WO2012136016A1 - Hydrogels which can form covalent crosslink under mild condition rapidly and preparing method thereof - Google Patents

Abstract

Hydrogels which can form covalent crosslink under mild condition rapidly and preparing method thereof are provided. The hydrogels comprise two components of NHS ester of PEG and PEG containing N-cysteine end group. The two components are dissolved and mixed, and the hydrogels are obtained. The hydrogels can form covalent crosslink under mild condition rapidly, and are particularly suitable for surgical sealant.hydrogels which can form covalent crosslink under mild condition rapidly and preparing method thereof

Description

[Invention name established by ISA according to Rule 37.2] Rapid formation of covalently crosslinked hydrogel under mild conditions and preparation method thereof

 Technical field

 The invention relates to a hydrogel and a preparation method thereof, in particular to a hydrogel capable of rapidly forming covalent cross-linking under mild conditions and a preparation method thereof.

 Background technique

 Hydrogel It is a network of hydrophilic polymers, insoluble in water, but can absorb and retain a large amount of water, is highly expanded in aqueous solution, and has good biocompatibility and similarity. They are used in surgical biologics for surgical sealants and adhesives, drug release, tissue repair and tissue engineering.

Existing hydrogel systems are typically formed by chemical or physical action. Gels that chemically crosslink to form water often involve the use of toxic crosslinkers and free radicals, and the resulting hydrogels are generally not biodegradable. On the other hand, physical hydrogels are formed by the action of non-covalent bonds, such as hydrogen bonding, ionic interaction, hydrophobic interaction, and phase transformation. The physical hydrogel thus formed is relatively fragile. Therefore, to meet the clinical needs, hydrogels that form covalent crosslinks without using toxic reagents under mild conditions still face challenges.

Hydrogels have been used clinically as surgical sealants. As an auxiliary means of surgery, surgical sealants are used in surgery for hemostasis, anti-adhesion and plugging, and are widely used in brain surgery, cardiac surgery, chest, abdominal surgery, neurosurgery and the like.

At present, surgical sealant products using hydrogels can be divided into three categories: one is fibrin based on fibrinogen extracted from mammalian blood (bovine blood, pig blood, or human blood). A surgical sealant, or a gelatin protein of cattle, is a blocking agent for the ingredients. Fibrinogen extracted from the blood of mammals (fibrinogen) forms a fibrin gel, such as TisseelTM, catalyzed by thrombin (thrombin) extracted from mammalian blood. , Anke glue, double embroidery. FloSealTM Use bovine gelatin and thrombin. Such products are products derived from blood and are at risk of being contaminated by pathogenic organisms. For example, there may be viruses and pathogens that cause diseases such as AIDS, hepatitis B, mad cow disease, and swine flu. In addition, proteins and thrombin derived from pigs and cattle are heterologous proteins, which may be used in the human body to develop a serious immune response.

Another type of surgical sealant product is the cross-linking of a protein of animal origin under the action of a small molecule aldehyde crosslinker to form a hydrogel, for example, cross-linking using gelain and bovine-derived gelatin to form a hydrogel. US patent US5385606 A method of forming a hydrogel by crosslinking bovine blood-derived albumin and adipaldehyde is described. In addition to the risk of pathogenic biological contamination and allergy to heterologous proteins in animals, the use of small molecular aldehyde crosslinkers is also toxic.

The third type of surgical sealant products are all composed of synthetic biocompatible polymer biomaterials. These products are not at risk of being contaminated by pathogenic organisms and allergic to foreign proteins.

No. 5,410,016 describes the preparation of linear block copolymers using polyethylene glycol and polylactic acid, followed by polymerization of the crosslinked acrylic acid moiety under excitation of light by ester linkage at both ends of the molecule. An aqueous solution of a block copolymer containing acrylate at both ends of the molecule is applied to the site of use to rapidly form a hydrogel (FocalSeal ^® ) in situ under catalyst and light. The downside of this FocalSeal ^® hydrogel system as a surgical sealer is that it is somewhat difficult to control in practice.

 US 6566406 A hydrogel consisting of two solutions is disclosed: a solution of an activated acid derivative of a tetra-branched polyethylene glycol and a short peptide trilysine (Lys-Lys-Lys) a surgical sealant formed by in-situ polymerization of the solution after mixing. This in-situ hydrogel system requires a pH of 9.5 (pH) of the solution. Left and right to form a hydrogel quickly. Under such alkaline conditions, it is easy to produce an effect on local stimuli and an inflammatory reaction.

