Ultrafast gelation, sprayable and fast on-demand degradation polyethylene glycol hemostatic hydrogel as well as preparation method and application of ultrafast gelation, sprayable and fast on-demand degradation polyethylene glycol hemostatic hydrogel

Abstract

The invention provides a polyethylene glycol hemostatic hydrogel. The polyethylene glycol hemostatic hydrogel is prepared by mixing three polyethylene glycol derivatives in an aqueous medium, the three polyethylene glycol derivatives comprise a repeating unit with a structure as shown in a formula (I) and a terminal group with a structure as shown in a formula (II), (III) and (IV). The polyethylene glycol hemostatic hydrogel provided by the invention has the advantages of ultrafast gelation, high mechanical strength, fast degradation as required, simple use and the like. The polyethylene glycol hemostatic hydrogel provided by the invention can be used for sealing and hemostasis of different tissue defects of different animals, especially for sealing and hemostasis of non-compressible tissue wounds; and the material can also be used as a drug sustained-release carrier to be applied to the field of tissue repair engineering materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a polyethylene glycol hemostatic hydrogel with ultra-fast gelation, sprayability, and rapid on-demand degradation, a preparation method thereof, and an application thereof. Background Art

[0002] Uncontrolled bleeding is the main cause of trauma-related deaths, highlighting the urgent need for rapid and effective hemostasis. Traditional hemostatic materials such as gauze and lint have a slow hemostatic speed, are prone to secondary infections after being impregnated, and will also cause secondary injuries due to excessive adhesion in the later stage; among new hemostatic materials, hemostatic powders are prone to form thrombi after hemostasis, and hemostatic sponges absorb blood components to promote blood coagulation, but they will become thick, thus causing compression on surrounding tissues and nerves, leading to complications, and their removal from the body is also a problem. Hydrogels can increase the concentration of local platelets and coagulation factors by adhesion, shortening the coagulation time. At the same time, they can also effectively adhere to tissues, keep the wound site moist, and promote healing. Accidental injuries often result in large-area, complex, and multi-site wounds, which can endanger the lives of patients due to difficult wound treatment. Traditional prefabricated hydrogels form specific macroscopic shapes through cross-linking, which may not be sufficient to handle the above-mentioned extensive and complex tissue injuries. Sprayable hydrogels can be pumped through a spray syringe to form a hydrogel film to protect the wound site, with the advantages of portability and rapid in-situ action. The ability to quickly spray and form a film can form closer contact with surrounding tissues, provide good flexibility for treating extensive and irregular wounds, and achieve rapid sealing. Currently, the hydrogel materials used for hemostasis have limitations such as slow gelation and weak mechanical strength. There is no instant gelation speed, and the hydrogel precursor solution will be diluted or even washed away by the blood at the wound site, making it impossible to form a hydrogel. Hydrogels with insufficient mechanical strength and adhesion performance cannot withstand a large enough bursting pressure and are easily burst open by the large pressure of arterial blood vessels or the heart, resulting in hemostasis failure. In addition, the biocompatibility and degradation performance of bioadhesive hydrogels will also affect their adhesion to wet tissues and wound sealing performance.

[0003] Therefore, it has become a problem to be solved to provide a hydrogel material that can achieve extremely rapid gelation and controllable degradation, while also having strong mechanical strength and excellent tissue adhesion performance. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a polyethylene glycol hemostatic hydrogel with ultra-fast gelation, sprayability, and rapid on-demand degradation, a preparation method thereof, and an application thereof. The hydrogel provided by the present invention can achieve extremely rapid gelation and controllable degradation, while also having strong mechanical strength and excellent tissue adhesion performance.

[0005] The present invention provides a polyethylene glycol hemostatic hydrogel and a preparation method thereof, which are prepared by mixing three polyethylene glycol derivatives in an aqueous medium; the three polyethylene glycol derivatives include repeating units with the structure of formula (I) and end groups with the structures of formula (II), (III), and (IV). The polyethylene glycol hemostatic hydrogel provided by the present invention has the advantages of ultra-fast gelation, high mechanical strength, rapid degradation on demand, and simple use. The polyethylene glycol hemostatic hydrogel provided by the present invention can be used for sealing and hemostasis of different tissue defects in different animals, especially for sealing and hemostasis of non-compressible tissue wounds; it can also be used as a drug sustained-release carrier in the field of tissue repair engineering materials. In addition, the hydrogel provided by the present invention also has a high adjustable space, and the physicochemical properties of the hydrogel can be regulated by adjusting the molecular weight of polyethylene glycol, the grafting rate of phthalaldehyde groups and 2-formylphenylboronic acid groups, the concentration of the precursor solution, etc., to meet the usage requirements of different scenarios.

[0006] Specifically, the present invention provides a polyethylene glycol hemostatic hydrogel with ultra-fast gelation, sprayability, and rapid degradation on demand, which includes component A, component B, and component C; component A includes a main chain with the structure of formula (I) and an end group with the structure of formula (II); component B includes a main chain with the structure of formula (I) and an end group with the structure of formula (III); component C includes a main chain with the structure of formula (I) and an end group with the structure of formula (IV);

[0007]

[0008] Preferably, component A, component B, and component C are linear polyethylene glycol derivatives or multi-arm polyethylene glycol derivatives.

[0009] Preferably, component A, component B, and component C have any one of the structures of formula (Va), formula (Vb), formula (Vc), formula (Vd), and formula (Ve);

[0010]

[0011]

[0012] wherein, a, b, c, d, and e are degrees of polymerization, and R is an end group with the structure of formula (II), (III), or (IV).

[0013] Preferably, the value range of a is 1 to 1000, preferably 40 to 400; the value range of b is 1 to 333, preferably 30 to 150; the value range of c is 1 to 250, preferably 25 to 100; the value range of d is 1 to 166, preferably 20 to 80; the value range of e is 1 to 125, preferably 10 to 50.

