A polyethylene glycol derivative, a method for preparing the same and a polyethylene glycol hydrogel

Abstract

The application provides a polyethylene glycol derivative, which comprises a main chain with the structure of formula (IIa) and an end group with the structure of formula (IIb). The application utilizes the esterification reaction of the hydroxyl group and the carboxyl group of polyethylene glycol to generate a polyethylene glycol derivative with an adjacent phthaldehyde end group, and finally forms a chemical cross-linking hydrogel with a polyethylene glycol with an amino end group. Due to the introduction of the ester bond, the application not only accelerates the degradation time of the hydrogel, but also improves the gelation rate of the hydrogel, and meanwhile, the hydrogel has high adhesion and a simple preparation process, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of materials technology, and in particular to a polyethylene glycol derivative, its preparation method, and a polyethylene glycol hydrogel. Background Technology

[0002] Hydrogels are materials with a three-dimensional network structure formed through cross-linking. Due to their physicochemical properties similar to the extracellular matrix and their good biocompatibility, hydrogels are widely used in the biomedical field. Polyethylene glycol (PEG) is a commonly used raw material for preparing hydrogels, including linear PEG and multi-arm PEG. Because the terminal hydroxyl groups of PEG have low reactivity, PEG usually needs to be derivatized to generate end groups with different functions, such as carboxyl, amino, thiol, and aldehyde groups. Through the chemical reactions of these end groups, chemically cross-linked hydrogels can be easily prepared, and other molecules can be coupled to functionalize the hydrogels.

[0003] Polyethylene glycol derivatives with succinimide end groups are commonly used to prepare chemically cross-linked hydrogels (CN108525016A, KR20020089772A). When an aqueous solution of this polyethylene glycol derivative is mixed with an aqueous solution of polyethylene glycol with amino end groups, the succinimide end groups react with the amino groups to form amide bonds, thus forming a chemically cross-linked hydrogel. However, the reaction produces N-hydroxysuccinimide as a byproduct, which may cause toxicity; additionally, the succinimide end groups gradually hydrolyze in aqueous solution, losing their reactivity. Polyethylene glycol derivatives with benzaldehyde end groups have also been used to prepare chemically cross-linked hydrogels (WO2020029432A1). When an aqueous solution of this polyethylene glycol derivative is mixed with a solution of a polyamino compound, the benzaldehyde end groups react with the amino groups to form Schiff base bonds, thus forming a chemically cross-linked hydrogel. Because Schiff base bonds are easily hydrolyzed, the degree of cross-linking reaction is low, the cross-linking density is low, and therefore the mechanical strength of the hydrogel is low.

[0004] Existing technology 202010455951.3 prepared a polyethylene glycol derivative with o-phthalaldehyde end groups, which was cross-linked with a polyethylene glycol derivative with amino end groups through chemical bonds to form a hydrogel. However, its preparation process is complex, the degradation rate is slow, and the gelation rate is relatively slow at the same concentration.

[0005] Therefore, it is essential to provide a polyethylene glycol derivative that is simple to prepare, has high synthesis efficiency, and shortens gelation and degradation times. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a polyethylene glycol derivative, its preparation method and polyethylene glycol hydrogel. The hydrogel prepared by the polyethylene glycol derivative provided by the present invention has a short degradation time, fast gelation rate and high adhesion performance, and the preparation process is simple.

[0007] First, this invention simplifies the preparation process, reducing the synthetic steps of succinimide ester of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid. By adjusting the acid-base balance and extracting 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid, the material is purified, greatly improving the synthesis efficiency. Second, compared with earlier patents, the grafting rate of o-phthalaldehyde is increased, which not only shortens the gelation time of the material but also improves its adhesion to the skin. Finally, compared with earlier synthesis methods, the introduction of ester bonds shortens the degradation time of the hydrogel, making it more compatible with the rate of human healing.

[0008] The present invention provides a polyethylene glycol derivative comprising a main chain having a structure of formula (IIa) and end groups having a structure of formula (IIb).