US 6312725 discloses a hydrogel surgical sealant product (CoSeal ^® ) formed by mixing two solutions in situ. The two solutions are respectively a solution of a tetra-branched polyethylene glycol having a terminal acid group as an activated acid derivative and a solution of a tetra-branched polyethylene glycol having a terminal group of a thiol group, which are mixed and formed by in-situ polymerization by forming a sulfur ester. Hydrogels. The disadvantages of this hydrogel CoSeal ^® are that in addition to the pH of the solution at pH 9.6, the hydrogel can form a hydrogel with local irritations and inflammatory reactions, due to the easy hydrolysis of sulphur bonds in aqueous solution. However, it is unstable, and the hydrogel formed by cross-linking of thioester bonds has a poor stability and a short period of time.

The thioester compound and the N -terminal half-photo-acid compound are mixed under mild conditions (phosphate buffer solution, pH 7-8) to form a new amide bond. This chemical reaction is called natural chemical bonding. WO 2008/131325 describes the formation of hydrogels using a natural chemical linkage as a crosslinking reaction. A solution of a tetra-branched polyethylene glycol having a thiol end group and a solution of a tetra-branched polyethylene glycol having a cysteine end group are used. The two solutions are mixed and in situ polymerized under mild conditions (pH 7-8) by formation of an amide bond to form a hydrogel. As a method of covalent cross-linking of hydrogels, natural chemical linkages have several distinct advantages: 1 Chemically selective, sulphur-only compounds with cysteamine-containing structures, or N -terminal half-leucine The compound reacts to form a new amide bond, which is not interfered by the presence of other thiols and sulfhydryl groups; 2 under mild conditions, does not use catalysts, initiators and other potentially toxic compounds to react efficiently; 3 with other chemical linkages using sulfhydryl groups Unlike the hydrogels formed, the natural chemical ligation reaction creates a new amide bond while also producing a sulfhydryl group on the backbone, thereby providing the resulting hydrogel with better bioadhesion. However, since the two solutions disclosed in WO 2008/131325 form a hydrogel that is slow to form (about 3 minutes), it is not suitable as a surgical sealant.

 Summary of the invention

 It is an object of the present invention to provide a novel hydrogel.

 The technical solution adopted by the present invention is:

 A hydrogel containing two components, A and B, and the structural formula of component A is as shown in formula I

 Formula (I)

 The structural formula of component B is as shown in formula II.

 Formula (II)

 Wherein Y is a linking atom or a linking group, and X is a carbon or a molecular mother core having a branch number of not less than 2, and n is 0 to 200 An integer between , m is an integer between 2 and 32.

 Preferably, the structural formula of component B is as shown in formula III.

 Formula (III)

Wherein R = -H , -CH ₂ CH ₂ COOH or -(CH ₂ ) ₃ NH(NH)CNH ₂ , Y is a linking atom or a linking group, and X is a carbon or a molecule having a branch number of not less than 2 Nucleus, n is an integer between 0 and 200, and m is an integer between 2 and 32.

 Preferably, n is an integer between 50 and 70. In particular, n=57.

 Preferably, m is an integer between 4 and 16. In particular, m=4 and X is C.

 The connecting atom is O , N or C .

 The preparation method of the above hydrogel comprises the following steps:

1.  1) Dissolve the two components A and B in a solution with a pH of 7 to 8 to obtain a mixed solution of components A and B respectively;

1.  2) Mix the mixed solutions of components A and B to obtain a hydrogel.

 Preferably, the mixed solution of the components A and B has a mass volume concentration of 5% to 20%.

 Preferably, the components A and B are mixed in a molar ratio of 4:1 to 1:4.

 Preferably, the solution having a pH of from 7 to 8 is a sodium phosphate buffer.

 The hydrogel of the present invention, after mixing the aqueous solutions of the A and B components, can rapidly react under mild conditions to form a new amide bond, which is not mixed. A covalently crosslinked hydrogel is obtained in 10 seconds. The hydrogel of the present invention is structurally stable, has excellent viscoelasticity, and can be used as a surgical sealant.