[0014] Preferably, it includes 1 to 5 parts by mass of component A, 1 to 10 parts by mass of component B, and 1 to 10 parts by mass of component C.

[0015] Preferably, it further includes an aqueous medium, and the aqueous medium is preferably water, physiological saline or a buffer solution; the buffer solution is preferably PBS buffer solution.

[0016] Preferably, the preparation method of component A includes the following steps:

[0017] (1) React 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid with polyethylene glycol having a hydroxyl group at the end;

[0018] (2) Deprotect the reaction product to obtain component A.

[0019] Preferably, the preparation method of component B includes the following steps:

[0020] a) React polyethylene glycol with 2-formylphenylboronic acid pinacol ester small molecule containing a carboxyl group under the action of a condensing agent and a catalyst to obtain an intermediate product;

[0021] b) Dialyze the intermediate product in water and freeze-dry to obtain component B.

[0022] Preferably, the preparation method of component C includes the following steps:

[0023] a) React p-nitrophenyl chloroformate with polyethylene glycol to obtain an intermediate product;

[0024] b) React the intermediate product with hydrazine monohydrate to obtain a reaction product, dialyze it in water and freeze-dry to obtain component C.

[0025] The present invention also provides a preparation method of the above hydrogel, including the following steps:

[0026] i) Mix component A, component B and component C with an aqueous medium respectively to obtain solution A, solution B and solution C.

[0027] ii) Mix solution A and solution B to obtain solution D.

[0028] iii) Mix solution C and solution D to obtain a hydrogel.

[0029] The present invention also provides an application of the above hydrogel in the preparation of a hemostatic drug.

[0030] Preferably, the hemostatic drug is used for hemostasis of blood vessels and organs.

[0031] Compared with the prior art, the present invention provides a poly(ethylene glycol) hemostatic hydrogel with ultrafast gelation, sprayability, and rapid on-demand degradation, which includes component A, component B, and component C; component A includes a main chain with the structure of formula (I) and an end group with the structure of formula (II); component B includes a main chain with the structure of formula (I) and an end group with the structure of formula (III); component C includes a main chain with the structure of formula (I) and an end group with the structure of formula (IV). The hydrogel network main body of the present invention, poly(ethylene glycol), has good biocompatibility and is convenient for end-group chemical modification. In the present invention, the synergistic effect of phthalaldehyde end groups and 2-formylphenylboronic acid end groups can achieve extremely rapid gelation and controllable degradation, while ensuring that the hydrogel material has strong mechanical strength and excellent tissue adhesion properties. Description of the Drawings

[0032] Figure 1 1H NMR spectrum of tetra-arm poly(ethylene glycol) with phthalaldehyde end groups provided in Example 1 of the present invention;

[0033] Figure 2 1H NMR spectrum of 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyric acid provided in Example 2 of the present invention;

[0034] Figure 3 1H NMR spectrum of tetra-arm poly(ethylene glycol) with 2-formylphenylboronic acid end groups provided in Example 3 of the present invention;

[0035] Figure 4 Schematic diagram of the gelation of FON hydrogel;

[0036] Figure 5 Spraying conditions of hydrogels formed by FPBA-N2H3 hydrogel, OPA-N2H3 hydrogel, and 4aPEG-FPBA solution and 4aPEG-OPA solution at different mixing volume ratios with 4aPEG-N2H3 solution;

[0037] Figure 6 Statistical chart of the gelation time of hydrogels formed by FPBA-N2H3 hydrogel, OPA-N2H3 hydrogel, and 4aPEG-FPBA solution and 4aPEG-OPA solution at different mixing volume ratios with 4aPEG-N2H3 solution;

[0038] Figure 7 Statistical chart of the strength of pigskin-mounted shear and tensile tests of hydrogels formed by FPBA-N2H3 hydrogel, OPA-N2H3 hydrogel, and 4aPEG-FPBA solution and 4aPEG-OPA solution at different mixing volume ratios with 4aPEG-N2H3 solution;

[0039] Figure 8 The diagram showing the state change of the hydrogel provided in Example 4 when sprayed on pigskin under cysteine treatment;

[0040] Figure 9 The degradation curve of the hydrogel provided in Example 4 under the subcutaneous tissue of rats;

[0041] Figure 10 The representative pictures of the hydrogel provided in Example 4 for liver hemostasis, as well as the hemostasis time and blood loss within 3 minutes;

[0042] Figure 11 The schematic diagram of the hydrogel provided in Example 4 for hemostasis of rat abdominal aortic rupture;

[0043] Figure 12 The schematic diagram of the hydrogel provided in Example 4 for hemostasis of rabbit abdominal aortic rupture;

[0044] Figure 13 The schematic diagram of the hydrogel provided in Example 4 for hemostasis of rabbit carotid artery rupture;

[0045] Figure 14 The schematic diagram of three different hydrogels for hemostasis of rabbit abdominal aortic rupture. Detailed implementation mode

[0046] The present invention provides a poly(ethylene glycol) hemostatic hydrogel with ultrafast gelation, sprayability, and rapid on-demand degradation, including component A, component B, and component C; the component A includes a main chain with the structure of formula (I) and end groups with the structure of formula (II); the component B includes a main chain with the structure of formula (I) and end groups with the structure of formula (III); the component C includes a main chain with the structure of formula (I) and end groups with the structure of formula (IV);

[0047]

[0048] In the present invention, the component A, component B, and component C are linear poly(ethylene glycol) derivatives or multi-arm poly(ethylene glycol) derivatives.

[0049] In some specific implementation modes of the present invention, the component A, component B, and component C have any one of the structures of formula (Va), formula (Vb), formula (Vc), formula (Vd), and formula (Ve);

[0050]

[0051]

[0052] Among them, a, b, c, d, e are the degrees of polymerization, and R is the end group of the structure of formula (II)(III)(IV).