[0009]

[0010] According to the present invention, the polyethylene glycol derivative is any one of formula (IIc), formula (IId), formula (IIe), formula (IIIf), and formula (IIg):

[0011]

[0012]

[0013] Where a is the degree of polymerization, 1≤a≤1000;

[0014] b represents the degree of aggregation, where 1 ≤ b ≤ 1000

[0015] c represents the degree of aggregation, where 1 ≤ c ≤ 1000

[0016] d represents the degree of aggregation, where 1 ≤ d ≤ 1000

[0017] e represents the degree of aggregation, where 1 ≤ e ≤ 1000

[0018] The present invention also provides a method for preparing the polyethylene glycol derivative as described in claim 1 or 2, comprising the following steps:

[0019] A) React 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid with polyethylene glycol having a hydroxyl end group to obtain the reaction product;

[0020] B) Deprotect the reaction product to obtain a polyethylene glycol derivative.

[0021] The reaction formula is as follows:

[0022]

[0023] This invention first dissolves 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid and polyethylene glycol with hydroxyl end groups in an organic solvent, and then directly obtains the product through esterification, deprotection with trifluoroacetic acid, dialysis, and lyophilization.

[0024] The molar equivalent of the 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is preferably 1 to 3 times that of the hydroxyl group; more preferably 2 times; the organic solvent is preferably anhydrous dichloromethane; and the selected esterification catalyst is preferably EDCI and DMAP.

[0025] The reaction time is 24 to 72 hours, preferably 48 hours; the reaction temperature is 10 to 40°C, preferably 25°C.

[0026] After obtaining the reaction product, the present invention deprotects the reaction product, dialyzes it and freeze-dries it to obtain a polyethylene glycol derivative.

[0027] The deprotection process can be performed using techniques well known to those skilled in the art. Preferably, a mixed solvent of trifluoroacetic acid and water is used; the volume of the mixed solvent is 5 to 10 times the mass of the reaction product, preferably 5 times; the deprotection time is 0.5 to 2 hours, preferably 1 hour; and the deprotection temperature is 15 to 40°C, preferably 25°C.

[0028] The dialysis and freeze-drying can be performed using techniques well known to those skilled in the art.

[0029] According to the present invention, the 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is prepared by the following method:

[0030] 1) 3,4-Di(dibromomethyl)benzoic acid was prepared by bromination of 3,4-dimethylbenzoic acid;

[0031] 2) Hydrolyze 3,4-bis(dibromomethyl)benzoic acid to obtain 3,4-dicarboxybenzoic acid;

[0032] 3) 3,4-Dicarboxybenzoic acid is reacted with methanol and a catalyst to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

[0033] The reaction formula is as follows:

[0034]

[0035] The preparation method of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is to first prepare 3,4-di(dibromomethyl)benzoic acid by bromination of 3,4-dimethylbenzoic acid.

[0036] The bromination reaction described in this invention can be carried out using techniques well known to those skilled in the art. This invention uses N-bromosuccinimide as the brominating reagent, benzoyl peroxide as the free radical initiator, and carbon tetrachloride as the solvent to carry out the bromination reaction.

[0037] The molar equivalent of the N-bromosuccinimide is 3 to 5 times that of 3,4-dimethylbenzoic acid, preferably 4 times; the molar equivalent of the benzoyl peroxide is 0.05 to 0.5 times that of 3,4-dimethylbenzoic acid, preferably 0.1 times; the volume (mL) of the carbon tetrachloride is 10 to 50 times the mass (g) of 3,4-dimethylbenzoic acid, preferably 20 times.

[0038] The bromination reaction is carried out at a temperature of 70–90°C, preferably 81°C; the bromination reaction is carried out for a time of 10–20 h, preferably 15 h.

[0039] After the bromination reaction was completed, the reaction mixture was filtered; the filter cake was washed with diethyl ether; all the filtrates were combined and concentrated, and then dried under vacuum; the solid product was recrystallized in acetonitrile to give 3,4-di(dibromomethyl)benzoic acid.