 DRAWINGS

 1 and 2 are rheological behavior diagrams of the hydrogel of the present invention.

 detailed description

 A hydrogel containing two components, A and B, and the structural formula of component A is as shown in formula I

 Formula (I)

 The structural formula of component B is as shown in formula II.

 Formula (II)

 Wherein Y is a linking atom or a linking group, and X is a carbon or a molecular mother core having a branch number of not less than 2, and n is 0 to 200 An integer between , m is an integer between 2 and 32.

 Preferably, the structural formula of component B is as shown in formula III.

 Formula (III)

Wherein R = -H , -CH ₂ CH ₂ COOH or -(CH ₂ ) ₃ NH(NH)CNH ₂ , Y is a linking atom or a linking group, and X is a carbon or a molecule having a branch number of not less than 2 Nucleus, n is an integer between 0 and 200, and m is an integer between 2 and 32. Of course, according to the basic common knowledge of those skilled in the art, the number n of repeating units of PEG may be different to meet different needs. The number of branches of the multi-branched molecular nucleus is at least 2, preferably at least 3, so that the A and B components can form a three-dimensional network by living cross-linking reaction. Achieve the purpose of forming a hydrogel. These multi-branched molecular nucleuses can be molecular nucleuses commonly used in the art. When the hydrogel of the present invention is used in a human body, the molecular mother core is preferably those which are degradable in the body and which are not toxic to the human body.

 Preferably, n is an integer between 50 and 70. In particular, n=57.

 Preferably, m is an integer between 4 and 16. In particular, m=4 and X is C . In this case, each branch can form a hydrogel more uniformly.

 The connecting atom is O, N or C . Of course, it can also be other divalent linking groups which can be degraded in the body and have no toxic effect on the human body. The choice of these groups is a routine choice for those skilled in the art.

 The preparation method of the above hydrogel comprises the following steps:

1.  1) Dissolve the two components A and B in a solution with a pH of 7 to 8 to obtain a mixed solution of components A and B respectively;

1.  2) Mix the mixed solutions of components A and B to obtain a hydrogel.

 Preferably, the mixed solution of the components A and B has a mass volume concentration of 5% to 20%.

 Preferably, the components A and B are mixed in a molar ratio of 4:1 to 1:4.

 Preferably, the solution having a pH of from 7 to 8 is a sodium phosphate buffer. Of course, other buffers can also be used.

 The invention will now be further described in conjunction with the examples.

 Synthesis of component A

 The reaction equation is as follows:

 Synthesis of PEG4A-SA:

 Take succinic anhydride 80 mg (molecular weight 100, 0.8 mmol), four-branched polyethylene glycol PEG4A with terminal amino group 1 g (average molecular weight 10k, amino acid 0.4mmol), dissolved in 2 ml of dichloromethane, stirred overnight, the reaction solution was detected, and the ninhydrin reaction was negative (yellow);

 Dry the solvent with nitrogen, add 50 ml of methanol to dissolve all solids, freeze the solution overnight (-20 °C), remove the centrifugation (-9 °C, At 6000 rpm, 20 minutes), remove the supernatant. Repeat the above steps in methanol, freeze, centrifuge, and remove the supernatant for 3 times. Add 50 ml of ether, shake for 10 minutes, and centrifuge ( The supernatant was removed at 20 ° C, 6000 rpm for 20 minutes, and the above purification process was repeated twice, and then dried under vacuum for two days to obtain PEG4A-SA.

¹ H NMR (CDCl ₃ , 500 MHz) δ 3.2-3.8 (m, -O-CH ₂ -CH ₂ -O-), 2.61 (2H, t, J = 7 Hz, -NH-CO-C H ₂ -), 2.48 (2H, t, J = 7 Hz, -C H ₂ -COOH).

 Synthesis of component A ( PEG4A-SA-Osu ):

Take PEG4A-SA (0.4 mmol), N -hydroxysuccinimide (HOSu) 0.115 g (1 mmol), dissolve in 1 ml of acetonitrile, add DCC 206 mg (1 mmol) / 1 ml of dichloromethane, shake overnight and filter The solid precipitate was removed, and the filtrate was evaporated to dryness under reduced pressure. 50 ml of isopropyl alcohol was added to dissolve the residue, and the solution was chilled overnight (-20 ° C), and centrifuged (-9 ° C, 6000 rpm, 20 minutes) to remove the supernatant;

 Repeat the above steps to dissolve in isopropanol, freeze, centrifuge, and remove the supernatant for 3 times. Add 50 ml of ether and shake. After centrifugation (20 ° C, 6000 rpm, 20 minutes, the supernatant was removed, and the above purification process was repeated twice, and then vacuum dried for two days.