[0053] The value range of a is from 1 to 1000, preferably from 40 to 400;

[0054] The value range of b is from 1 to 333, preferably from 30 to 150;

[0055] The value range of c is from 1 to 250, preferably from 25 to 100;

[0056] The value range of d is from 1 to 166, preferably from 20 to 80;

[0057] The value range of e is from 1 to 125, preferably from 10 to 50.

[0058] In the present invention, the hydrogel comprises 1 to 5 parts by mass of component A, 1 to 10 parts by mass of component B, and 1 to 10 parts by mass of component C.

[0059] Among them, the hydrogel comprises 1 to 5 parts by mass of component A, which can be 1, 2, 3, 4, 5, or any value between 1 and 5 parts by mass;

[0060] The hydrogel further comprises 1 to 10 parts by mass of component B, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 1 and 10 parts by mass.

[0061] The hydrogel further comprises 1 to 10 parts by mass of component C, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 1 and 10 parts by mass.

[0062] The mass ratio of component A, component B, and component C is preferably (1 to 2):(2 to 5):(3 to 7), and most preferably 1:4:5.

[0063] The hydrogel further comprises an aqueous medium, and the aqueous medium is preferably water, physiological saline, or a buffer solution; the buffer solution is preferably PBS buffer solution.

[0064] In the present invention, the ratio of the mass of each of component A, component B, and component C to the volume of the aqueous medium is preferably (1 to 1000) mg:1 mL, more preferably (100 to 200) mg:1 mL, and specifically can be 100 mg:1 mL, 120 mg:1 mL, 150 mg:1 mL, 200 mg:1 mL.

[0065] In the present invention, the preparation method of component A comprises the following steps:

[0066] (1) React 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid with polyethylene glycol having a hydroxyl group at the end;

[0067] (2) Deprotect the reaction product to obtain component A.

[0068] Specifically, the present invention first prepares 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid, wherein the 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is prepared according to the following method:

[0069] (1) Prepare 3,4-bis(dibromomethyl)benzoic acid by bromination of 3,4-dimethylbenzoic acid;

[0070] (2) Carry out a hydrolysis reaction on 3,4-bis(dibromomethyl)benzoic acid to obtain 3,4-diformylbenzoic acid;

[0071] (3) React 3,4-diformylbenzoic acid with methanol and a catalyst to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid;

[0072] The present invention prepares 3,4-bis(dibromomethyl)benzoic acid by bromination of 3,4-dimethylbenzoic acid.

[0073] The bromination reaction can adopt the technical solutions well-known to those skilled in the art. The present invention preferably uses N-bromosuccinimide as the bromination reagent, benzoyl peroxide as the free radical initiator, and carbon tetrachloride as the solvent for the bromination reaction.

[0074] The molar equivalent of the N-bromosuccinimide is preferably 3 to 5 times, preferably 4 times, that of the 3,4-dimethylbenzoic acid; the molar equivalent of the benzoyl peroxide is preferably 0.05 to 0.5 times, preferably 0.1 times, that of the 3,4-dimethylbenzoic acid; the volume of the carbon tetrachloride is preferably 10 to 50 times, preferably 20 times, the mass of the 3,4-dimethylbenzoic acid.

[0075] The temperature of the bromination reaction is 70 to 90 °C, preferably 81 °C; the time of the bromination reaction is 10 to 20 h, preferably 15 h.

[0076] After the bromination reaction is completed, filter the reaction mixture; wash the filter cake with ether; combine and concentrate all the filtrates, and then vacuum dry; recrystallize the solid product in acetonitrile to obtain 3,4-bis(dibromomethyl)benzoic acid.

[0077] The present invention preferably uses a rotary evaporator for concentration; the preferred concentration temperature is 30 °C; the preferred concentration is to 10% of the liquid volume.

[0078] The recrystallization can adopt the technical solutions well-known to those skilled in the art.

[0079] The present invention carries out a hydrolysis reaction on 3,4-bis(dibromomethyl)benzoic acid to obtain 3,4-diformylbenzoic acid.

[0080] The hydrolysis reaction can adopt the technical solutions well-known to those skilled in the art. Preferably, the present invention dissolves 3,4-bis(dibromomethyl)benzoic acid in an aqueous solution of sodium carbonate for the hydrolysis reaction.

[0081] The mass-volume concentration of the aqueous sodium carbonate solution is 10%; the volume of the aqueous sodium carbonate solution is preferably 5 to 20 times, preferably 10 times that of 3,4-bis(dibromomethyl)benzoic acid.

[0082] The temperature of the hydrolysis reaction is 60 to 80 °C, preferably 70 °C; the time of the hydrolysis reaction is 3 to 5 h, preferably 4 h.

[0083] After the hydrolysis reaction, concentrated hydrochloric acid is selected to adjust the pH of the reaction solution to 0 to 3, preferably 1; ethyl acetate is selected for extraction; after concentration and vacuum drying, 3,4-diformylbenzoic acid is obtained.

[0084] The present invention reacts 3,4-diformylbenzoic acid with methanol and a catalyst to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

[0085] The catalyst is preferably scandium trifluoromethanesulfonate; the temperature of the reaction is 10 to 40 °C, preferably 25 °C; the time of the reaction is 6 to 24 h, preferably 12 h.

[0086] After the reaction, the crude product can be purified by any one of the following two methods:

[0087] 1. Purification is carried out by silica gel column chromatography to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid. The mobile phase of the column chromatography is preferably n-hexane and ethyl acetate, and the volume ratio is 1:5 to 1:1, preferably 1:3.

[0088] 2. The obtained crude product is dissolved with 5% sodium hydroxide solution using 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid to convert the carboxylic acid group in the crude product into carboxylate sodium, and separated with ethyl acetate, and the aqueous phase is reserved. The aqueous phase is adjusted to pH = 5 again with concentrated hydrochloric acid, separated with ethyl acetate three times, the organic phases are combined, and concentrated by rotary evaporation to obtain the purified 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

[0089] After obtaining 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid, the present invention dissolves 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid and polyethylene glycol with a hydroxyl group at the end in an organic solvent, and obtains component A through esterification reaction, deprotection with trifluoroacetic acid, dialysis and freeze-drying.