[0040] The present invention preferably uses a rotary evaporator for concentration; the preferred concentration temperature is 30°C; and the preferred concentration is to 10% of the liquid volume.

[0041] The recrystallization can be performed using a technique well-known to those skilled in the art.

[0042] The present invention describes the hydrolysis of 3,4-bis(dibromomethyl)benzoic acid to obtain 3,4-dicarboxybenzoic acid.

[0043] The hydrolysis reaction can be carried out using techniques well known to those skilled in the art. Preferably, the hydrolysis reaction is performed by dissolving 3,4-bis(dibromomethyl)benzoic acid in an aqueous solution of sodium carbonate.

[0044] The sodium carbonate aqueous solution has a mass-volume concentration of 10%; the volume of the sodium carbonate aqueous solution is 5-20 times the mass of 3,4-bis(dibromomethyl)benzoic acid, preferably 10 times.

[0045] The hydrolysis reaction is carried out at a temperature of 60–80°C, preferably 70°C; the hydrolysis reaction is carried out for a time of 3–5 hours, preferably 4 hours.

[0046] After the hydrolysis reaction is completed, the pH of the reaction solution is adjusted to 0-3, preferably 1, using concentrated hydrochloric acid; ethyl acetate is used for extraction; after concentration and vacuum drying, 3,4-dicarboxybenzoic acid is obtained.

[0047] This invention reacts 3,4-dicarboxybenzoic acid with methanol and a catalyst to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

[0048] The catalyst is preferably scandium trifluoromethanesulfonate; the reaction temperature is 10–40°C, preferably 25°C; the reaction time is 6–24 h, preferably 12 h.

[0049] After the reaction is complete, the crude product can be purified using either of the following two methods, with method 2 being the preferred choice:

[0050] 1. Purification was performed by silica gel column chromatography to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid. The preferred mobile phase for column chromatography was n-hexane and ethyl acetate in a volume ratio of 3:1.

[0051] 2. The crude product was dissolved in 5% sodium hydroxide solution, at which point the carboxylic acid was converted to sodium carboxylate. The solution was washed with ethyl acetate, and the aqueous phase was retained. The aqueous phase was adjusted to pH 5 with concentrated hydrochloric acid and extracted three times with ethyl acetate. The organic phases were combined and concentrated by rotary evaporation to obtain purified 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

[0052] The preparation of phthalaldehyde-terminated polyethylene glycol containing ester bonds can be carried out using methods well known to those skilled in the art. Dichloromethane is preferred as the organic solvent used in the esterification reaction, and a 50% trifluoroacetic acid aqueous solution is selected for deprotection. The preferred mass-to-volume ratio of solution volume to solid mass is 1 g / 10 mL, and the preferred reaction temperature is 25 °C.

[0053] The present invention also provides a polyethylene glycol hydrogel, which is formed by chemically bonding a polyethylene glycol derivative and a polyethylene glycol with an amino-terminated end group, wherein the polyethylene glycol derivative is the polyethylene glycol derivative described in claim 1 or 2.

[0054] The end-amino-containing polyethylene glycol comprises a main chain having a structure of formula (IVa) and end groups having any one of the structures of formula (IVb), formula (IVc) and formula (IVd);

[0055]

[0056] According to the present invention, the main chain of the polyethylene glycol with amino-terminal groups is any one of formula (Va), formula (Vb), formula (Vc), formula (Vd), and formula (Ve):

[0057]

[0058]

[0059] Where a is the degree of polymerization, 1≤a≤1000;

[0060] b represents the degree of aggregation, where 1 ≤ b ≤ 1000

[0061] c represents the degree of aggregation, where 1 ≤ c ≤ 1000

[0062] d represents the degree of aggregation, where 1 ≤ d ≤ 1000

[0063] e represents the degree of aggregation, where 1 ≤ e ≤ 1000

[0064] R is any one of the formulas IVb, IVc, and IVd.