¹ H NMR (CDCl ₃ , 500 MHz) δ 3.3-3.8 (m, -O-CH ₂ -CH ₂ -O-), 2.95 (2H, t, J = 7 Hz, -NH-CO-C H ₂ -), 2.80 (4H, s, -N-CO-C H ₂ -C H ₂ -CO-N-), 2.57 (2H, t, J = 7 Hz, -C H ₂ -CO-ON-).

 Of course, other well-known methods can also be used, such as preparing component A.

 Synthesis of component B:

 Component B can be protected by a cysteine such as Boc-Cys(Trt)-OH, or L- Thioproline is obtained by reacting a polymer containing an amino group, a hydroxyl group, or a functional group, and removing a protecting group.

 For example, when using a 4-branched polyethylene glycol (PEG4A) having an amino group as the core of the polymer molecule, Boc-Cys(Trt)-OH is directly reacted, and then the protecting group is removed to obtain component B. The reaction equation is as follows:

[Correct according to Rule 26 25.07.2011]

 Component B can also pass a fully protected cysteine-containing dipeptide, such as Boc-Cys(Trt)-Gly-OH, Boc-Cys(Trt)-Glu(OtBu)-OH and Boc-Cys(Trt)-Arg(Pbf)-OH It is reacted with a polymer containing an amino group, a hydroxyl group, or which can be converted into these functional groups, and a protective group is removed to obtain a reaction.

 A fully protected cysteine-containing dipeptide can be obtained by solid phase synthesis of the Fmoc polypeptide. The reaction equation is as follows:

[Correct according to Rule 26 25.07.2011]

 For example, when using a 4-branched polyethylene glycol (PEG4A) having an amino group as the core of the polymer molecule, The fully protected cysteine-containing dipeptide is directly reacted, and the protecting group is removed to obtain component B. The reaction equation is as follows.

 Synthesis of B1 (Cys-PEG4A)

 Take Boc-Cys(Trt)-OH (0.25 mmol), PEG4A (0.5g, amino 0.2 Methyl), BOP (0.11g, 0.25 mmol) dissolved in dichloromethane (2ml), then added DIEA (44 μL, 0.25 mmol), shake 2 hours, dry the solvent with nitrogen;

 Add 50 ml of methanol to dissolve all solids, freeze the solution overnight (-20 ° C), remove the centrifugation (-9 ° C, 6000 rpm, 20 minutes), remove the supernatant;

 Repeat the above steps for the precipitation, dissolve in methanol, freeze, centrifuge, and remove the supernatant for 3 times. Add 50 ml of ether and shake. Minute, centrifuge (20 ° C, 6000 rpm, 20 minutes, remove the supernatant, repeat the above purification process 2 times, vacuum dry for two days, get Boc-Cys(Trt)-PEG4A .

Silica gel thin layer chromatography (solvent system DCM-MeOH-HOAC = 100:3:1) detected the absence of Boc-Cys(Trt)-OH. The ninhydrin test was pale yellow. ¹ H NMR (CDCl ₃ , 500 MHz) δ 7.40-7.26 (15H, m, -CPh ₃ ), 3.8-3.4 (m, -O-CH ₂ -CH ₂ -O-), 1.41 (9H, s, t-Bu ).

 Add the dried Boc-Cys(Trt)-PEG4A sample to the triisopropylsilane (TIS) containing 1 ml and 1 , 2-dimercaptoethane (EDT) 1 ml of trifluoroacetic acid (30 ml), stirred at room temperature for 2 hours, then the solvent was evaporated under reduced pressure;

 The residue was dissolved in methanol (50 ml), and the methanol solution was frozen overnight (-20 ° C) and centrifuged (-9 ° C, 6000 rpm, 20 minutes), remove the supernatant;

Repeat the above steps, dissolve in methanol, freeze, centrifuge, remove the supernatant three times, add 50 ml of ether, shake for 10 minutes, centrifuge (20 ° C, 6000 rpm, 20 minutes, remove the supernatant, repeat the above purification After 2 passes, it was vacuum dried for two days to give the trifluoroacetate salt of component B1 ( Cys-PEG4A ).