[0090] The molar equivalent of the 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is preferably 2 times that of the hydroxyl group; the organic solvent is preferably anhydrous dichloromethane; the selected esterification catalyst is preferably EDCI and DMAP.

[0091] The reaction time is preferably 48 h; the reaction temperature is preferably 25 °C.

[0092] After obtaining the reaction product, the present invention subjects the reaction product to deprotection, dialysis and freeze-drying to obtain the polyethylene glycol derivative.

[0093] The deprotection can adopt the technical solutions well-known to those skilled in the art. The present invention preferably adopts a mixed solvent of trifluoroacetic acid and water. Preferably, the organic acid selected for deprotection is 50% aqueous trifluoroacetic acid solution; the volume of the mixed solvent is preferably 5-10 times (mL / g) of the mass of the reaction product; the deprotection time is preferably 1 h; the deprotection temperature is preferably 25 °C.

[0094] The dialysis and freeze-drying can adopt the technical solutions well-known to those skilled in the art.

[0095] In the present invention, the preparation method of the component B includes the following steps:

[0096] a) Polyethylene glycol reacts with a small molecule of 2-formylphenylboronic acid pinacol ester containing a carboxyl group under the action of a condensing agent and a catalyst to obtain an intermediate product;

[0097] b) The intermediate product is dialyzed in water and freeze-dried to obtain the component B.

[0098] In step a), the polyethylene glycol can be linear polyethylene glycol or multi-arm polyethylene glycol, such as tri-arm polyethylene glycol, tetra-arm polyethylene glycol, hexa-arm polyethylene glycol or octa-arm polyethylene glycol; the number average molecular weight of the polyethylene glycol is preferably 44-44000, more preferably 5000-20000, and most preferably 10000.

[0099] In the above-mentioned preparation steps of the component B provided by the present invention, in step a), the small molecule of 2-formylphenylboronic acid pinacol ester containing a carboxyl group can specifically be 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyric acid; the molar equivalent of the small molecule of 2-formylphenylboronic acid pinacol ester containing a carboxyl group is preferably 1.2-5 times, more preferably 2 times, that of the hydroxyl group in the polyethylene glycol.

[0100] In the above preparation step of component B provided by the present invention, in step a), the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the molar equivalent of the condensing agent is preferably 5 to 10 times, more preferably 6 times, that of the hydroxyl groups in polyethylene glycol; the catalyst is preferably 4-dimethylaminopyridine; the molar equivalent of the catalyst is preferably 2 to 5 times, more preferably 3 times, that of the hydroxyl groups in polyethylene glycol.

[0101] In the above preparation step of component B provided by the present invention, in step a), the reaction temperature is preferably 10 to 40 °C, more preferably 25 °C (room temperature); the reaction time is preferably 24 to 72 h, more preferably 48 h.

[0102] In the above preparation step of component B provided by the present invention, in step a), the reaction is carried out in an organic solvent; the organic solvent is preferably anhydrous dichloromethane; after the reaction is completed, the solvent is dried by suction.

[0103] In the above preparation step of component B provided by the present invention, in step b), it is preferred not to directly dialyze the intermediate product, but to first dissolve the intermediate product in water, filter, and then dialyze the obtained filtrate; the dialysis time is preferably 1 to 5 days, more preferably 3 days.

[0104] In the present invention, the preparation method of component C includes the following steps:

[0105] a) React p-nitrophenyl chloroformate with polyethylene glycol to obtain an intermediate product;

[0106] b) React the intermediate product with hydrazine monohydrate, then dialyze the reaction product in water and freeze-dry to obtain component C.

[0107] In step a), the polyethylene glycol can be linear polyethylene glycol or multi-arm polyethylene glycol, such as tri-arm polyethylene glycol, tetra-arm polyethylene glycol, hexa-arm polyethylene glycol or octa-arm polyethylene glycol; the number-average molecular weight of the polyethylene glycol is preferably 44 to 44000, more preferably 5000 to 20000, and most preferably 10000.

[0108] In the above preparation step of component C provided by the present invention, in step a), the present invention dissolves p-nitrophenyl chloroformate and polyethylene glycol in an organic solvent and reacts in the presence of an acid-binding agent, and then precipitates to obtain an intermediate product.

[0109] In the above preparation step of component C provided by the present invention, in step a), the molar equivalent of p-nitrophenyl chloroformate is 4 to 6 times, preferably 5 times, that of the hydroxyl groups in polyethylene glycol; the organic solvent is preferably dichloromethane; the acid-binding agent is preferably pyridine.

[0110] In the above-mentioned preparation step of component C provided by the present invention, in step a), the reaction time is 12 to 48 h, preferably 24 h; the reaction temperature is 10 to 40 °C, preferably 25 °C.

[0111] In the above-mentioned preparation step of component C provided by the present invention, in step a), the present invention preferably uses anhydrous ether for sedimentation; the sedimented solid is filtered and vacuum dried to obtain an intermediate product.

[0112] In the above-mentioned preparation step of component C provided by the present invention, in step b), the intermediate product obtained in step a) and hydrazine monohydrate are dissolved in an organic solvent and reacted to obtain a reaction product. The molar equivalent of hydrazine monohydrate is 5 to 20 times, preferably 10 times, that of the p-nitrobenzene carbonate group in the intermediate product; the organic solvent is preferably dichloromethane.

[0113] In the above-mentioned preparation step of component C provided by the present invention, in step b), the reaction time is 8 to 24 h, preferably 12 h; the reaction temperature is 0 °C.

[0114] In the above-mentioned preparation step of component C provided by the present invention, in step a), after obtaining the reaction product, the present invention dialyzes and freeze-dries the reaction product to obtain a polyethylene glycol derivative.