[0065] The present invention also provides a method for preparing a polyethylene glycol hydrogel, which is prepared from a polyethylene glycol derivative, a polyethylene glycol with amino-terminated ends, and a solvent.

[0066] In this invention, the preparation process of the polyethylene glycol hydrogel includes:

[0067] (1) Prepare solution 1 of the polyethylene glycol derivative;

[0068] (2) Prepare a solution 2 of the polyethylene glycol with amino-terminated end groups;

[0069] (3) Mix the solution 1 and the solution 2 to obtain the polyethylene glycol hydrogel through a chemical reaction.

[0070] In solution 1, the mass-volume concentration of the polyethylene glycol derivative is 1 to 1000 mg / mL, preferably 10 to 200 mg / mL.

[0071] In solution 2, the mass-volume concentration of the amino-terminated polyethylene glycol is 1–1000 mg / mL, preferably 10–200 mg / mL.

[0072] The solvents used in the preparation of solutions 1 and 2 are selected from water, physiological saline, or buffer solutions, and the buffer solution is selected from phosphate buffer solutions.

[0073] The mass ratio of the polyethylene glycol derivative to the amino-terminated polyethylene glycol is 1:0.01 to 100, preferably 1:0.1 to 10.

[0074] The polyethylene glycol derivatives provided by this invention have good water solubility and biocompatibility, and their terminal o-phthalaldehyde can react chemically with a variety of groups.

[0075] Due to the high reactivity of the o-phthalaldehyde group with amino, (acyl)hydrazine, and aminooxy groups, chemically cross-linked hydrogels can be prepared by mixing the polyethylene glycol derivative provided in this invention with polyethylene glycol containing amino groups at the end. Compared with previous work, this invention utilizes the direct esterification reaction between hydroxyl and carboxyl groups to increase the grafting rate of the groups, thereby increasing the gelation rate of the hydrogel. Simultaneously, the introduction of ester bonds also accelerates the degradation time of the hydrogel.

[0076] This invention relates to a polyethylene glycol derivative hydrogel with o-phthalaldehyde end groups, which can undergo a highly efficient coupling reaction with polyethylene glycols with amino, (acyl)hydrazine, or aminooxy end groups. It exhibits rapid gelation and high mechanical strength. In practical applications, it can be used clinically as a tissue adhesive. An ideal tissue adhesive should possess strong tissue adhesion, good biocompatibility, controllable degradation, rapid gelation, and rapid hemostasis. This patent, through process optimization, improves the grafting rate of o-phthalaldehyde (by 13%) compared to an earlier patent (application number: 202010455951.3), shortens the gelation and degradation time of the material, and increases the adhesive strength of the hydrogel. Attached Figure Description

[0077] Figure 1 The NMR spectrum of 3,4-di(dibromomethyl)benzoic acid;

[0078] Figure 2 The NMR spectrum of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid;

[0079] Figure 3 NMR spectrum of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid succinimide ester prepared for Comparative Example 1;

[0080] Figure 4 The NMR spectrum of 4aPEG-OPA containing ester bonds prepared in Example 6;

[0081] Figure 5 The NMR spectrum of 4aPEG-OPA with amide bonds at the 2-terminal group is shown in the comparative example.

[0082] Figure 6 NMR comparison of 4aPEG-OPA containing amide bonds in Comparative Example 2 and 4aPEG-OPA containing ester bonds in Example 11;

[0083] Figure 7 Statistical graph of grafting rates for 4aPEG-OPA containing ester bonds and 4aPEG-OPA containing amide bonds;

[0084] Figure 8Example 12: Comparison of gelation time between 4aPEG-OPA hydrogel containing ester bonds and 4aPEG-OPA hydrogel containing amide bonds in Comparative Example 3;