The ninhydrin test showed a dark blue color. The Ellman reagent reacted bright yellow. Indicates that the protecting group has been removed. The trifluoroacetic acid salt was dissolved in an ammonium hydrogencarbonate solution (0.1 M, 25 ml) and lyophilized to give component B1 ( Cys-PEG4A ).

 Synthesis of protected cysteine dipeptides

 Boc-Cys(Trt)-Gly-OH, or Boc-Cys(Trt)-Glu(OBu)-OH, or The synthesis of Boc-Cys(Trt)-Arg(Pbf)-OH uses peptide solid phase synthesis, Fmoc chemistry and tris(2-chlorophenyl)chloromethane resin (2-chlorotrityl). Chloride resin ). Proceed as follows:

 Take resin (1g, 1.55mmol/g), add Fmoc protected amino acid, Fmoc-Gly-OH, or Fmoc-Glu(OBu)-OH, or Fmoc-Arg(Pbf)-OH in dichloromethane (1mmol, 10ml) with diisopropylethylamine (DIEA) (1 ml), after shaking for 30 minutes, the resin was washed three times with dimethylformamide (DMF);

 Add DCM-MeOH-DIEA (8:1:1) 20 ml, shake for 20 minutes with dimethylformamide (DMF The resin was washed three times. Add 20 ml of 20% hexahydropyridine in dimethylformamide, shake for 20 minutes, remove the solvent, use dimethylformamide (DMF) Washing the resin four times, the ninhydrin test is dark blue;

 Take Boc-Cys(Trt)-OH (0.92 g, 2 mmol), BOP (0.88 g, 2 Methyl), dissolved in 8 ml of dichloromethane, then added to DIEA 522 μl (3 mmol), placed in the resin for 10 minutes, shaken for 2 hours;

 The resin was washed three times with dimethylformamide (DMF) and methanol, and the resin was dried under vacuum, and the ninhydrin test was pale yellow;

 The polypeptide resin obtained above was added to a solution of 1% trifluoroacetic acid in dichloromethane, 30 ml, and shaken 20 Minute, collect the solution;

 Repeat to add 30ml of 1% trifluoroacetic acid in dichloromethane, shake for 20 minutes, collect the solution step 4 Then, the solution was combined, 2 ml of pyridine was added, the solvent was removed under reduced pressure, and water was added to precipitate product.

 The precipitate was washed with water until the eluate was neutral and dried under vacuum to give a protected cysteine dipeptide.

 Synthesis of B2

 Take Boc-Cys(Trt)-Gly-OH or Boc-Cys(Trt)-Glu(OBu)-OH , or Boc-Cys(Trt)-Arg(Pbf)-OH (0.25 mmol), PEG4A (0.5g, amino 0.2 mmol), BOP (0.11 g, 0.25 mmol) dissolved in dichloromethane (2 ml), then added DIEA (44 μL, 0.25 mmol), shake 2 Hour, dry the solvent with nitrogen, add 50 ml of methanol to dissolve all solids, freeze the solution overnight (-20 ° C), remove the centrifugation (-9 ° C, 6000 rpm, 20 Minutes), remove the supernatant. Repeat the above steps, dissolve in methanol, freeze, centrifuge, remove the supernatant for 3 times, add 50 ml of ether, shake for 10 minutes, centrifuge (20 ° C, 6000 rpm) , 20 minutes, remove the supernatant, repeat the above purification process 2 times, and then dry in vacuum for two days to obtain Boc-Cys(Trt)-Gly-PEG4A, or Boc-Cys(Trt)-Glu-PEG4A, or Boc-Cys(Trt)-Arg(Pbf)-PEG4A, silica gel thin layer chromatography (solvent system) DCM-MeOH-HOAC = 100:3:1) The presence of unprotected dipeptide was detected, the ninhydrin test was pale yellow, and the structure was confirmed by nuclear magnetic resonance spectroscopy;

 Dry the Boc-Cys(Trt)-Gly-PEG4A, or Addition of trifluoroacetic acid to Boc-Cys(Trt)-Glu-PEG4A, or Boc-Cys(Trt)-Arg(Pbf)-PEG4A samples (30 ML) containing 1 ml of triisopropylsilane (TIS) and 1 ml of 1,2-dimercaptoethane (EDT), stirred at room temperature for 2 hours, and then the solvent was removed under reduced pressure;