[0115] The dialysis and freeze-drying can adopt the technical solutions well-known to those skilled in the art.

[0116] The present invention also provides a preparation method of the above hydrogel, comprising the following steps:

[0117] i) Mix the component A, component B and component C with an aqueous medium respectively to obtain solution A, solution B and solution C.

[0118] ii) Mix solution A and solution B to obtain solution D.

[0119] iii) Mix solution C and solution D to obtain a hydrogel.

[0120] Among them, the mass-volume concentration of component A in solution A can be 1-1000 mg / mL, preferably 10-200 mg / mL, more preferably 100-150 mg / mL; the mass-volume concentration of component B in solution B can be 1-1000 mg / mL, preferably 10-200 mg / mL, more preferably 50-100 mg / mL; the mass-volume concentration of component C in solution C can be 1-1000 mg / mL, preferably 10-200 mg / mL, more preferably 50-100 mg / mL.

[0121] In step ii), the mixing volume ratio of solution A and solution B is 1:1 - 1:5, which can be 1:1, 1:2, 1:3, 1:4, 1:5, or any value between 1:1 - 1:5, preferably 1:4.

[0122] In step iii), solution D and solution C are preferably mixed using a double-barrel syringe, and the resulting hydrogel is then sprayed out via the double-barrel syringe.

[0123] The hydrogel provided by the present invention includes a polyethylene glycol derivative with a 2-formylphenylboronic acid end group and a polyethylene glycol derivative with a phthalaldehyde end group. The polyethylene glycol derivatives provided by the present invention can all undergo efficient coupling reactions with various α-nucleophilic groups such as (acyl)hydrazide groups and amineoxy groups. The polyethylene glycol derivative with a 2-formylphenylboronic acid end group of the present invention can instantaneously form a hydrogel, with mild reaction conditions and controllable degradation using cysteine; the hydrogel formed by the polyethylene glycol derivative with a phthalaldehyde end group can increase tissue adhesion and enhance stability. The three-component polyethylene glycol hydrogel provided by the present invention can be used for sealing and hemostasis of different tissue defects in different animals, especially for sealing and hemostasis of non-compressible tissue wounds.

[0124] By combining the advantages of the polyethylene glycol derivative with a phthalaldehyde end group and the polyethylene glycol derivative with a 2-formylphenylboronic acid end group, a hydrogel tissue adhesive with the polyethylene glycol derivative with a phthalaldehyde end group, the polyethylene glycol derivative with a 2-formylphenylboronic acid end group, and the polyethylene glycol derivative with a hydrazide end group as three components is prepared. By adjusting and optimizing the ratio between the three components, a new hydrogel tissue adhesive material with both ultra-fast gelation and good tissue adhesion performance is obtained.

[0125] The present invention also provides an application of the above hydrogel in the preparation of a hemostatic drug.

[0126] The hemostatic drug is used for hemostasis of organs such as blood vessels, liver, and spleen, especially for hemostasis of arterial blood vessels.

[0127] The hydrogel network main body of the present invention, polyethylene glycol, has good biocompatibility and is convenient for end-group chemical modification. In the present invention, the synergistic effect of the phthalaldehyde end group and the 2-formylphenylboronic acid end group can achieve extremely fast gelation and controllable degradation, while ensuring that the hydrogel material has strong mechanical strength and excellent tissue adhesion performance.

[0128] To further understand the present invention, the following examples are used to illustrate the ultra-fast gelation, sprayable, and rapidly degradable on-demand polyethylene glycol hemostatic hydrogel provided by the present invention, its preparation method, and its application. The protection scope of the present invention is not limited by the following examples.

[0129] Example 1

[0130] Dissolve 1,3 - dimethoxy - 1,3 - dihydroisobenzofuran - 5 - carboxylic acid (5 g) in 300 mL of dichloromethane, and add EDCI (6.4 g) and DMAP (0.7 g) for pre - reaction for 30 min. Subsequently, add 200 mL of a dichloromethane solution of tetra - arm polyethylene glycol with a hydroxyl - terminated end (number - average molecular weight 20000, 167.2 g), and react for 48 h. After the reaction is completed, concentrate and rotary - evaporate to dryness, dissolve with dichloromethane (400 mL), wash with water, rotary - evaporate the organic phase to dryness again, add 180 mL of an aqueous solution of trifluoroacetic acid (V trifluoroacetic acid:V water = 1:1) and react for 1.5 h to deprotect. After the reaction is completed, transfer to a dialysis bag for dialysis and lyophilization to obtain a white solid (48 g, 87%). Perform nuclear magnetic resonance hydrogen spectrum analysis on the tetra - arm polyethylene glycol with phthalaldehyde - terminated end prepared in this example, and the results are as Figure 1 shown. Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the tetra - arm polyethylene glycol with phthalaldehyde - terminated end provided in Example 1 of the present invention.

[0131] Example 2

[0132] Synthesize 4 - (3 - formyl - 4 - (4,4,5,5 - tetramethyl - 1,3,2 - dioxaborolan - 2 - yl)phenoxy)butyric acid according to the following chemical reaction route:

[0133]

[0134] The specific preparation process is as follows:

[0135] 1) Dissolve 2 - bromo - 5 - hydroxybenzaldehyde (8.0 g) and ethyl 4 - bromobutyrate (9.4 g) in 80 mL of anhydrous dimethylformamide, add potassium carbonate (8.2 g), and react at room temperature for 12 h; after the reaction is completed, mix the reaction solution with 1000 mL of n - hexane / ethyl acetate (1 / 1, v / v), wash three times with water, and dry the organic phase with anhydrous sodium sulfate; purify the crude product by silica gel column chromatography, and the eluent is n - hexane / ethyl acetate (9 / 1, v / v) to obtain a colorless viscous liquid of ethyl 4 - (4 - bromo - 3 - formylphenoxy)butyrate (3.3 g, yield 52.35%);

