-MEO-UPy at a solid content of 15wt% in a complete medium to obtain a polymer sol;

(2) The tumor cells are evenly mixed into the temperature-sensitive polymer sol at a concentration of 10 ⁶ cells/mL;

(3) Transfer the sol to an injection tool (medical syringe) and inject it into any part of the body of the experimental nude mouse to make a tumor model, and obtain a mature tumor model within seven days. The advantage is that the traditional method of manufacturing tumor models can only inject cancer cell suspension into the part with sufficient blood circulation, otherwise the model is very easy to fail, such as the cancer cell suspension injected subcutaneously in the back is difficult to form a tumor model. However, the micro-environment with good biocompatibility and bioactivity established by Gel-MEO-UPy hydrogel provides comfortable living conditions for cancer cells and creates conditions for it to establish a successful tumor model in any location.

The embodiments of the present invention are not limited by the above examples, and other changes, modifications, substitutions, combinations, and simplifications made under the spirit and principle of the present invention should be equivalent replacement methods and belong to the present invention. protected range.

Claims

A method for synthesizing temperature-sensitive polymers, which is characterized by the following steps:

1) GelMA synthesis: dissolve gelatin in water phase buffer system at 40～45℃, add methacrylic acid anhydride, control pH to 7.4～11.0; react for 1～5 hours, terminate the reaction, dissolve the product, purify the product, remove the solvent, The product methacrylic acid acylated gelatin GelMA is obtained;

2) UPyMA synthesis: dissolve aminopyrimidinone in anhydrous polar organic solvent at 150～180℃ to obtain aminopyrimidinone solution; add isocyanate ethyl methacrylate, cool to room temperature, and stir well until white precipitate appears; The solvent is removed to obtain the product UpyMA; the structural formula of the UPyMA is:

3) Polymerization: Disperse the three monomers of UPyMA, GelMA and MEO n MA with a mass ratio of 1: (1 to 5): (5 to 10) in an alkaline aqueous solution with a pH of 8 to 11 to remove oxygen. At room temperature, a redox initiator or a water-soluble initiator is added to cause a polymerization reaction until the reaction is sufficient; the structural formula of MEO n MA is:

n is an integer greater than or equal to 2;

4) Purify the product and remove the solvent to obtain a temperature-sensitive polymer.
The method for synthesizing a temperature-sensitive polymer according to claim 1, wherein the volume-mass ratio of the methacrylic anhydride to gelatin is 1: 0.5 to 10; the unit of mass is g; the unit of volume is ml; The molar ratio of said aminopyrimidinone to ethyl methacrylate isocyanate is 1:1~2.
The method for synthesizing a temperature-sensitive polymer according to claim 1, wherein the mass ratio of the aminopyrimidone to the anhydrous polar solvent is 1:20-30; the methacrylic acid anhydride is added dropwise The speed of addition is not faster than 0.4ml/min.
The method for synthesizing a temperature-sensitive polymer according to claim 1, wherein the termination reaction in step 1) is to use an aqueous buffer system with a pH of 7.4 to dilute the reaction system 3 to 10 times or adjust the pH of the system to 7.4 ; The dissolving product is dissolved at 40 ~ 45 ℃ for 0.5 ~ 2 hours using the water phase buffer system to terminate the reaction.
The method for synthesizing a temperature-sensitive polymer according to claim 1, wherein the purified product in step 1) is dialysis, ultrafiltration or chromatographic separation to remove solutes with a molecular weight of less than 3000-15000 and products insoluble in water ; Step 1) The removal of the solvent is to remove the solvent by lyophilization or reprecipitation to obtain a dry state of GelMA; Step 2) The removal of the solvent is repeated washing of the white precipitate with acetone, or the removal of the step 2) The solvent is achieved by chromatographic separation and then dried; the purified product described in step 4) is a solute with a molecular weight of 3000 to 15000 purified by dialysis, ultrafiltration or chromatography.
The method for synthesizing a temperature-sensitive polymer according to claim 1, wherein the aqueous buffer system in step 1) is Na 2 CO 3 -NaHCO 3 sodium bicarbonate aqueous solution or PBS; the gelatin and water The mass ratio of the phase buffer system is 1:9 to 1:20; the dissolution time is 1 to 2 hours until a pale yellow clear liquid.
The method for synthesizing a temperature-sensitive polymer according to claim 1, wherein the oxidant in the redox initiator is hydrogen peroxide, persulfate, hydroperoxide; the reducing agent is an inorganic reducing agent or Organic reducing agent; the inorganic reducing agent is one or more of NaHSO 3 , Na 2 SO 3 and Na 2 S 2 O 3 ; the organic reducing agent is one of alcohol, amine, oxalic acid and glucose One or more species; the molar ratio of the oxidant to UPyMA is 2:3 to 1:6.
A temperature-sensitive polymer, characterized in that it is obtained by the synthesis method according to any one of claims 1-7.
The temperature-sensitive injectable hydrogel prepared by using the temperature-sensitive polymer according to claim 8, characterized in that the temperature-sensitive polymer is dispersed in the aqueous phase system with a solid content of 5-20 wt% to form a temperature-sensitive polymer Aqueous phase dispersion system; the cells or adjuvant treatment materials are evenly mixed into the temperature-sensitive polymer aqueous phase dispersion system to obtain a bioactive substance sol encapsulation system; the bioactive substance sol encapsulation system is heated in situ to the low criticality of the sol Form a gel encapsulation system of bioactive substances above the phase transition temperature; or form a sol below the low critical phase transition temperature and above the solidification temperature of the bioactive substance sol encapsulation system, then move into the sol into the injection tool and inject until the temperature is higher than The low critical phase transition temperature is bonded in situ to form a gel.
The temperature-sensitive injectable hydrogel according to claim 9, wherein the cells are autologous stem cells, corneal endothelial cells, corneal epithelial cells or chondrocytes; or the cells are ethical Of allogenic cells used for treatment or research;

The substances for adjuvant therapy are antitumor drugs, differentiation promoting drugs, antibiotic drugs.