 The residue was dissolved in methanol (50 ml), and the methanol solution was frozen overnight (-20 ° C) and centrifuged (-9 ° C, 6000 rpm, 20 minutes), remove the supernatant;

 Repeat the above steps for the precipitation, dissolve in methanol, freeze, centrifuge, and remove the supernatant for 3 times. Add 50 ml of ether and shake. Minutes, centrifuge (20 ° C, 6000 rpm, 20 minutes, remove the supernatant, repeat the above purification process 2 times, vacuum dry for two days, get the component B2 (B-Gly, B-Glu Or B-Arg) trifluoroacetate.

 The ninhydrin test showed a dark blue color. The Ellman reagent reacted bright yellow. Indicates that the protecting group has been removed.

 Dissolve the trifluoroacetate salt of component B2 in ammonium bicarbonate solution (0.1M, 25 ml) and freeze-dry to obtain product B2. .

 Hydrogel formation and preparation

 Take one tube (100 mm length × 13 mm diameter) and place a stirrer (10 mm length × 3 mm) Diameter) into the test tube, control temperature 37 ° C, adjust the stirring speed to 2000 rpm, add 250 μl of component B1, or component B2 sodium phosphate buffer solution ( 0.1 M, pH 7.2) (20% w/v), continue to stir, then add 250 μl of component A sodium phosphate buffer solution (0.1 M, pH 7.2) (20% w/v) ,.

 Take Comparative Example 1, Comparative Example 2, and Comparative Example 3 (US6566406, US6312725, and The hydrogel product disclosed in WO 2008/131325 was tested under the same conditions at the same concentration.

After mixing, the chronograph is started. When the stirrer in the solution stops rotating, the timing is stopped, and the time, that is, the formation time of the hydrogel, is recorded. The results are shown in the following table.

 Table 1 Time of hydrogel formation

sample	Gel time
Component B1	> 100 minutes
Component A: Component B1 (1: 1)	4 seconds
Component A: Component B-Gly ( 1 : 1 )	4 seconds
Component A: Component B-Glu ( 1 : 1 )	10 seconds
Component A: Component B-Arg ( 1 : 1 )	2 seconds
Comparative example 1	20 seconds
Comparative Example 2 Monomer I: Monomer II = 1: 1	30 seconds
Comparative Example 3 Monomer I: Monomer II = 1: 1	4 minutes

 The component B1 of the present invention can form a hydrogel by crosslinking a thiol group to form a disulfide bond, but at a very slow rate.

Although the above-described method for judging the formation time of the hydrogel is relatively rough, the formed network structure may be broken due to the rotation of the stirrer, so that the recording time is longer than the actual hydrogel formation time. However, as long as the test is performed under the same control conditions, the measured time should have a relative reference value.

 Hydrogel dissolution test: 1 Mix equimolar equal volume of component A in sodium phosphate buffer solution (0.1 M, pH 7.2 ) (10%) and component B sodium phosphate buffer solution (0.1 M, pH 7.2) (10%), take two 100 μl portions to two plastic tubules, 10 After minute, check for hydrogel. Add 100 μl of 0.1 M aqueous hydroxylamine solution and 100 μl of 200 mM 2- in two small tubes. a solution of mercaptoethanol, shaking, and the hydrogel is not dissolved;

 2 Take two parts of 100 μl of 10% component B sodium phosphate buffer solution ( 0.1 M, pH 7.2 ) separately to two plastic tubes, after 5 hours are still the solution, overnight, for the hydrogel, treated with the above two solutions, the whole solution of the hydrogel.

 The results show that the hydrogel provided by the present invention is not crosslinked by sulphur bonds, nor is it formed by cross-linking by disulfide bonds.

 Rheological behavior of hydrogels

 Hydrogel formation experiment:

 Note: Since component A and component B are under the above gel formation conditions (20%, 3 7 °C) ) Rapid formation of hydrogels. Under such conditions, the gel formation time cannot be measured on the rheometer and there is not enough operation time. Therefore, in the rheological experiment, the lower polymer concentration and reaction temperature are used (5%, 20 °C) The rheological behavior was studied by slowing down the formation rate of the hydrogel by prolonging the gel formation time.