[0136] 2) Ethyl 4-(4-bromo-3-formylphenoxy)butyrate (5.4 g), bis(pinacolato)diboron (4.73 g), dichlorobis[(1,1'-bis(diphenylphosphino)ferrocene)palladium(II)] (0.37 g), and potassium acetate (4.9 g) were dispersed and dissolved in 67.5 mL of anhydrous dioxane, and the mixture was refluxed at 100 °C for 2 h; after the reaction was completed, the solvent was evaporated to dryness, the residue was dissolved in dichloromethane, washed with water three times, and dried over anhydrous sodium sulfate; the crude product was purified by silica gel column chromatography with the eluent of n-hexane / ethyl acetate (9 / 1, v / v) to obtain ethyl 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyrate as a colorless viscous liquid (5.14 g, yield 81.31%);

[0137] 3) Ethyl 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyrate (5.14 g) was stirred and mixed evenly with 3.43 mL of trifluoroacetic acid and 30 mL of water, and the reaction was carried out at 90 °C for 3 h; after the reaction was completed, the solvent was evaporated to dryness, and the crude product was purified by silica gel column chromatography with the eluent of dichloromethane / methanol (19 / 1, v / v) to obtain 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyric acid as a colorless viscous liquid (4.39 g, yield 38%). The 1H NMR analysis of 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyric acid prepared in this example was carried out, and the results are as Figure 2 shown, Figure 2 which is the 1H NMR spectrum of 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyric acid provided in Example 2 of the present invention.

[0138] Example 3

[0139] Synthesize a four-armed polyethylene glycol with 2-formylphenylboronic acid at the end according to the following chemical reaction route:

[0140]

[0141] wherein x represents the degree of polymerization and y represents the number of arms of the multi-armed polyethylene glycol. In this example, y = 4.

[0142] The specific preparation process is as follows:

[0143] Dissolve tetra-arm polyethylene glycol (number-average molecular weight 10,000, 4.17 g) and 4-(3-formyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butyric acid (4.39 g) in 56 mL of dichloromethane. Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.92 g) and 4-dimethylaminopyridine (0.611 g), and react at room temperature for 48 h. After the reaction, drain the solvent, dissolve the residue in water, filter, dialyze the filtrate in deionized water for three days, and freeze-dry to obtain tetra-arm polyethylene glycol with 2-formylphenylboronic acid as the terminal group (4.0 g, yield close to 100%);

[0144] The chemical structure of the tetra-arm polyethylene glycol with 2-formylphenylboronic acid as the terminal group prepared in this example is shown in formula (Vc), where c = 57 and R is the structure of formula (III).

[0145] Perform nuclear magnetic resonance hydrogen spectrum analysis on the tetra-arm polyethylene glycol with 2-formylphenylboronic acid as the terminal group prepared in this example. The results are as Figure 3 shown, Figure 3 which is the nuclear magnetic resonance hydrogen spectrum of the tetra-arm polyethylene glycol with 2-formylphenylboronic acid as the terminal group provided in Example 3 of the present invention.

[0146] Example 4

[0147] Dissolve tetra-arm polyethylene glycol with hydrazine group as the terminal group (4aPEG-N2H3) and tetra-arm polyethylene glycol with 2-formylphenylboronic acid as the terminal group (4aPEG-FPBA) in PBS solution respectively to prepare solutions with a mass fraction of 10%. Dissolve tetra-arm polyethylene glycol with phthalaldehyde as the terminal group (4aPEG-OPA) in PBS solution to prepare a solution with a mass fraction of 15%. Mix the 4aPEG-FPBA solution and the 4aPEG-OPA solution evenly according to a volume ratio of 4:1 to form solution A. Add solution A and the 4aPEG-N2H3 solution in equal volume to a glass bottle and mix evenly. Invert the glass bottle. If there is no flowing liquid in the bottle, the hydrogel (FON hydrogel) has been formed. Figure 4 It is a schematic diagram of the gel formation of the FON hydrogel.

[0148] Example 5

[0149] Dissolve the four-armed polyethylene glycol with a hydrazine group at the end (4aPEG-N2H3) and the four-armed polyethylene glycol with 2-formylphenylboronic acid at the end (4aPEG-FPBA) in PBS solution respectively to prepare a 10% (mass fraction) solution; dissolve the four-armed polyethylene glycol with o-phthalaldehyde at the end (4aPEG-OPA) in PBS solution to prepare a 15% (mass fraction) solution. Mix the 4aPEG-FPBA solution and the 4aPEG-OPA solution in different volume ratios of 1:1, 2:1, 3:1, and 4:1 evenly, and then add them to a double-barreled syringe in equal volume with the 4aPEG-N2H3 solution and spray them onto a glass plate perpendicular to the spraying direction through a nozzle; add the 4aPEG-FPBA solution and the 4aPEG-OPA solution to a double-barreled syringe in equal volume with the 4aPEG-N2H3 solution respectively and spray them onto a glass plate perpendicular to the spraying direction through a nozzle. Figure 5 The spraying conditions of the FPBA-N2H3 hydrogel, the OPA-N2H3 hydrogel, and the hydrogels formed by the 4aPEG-FPBA solution and the 4aPEG-OPA solution in different mixing volume ratios with the 4aPEG-N2H3 solution.

[0150] From Figure 5 It can be seen that there is a situation of the precursor solution sliding down after the hydrogels of the latter four ratios are sprayed onto the vertical glass plate, while the hydrogels with a volume ratio of 4:1:5 and the FPBA-N2H3 hydrogel have no liquid sliding, indicating that these two hydrogels can achieve instantaneous gelation, which is beneficial for rapid hemostasis.