[0085] Figure 9 Degradation curves of 4aPEG-OPA hydrogels containing ester bonds and 4aPEG-OPA hydrogels containing amide bonds in PBS solution in Example 13, Comparative Example 4 and Verification Example 4;

[0086] Figure 10 Degradation curves of 4aPEG-OPA hydrogels containing ester bonds and 4aPEG-OPA hydrogels containing amide bonds in PBS solution containing proteinase K in Examples 14, Comparative Example 5, and Validation Example 4;

[0087] Figure 11 The chart shows the statistical results of shear and tensile strength tests on pigskin made of different materials in Example 15, Comparative Example 6, Comparative Example 7, and Verification Example 5. Detailed Implementation

[0088] This invention provides a polyethylene glycol derivative, its preparation method, and a polyethylene glycol hydrogel. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0089] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0090] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0091] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0092] The numerical ranges and parameters involved in this invention have been presented as accurately as possible to the relevant values ​​in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, for example, within 1% or 0.5%.

[0093] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0094] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a polyethylene glycol derivative, its preparation method, and a polyethylene glycol hydrogel provided by the present invention.

[0095] Example 1

[0096]

[0097] 3,4-Dimethylbenzoic acid (50 g) and N-bromosuccinimide (267 g) were dissolved in hot carbon tetrachloride (1200 mL), and then 7.25 g of benzoyl peroxide was slowly added. The mixture was refluxed at 81 °C for 15 h. After filtration, the filter cake was washed with diethyl ether (300 mL × 5). All filtrates were combined, concentrated by rotary evaporation, and then dried to solid under vacuum. The resulting solid was recrystallized in acetonitrile to give 3,4-di(dibromomethyl)benzoic acid (55 g, 35%). NMR characterization was performed using a 500 M 1H NMR spectrum with dimethyl sulfoxide-d6 as the deuteration reagent. Figure 1 The NMR spectrum of 3,4-bis(dibromomethyl)benzoic acid.

[0098] Example 2

[0099]

[0100] 55 g of 3,4-di(dibromomethyl)benzoic acid prepared in Example 1 was dissolved in 550 mL of a 10% (w / v) sodium carbonate aqueous solution and reacted at 70 °C for 4 h. The pH of the reaction solution was adjusted to 1 with concentrated hydrochloric acid, and then extracted with ethyl acetate (4 × 200 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried under vacuum to obtain 13.7 g (65%) of 3,4-dicarboxybenzoic acid.

[0101] Example 3

[0102]

[0103] 13.7 g of 3,4-dicarboxybenzoic acid prepared in Example 2 was dissolved in anhydrous methanol (400 mL), and then scandium trifluoromethanesulfonate (2 g) was added as a catalyst. The reaction was carried out for 12 h. The product was dried under vacuum to obtain crude 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid. The crude product was purified using any of the following methods:

[0104] 1. Obtained by column chromatography, the mobile phase is V. 石油醚 / V 乙酸乙酯 =5:1.

[0105] 2. Dissolve in 500 mL of 5% sodium hydroxide solution, wash with 300 mL × 3 ethyl acetate, and retain the aqueous phase. Adjust the pH of the aqueous phase to 6 with concentrated hydrochloric acid, extract with 300 mL × 3 ethyl acetate, retain the organic phase, dry with anhydrous magnesium sulfate, filter and evaporate to dryness.

[0106] The product was a white solid (15 g, 87%), characterized by 500 MHz 1H NMR spectroscopy. The deuteration reagent was dimethyl sulfoxide-d6. Figure 2 The NMR spectrum is for 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

[0107] Example 4

[0108] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of linear polyethylene glycol (number average molecular weight 2000, 33.4 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V 水 The reaction mixture was 1:1) and reacted for 1.5 h to remove protection. After the reaction was complete, the mixture was transferred to a dialysis bag for dialysis and lyophilization to obtain a white solid (31 g, 79%).