 Mix equimolar equal volumes of component A in sodium phosphate buffer solution (0.1 M, pH 7.2) (5% w/v And the component B1 sodium phosphate buffer solution (0.1 M, pH 7.2) (5% w/v), measure 500 μl of the solution, and quickly add the solution to the temperature-controlled sample plate (20 ° C) ), adjust the position of the plate (diameter 25 mm) to fill the solution with a 1 mm slit between the plates.

 Install the humidity control device at strain 1 % and frequency 1 Hz Under constant conditions, the oscillatory mode measures the storage modulus (G') and the loss modulus (G'). Take one data point every 15 seconds. Longest measurement time to 300 minutes.

 Frequency sweep experiment: After G' reaches a stable plateau value, the frequency sweep (0.01 Hz) under constant strain (1%) To 10 Hz).

 Tensile scanning experiment: strain scanning (1% to 100%) was performed at a constant frequency (1 Hz).

 The test results are shown in Figures 1 and 2.

 data analysis

 In hydrogel formation experiments, G' and G' The intersection is called the hydrogel formation point, and the corresponding time point is the hydrogel formation time. Stable G' in tension and frequency sweep experiments Values indicate that the hydrogel formed is a covalently crosslinked hydrogel. As can be seen from the figure, the hydrogel of the present invention has a very short formation time and is a covalently crosslinked hydrogel having excellent viscoelasticity.

 The crosslinking mechanism of the hydrogel of the present invention is as follows:

 Through this reaction, A, B of the present invention After the aqueous solution of the components is mixed, the hydrogel can be rapidly formed. According to this response, it is readily known in the art that components A and B are preferably 1 : 1 The molar ratio is mixed. Of course, other molar ratios can also be used according to specific needs, such as A : B = 4 : 1 ～ 1 : 4 , such as 0.25:1 , 0.5:1 , 0.75:1 . , 1:1, 1.25:1, 1.5:1, 1.75:1, 2:1.

 Depending on the components, hydrogels represented by the following structural formulas can be obtained separately.

 Crosslinking of PEG4A-SA-Osu and B-Gly

 Crosslinking of PEG4A-SA-Osu and B-Glu

 Crosslinking of PEG4A-SA-Osu and B-Arg

Of course, if other molecular cores are used, their structure will be slightly different, but they can also be covalently cross-linked to obtain the corresponding hydrogel. The structure is very readily available to those skilled in the art.

Claims (10)

1.  A hydrogel containing two components, A and B, and the structural formula of component A is as shown in formula I

   

   Formula (I)

   The structural formula of component B is as shown in formula II.

   

   Formula (II)

   Wherein Y is a linking atom or a linking group, X is a carbon or a molecular core having a branch number of not less than 2, n is an integer between 0 and 200, and m is An integer between 2 and 32.
2. A hydrogel according to claim 2, wherein: the structural formula of component B is as shown in formula III

   

   Formula (III)

   Wherein R = -H , -CH ₂ CH ₂ COOH or -(CH ₂ ) ₃ NH(NH)CNH ₂ , Y is a linking atom or a linking group, and X is a carbon or a molecule having a branch number of not less than 2 Nucleus, n is an integer between 0 and 200, and m is an integer between 2 and 32.
3. A hydrogel according to claim 1 or 2, wherein: n is an integer between 50 and 70.
4.  A hydrogel according to claim 1 or 2, wherein m is an integer between 4 and 16.
5. A hydrogel according to claim 4, wherein X is C and m = 4.
6. A hydrogel according to claim 1 or 2, wherein the linking atom is O, N or C.
7. A method of preparing a hydrogel according to any one of claims 1 to 6, comprising the steps of:

   Dissolving the two components A and B in a solution having a pH of 7-8, respectively, to obtain a mixed solution of components A and B;

   The mixed solutions of the components A and B were mixed to obtain a hydrogel.
8. The method for producing a hydrogel according to claim 7, wherein the mixed solution of the components A and B has a mass volume concentration of 5% to 20%.
9. The method for producing a hydrogel according to claim 7, wherein the components A and B are mixed in a molar ratio of 4:1 to 1:4.
10. The method for producing a hydrogel according to claim 7, wherein the solution having a pH of from 7 to 8 is a sodium phosphate buffer.

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