[0151] Example 6

[0152] The four-armed polyethylene glycol with a hydrazine group at the end (4aPEG-N2H3) and the four-armed polyethylene glycol with 2-formylphenylboronic acid at the end (4aPEG-FPBA) were separately dissolved in PBS solution to prepare solutions with a mass fraction of 10%; the four-armed polyethylene glycol with o-phthalaldehyde at the end (4aPEG-OPA) was dissolved in PBS solution to prepare a solution with a mass fraction of 15%. The 4aPEG-FPBA solution and the 4aPEG-OPA solution were respectively added to a glass bottle in equal volume with the 4aPEG-N2H3 solution and mixed evenly, then placed in a water bath at 37°C to keep constant temperature. Invert the glass bottle. If there is no flowing liquid in the bottle, a hydrogel has been formed (FPBA-N2H3 hydrogel and OPA-N2H3 hydrogel, where FPBA-N2H3 hydrogel is a gel prepared by mixing the 4aPEG-FPBA solution and the 4aPEG-N2H3 solution in a volume ratio of 1:1; OPA-N2H3 hydrogel is a gel prepared by mixing the 4aPEG-OPA solution and the 4aPEG-N2H3 solution in a volume ratio of 1:1). The 4aPEG-FPBA solution and the 4aPEG-OPA solution were mixed evenly in different volume ratios of 1:1, 2:1, 3:1, 4:1, and then added to a glass bottle in equal volume with the 4aPEG-N2H3 solution and mixed evenly, and placed in a water bath at 37°C to keep constant temperature. Taking 1 second as a time point, invert the glass bottle and observe the sample. If the solution in the bottle is still in a flowing state, repeat the previous steps. If it does not flow and remains unchanged for 30 seconds, it is determined that gelation has occurred at this time. Record the time from mixing to the transformation into a hydrogel for each sample, that is, the gelation time. Figure 6 It is a statistical chart of the gelation time of FPBA-N2H3 hydrogel, OPA-N2H3 hydrogel, and the hydrogels formed by mixing the 4aPEG-FPBA solution and the 4aPEG-OPA solution with the 4aPEG-N2H3 solution at different mixing volume ratios.

[0153] From Figure 6 it can be seen that the gelation time of the hydrogels in the latter four ratios is relatively long, which is not conducive to rapid gelation for hemostasis and sealing. However, the hydrogel with a volume ratio of 4:1:5 can achieve a gelation speed of about 1 s. The FPBA-N2H3 hydrogel can gel within 1 s, which is consistent with the situation in Example 5.

[0154] Example 7

[0155] Cut the pig skin into 2.5 cm × 2.5 cm squares and glue one side to a 2.5 cm wide rectangular wooden piece with 502 glue. Dissolve tetra-arm polyethylene glycol with hydrazide group at the end (4aPEG-N2H3) and tetra-arm polyethylene glycol with 2-formylphenylboronic acid at the end (4aPEG-FPBA) in PBS solution respectively to prepare 10% (mass fraction) solutions; dissolve tetra-arm polyethylene glycol with o-phthalaldehyde at the end (4aPEG-OPA) in PBS solution to prepare a 15% (mass fraction) solution. After mixing the 4aPEG-FPBA solution and the 4aPEG-OPA solution evenly according to the volume ratios of 1:1, 2:1, 3:1, and 4:1, take 25 μL and spread it evenly on a piece of pig skin. At the same time, take 25 μL of the 4aPEG-N2H3 solution and spread it on another piece of pig skin. After spreading, quickly pair them up, press and fix them with a 200 g weight, and place them in a 37 °C water bath for incubation for 2 h. After completion, use a universal testing machine to measure its tensile strength. Figure 7 It is a statistical chart of the shear tensile test strength of pig skin carrying hydrogels formed by FPBA-N2H3 hydrogel, OPA-N2H3 hydrogel, and hydrogels formed by 4aPEG-FPBA solution and 4aPEG-OPA solution at different mixing volume ratios with 4aPEG-N2H3 solution.

[0156] From Figure 7 it can be seen that the hydrogel with a volume ratio of 4:1:5 has better adhesion strength, which is beneficial to wound sealing.

[0157] Example 8

[0158] Spray the hydrogel provided in Example 4 on the surface of pig skin through a double-barreled syringe. Subsequently, cover half of the hydrogel with a gauze soaked in cysteine PBS solution, place it in a 37 °C shaker for incubation, take it out after 30 min and take pictures to observe the state of the hydrogel. Figure 8 It is a diagram of the state change of the hydrogel sprayed on pig skin in Example 4 under cysteine treatment.

[0159] From Figure 8 it can be seen that the hydrogel under the gauze soaked with cysteine solution is degraded within 30 minutes.

[0160] Example 9

[0161] Prepare the hydrogel in a cylindrical polytetrafluoroethylene mold through a double-barreled syringe with the hydrogel provided in Example 4. The specific preparation conditions are: 25 °C, humid condition, react for 1 h to obtain a cylindrical hydrogel sample; select 220 g male SD rats, anesthetize them, shave the hair on their backs, disinfect with iodophor, make an incision about 1 cm long with a scalpel, implant the prepared hydrogel sample subcutaneously, and suture; at a certain time, euthanize the rats and take pictures and weigh the remaining gel. The experimental results are asFigure 9 As shown Figure 9 This is the degradation curve of the hydrogel provided in Example 4 under the skin of rats. It can be seen from this figure that the degradation time of the hydrogel is 12 days.

[0162] Example 10

[0163] Male SD rats weighing 220 g were selected for the establishment of the model of massive hemorrhage in the rat liver. After anesthesia, the liver of the rat was exposed through an abdominal incision, and a wound 1 cm long and 3 mm deep was made on the liver with a scalpel. The rats were randomly divided into three groups for experiments, namely the blank group (without any hemostatic measures), the experimental group, and the commercially available fibrin glue group (porcine fibrin adhesive). When the liver was actively bleeding, the hydrogel provided in Example 4 was sprayed onto the liver wound through a double-barreled syringe, the bleeding situation was observed, and the hemostasis time and the blood loss of each rat within 3 min were recorded. Figure 10 This is a representative picture of the hydrogel provided in Example 4 for liver hemostasis, as well as the hemostasis time and the blood loss within 3 min. Among them, the blank group and the fibrin glue group were used as negative and positive controls respectively.

[0164] Example 11

[0165] In the experiment, male SD rats weighing 220 g were used for the hemostasis model of rat abdominal aortic rupture. After anesthesia, the abdominal aorta was dissected from the surrounding tissues, and a defect of about 1 mm was made with surgical scissors. The hydrogel provided in Example 4 was sprayed onto the vascular defect through a double-barreled syringe. After waiting for 2 min to stabilize, the arterial clamp was removed, and the hemostasis effect was observed. Figure 11 This is a schematic diagram of the hydrogel provided in Example 4 for hemostasis of rat abdominal aortic rupture. As Figure 11 can be seen, the FON hydrogel can achieve hemostasis of the rat abdominal aorta, and the hemostasis effect is good.

[0166] Example 12

[0167] In the experiment, New Zealand white rabbits weighing about 3 kg were used for the hemostasis model of rabbit abdominal aortic rupture. After anesthesia, the abdominal aorta was dissected from the surrounding tissues, and a defect of about 1 mm was made with surgical scissors. The hydrogel provided in Example 4 was sprayed onto the vascular defect through a double-barreled syringe. After waiting for 2 min to stabilize, the arterial clamp was removed, and the hemostasis effect was observed. Figure 12 This is a schematic diagram of the hydrogel provided in Example 4 for hemostasis of rabbit abdominal aortic rupture.

[0168] Example 13

[0169] In the experiment, New Zealand white rabbits weighing about 3 kg were used for the rabbit carotid artery rupture hemostasis model. After anesthesia, the carotid artery was dissected from the surrounding tissues, and a defect of about 1 mm was created with surgical scissors. The hydrogel provided in Example 4 was sprayed onto the vascular defect through a double-barreled syringe. After waiting for 2 minutes to stabilize, the artery clamp was removed, and the hemostasis effect was observed. Figure 13 It is a schematic diagram of the hydrogel provided in Example 4 for rabbit carotid artery rupture hemostasis.

[0170] Example 14

[0171] In the experiment, New Zealand white rabbits weighing about 3 kg were used for the rabbit abdominal aorta rupture hemostasis model. After anesthesia, the abdominal aorta was dissected from the surrounding tissues, and a defect of about 1 mm was created with surgical scissors. The FPBA-N2H3 hydrogel, OPA-N2H3 hydrogel, and the hydrogel provided in Example 4 were sprayed onto the vascular defect through a double-barreled syringe. After waiting for 2 minutes to stabilize, the artery clamp was removed, and the hemostasis effect was observed. Figure 14 It is a schematic diagram of three different hydrogels for rabbit abdominal aorta rupture hemostasis. As Figure 14 can be seen, the FPBA-N2H3 hydrogel and OPA-N2H3 hydrogel failed to stop bleeding, while the FON hydrogel was successful in hemostasis.

[0172] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A poly(ethylene glycol) hemostatic hydrogel with ultra-fast gelation, sprayability, and rapid on-demand degradation, characterized in that, It includes component A, component B and component C; Component A includes a main chain with the structure of formula (I) and an end group with the structure of formula (II); Component B includes a main chain with the structure of formula (I) and an end group with the structure of formula (III); Component C includes a main chain with the structure of formula (I) and an end group with the structure of formula (IV); 2. The hydrogel according to claim 1, wherein Component A, component B and component C are linear polyethylene glycol derivatives or multi-arm polyethylene glycol derivatives.

3. The hydrogel according to claim 1, characterized in that, Component A, component B and component C have any one of the structures of formula (Va), formula (Vb), formula (Vc), formula (Vd) and formula (Ve); Wherein, a, b, c, d, e are degrees of polymerization, and R is the end group of the structures of formula (II), (III) and (IV).

4. The hydrogel according to claim 3, wherein, The value range of a is 1 to 1000, preferably 40 to 400; the value range of b is 1 to 333, preferably 30 to 150; the value range of c is 1 to 250, preferably 25 to 100; the value range of d is 1 to 166, preferably 20 to 80; the value range of e is 1 to 125, preferably 10 to 50.

5. The hydrogel according to claim 1, wherein It includes 1 to 5 parts by mass of component A, 1 to 10 parts by mass of component B and 1 to 10 parts by mass of component C.

6. The hydrogel according to claim 1, wherein It also includes an aqueous medium, and the aqueous medium is preferably water, physiological saline or buffer solution; the buffer solution is preferably PBS buffer solution.

7. The hydrogel according to claim 1, wherein The preparation method of component A includes the following steps: (1) React 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid with polyethylene glycol having a hydroxyl group at the end; (2) Deprotect the reaction product to obtain component A.

8. The hydrogel according to claim 1, wherein The preparation method of component B includes the following steps: a) React polyethylene glycol with 2-formylphenylboronic acid pinacol ester small molecule containing a carboxyl group under the action of a condensing agent and a catalyst to obtain an intermediate product; b) Dialyze the intermediate product in water and freeze-dry it to obtain component B.

9. The hydrogel according to claim 1, wherein The preparation method of component C includes the following steps: a) React p-nitrophenyl chloroformate with polyethylene glycol to obtain an intermediate product; b) React the intermediate product with hydrazine monohydrate to obtain a reaction product, dialyze it in water and freeze-dry it to obtain component C.

10. A method for preparing a hydrogel according to any one of claims 1 to 9, characterized in that, It includes the following steps: i) Mix component A, component B and component C with the aqueous medium respectively to obtain solution A, solution B and solution C. ii) Mix solution A and solution B to obtain solution D. iii) Mix solution C and solution D to obtain a hydrogel.

11. Use of a hydrogel as described in any one of claims 1 to 9 in the preparation of a hemostatic drug.

12. The application according to claim 11, wherein The hemostatic drug is used for hemostasis of blood vessels and organs.

Images