[0109] Example 5

[0110] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of three-arm polyethylene glycol (number average molecular weight 10000, 111.5 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V水 The reaction mixture was 1:1) and reacted for 1.5 h to remove protection. After the reaction was complete, the mixture was transferred to a dialysis bag for dialysis and lyophilization to obtain a white solid (102 g, 87%).

[0111] Example 6

[0112]

[0113] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of tetra-arm polyethylene glycol (number average molecular weight 10000, 83.6 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V 水 The reaction mixture (1:1) was allowed to react for 1.5 h for deprotection. After the reaction, the mixture was transferred to a dialysis bag and lyophilized to obtain a white solid (4aPEG-OPA) (81.5 g, 92%). NMR characterization was performed using a 500 M 1H NMR spectrum. The deuteration reagent was D₂O. Figure 4 The NMR spectrum of 4aPEG-OPA containing ester bonds prepared in Example 6.

[0114] Example 7

[0115] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of tetra-arm polyethylene glycol (number average molecular weight 20000, 167.2 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V 水 The reaction mixture was 1:1) and reacted for 1.5 h to remove protection. After the reaction was complete, the solid was transferred to a dialysis bag for dialysis and lyophilization to obtain a white solid (164 g, 95%).

[0116] Example 8

[0117] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of six-arm polyethylene glycol (number average molecular weight 10000, 55.75 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V 水 The reaction mixture was 1:1) and reacted for 1.5 h to remove protection. After the reaction was complete, the mixture was transferred to a dialysis bag for dialysis and lyophilization to obtain a white solid (54 g, 88%).

[0118] Example 9

[0119] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of six-arm polyethylene glycol (number average molecular weight 20000, 111.5 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V 水 The reaction mixture was 1:1) and reacted for 1.5 h to remove protection. After the reaction was complete, the mixture was transferred to a dialysis bag for dialysis and lyophilization to obtain a white solid (106 g, 91%).

[0120] Example 10

[0121] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of octahedral polyethylene glycol (number average molecular weight 10000, 41.8 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V 水 The reaction mixture was 1:1) and reacted for 1.5 h to remove protection. After the reaction was complete, the mixture was transferred to a dialysis bag for dialysis and lyophilization to obtain a white solid (39 g, 82%).

[0122] Example 11

[0123] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 was dissolved in 300 mL of dichloromethane, and EDCI (19 g) and DMAP (2 g) were added for pre-reaction for 30 min. Then, 600 mL of a dichloromethane solution of octahedral polyethylene glycol (number average molecular weight 20000, 83.6 g) with hydroxyl-terminated groups was added, and the reaction was allowed to proceed for 48 h. After the reaction was complete, the solution was concentrated and evaporated to dryness, dissolved in 1000 mL of dichloromethane, washed with water, and the organic phase was evaporated to dryness again. 500 mL of an aqueous solution of trifluoroacetic acid (V...) was then added. 三氟乙酸 V 水 The reaction mixture was 1:1) and reacted for 1.5 h to remove protection. After the reaction was complete, the mixture was transferred to a dialysis bag for dialysis and lyophilization to obtain a white solid (82 g, 92%). Figure 6 The NMR spectra of 4aPEG-OPA containing amide bonds in Comparative Example 2 and 4aPEG-OPA containing ester bonds in Example 11 are shown.

[0124] Example 12

[0125] Tetra-arm polyethylene glycol (MAG) with amino-terminated groups (number average molecular weight 10,000) and tetra-arm MEG with o-phthalaldehyde-terminated groups containing ester bonds (MAG) were dissolved in PBS solution to prepare solutions with mass fractions of 5%, 10%, and 15%, respectively. At the same mass fraction, 150 μL of each solution was taken, thoroughly mixed in a centrifuge tube, and placed in a 37°C water bath to gel. The gelation time was recorded, and samples were collected at least three times for each mass fraction. The results were statistically analyzed. The final data are shown below. Figure 8 As shown. Figure 8 This is a comparison chart showing the gelation time of the 4aPEG-OPA hydrogel containing ester bonds in Example 12 and the 4aPEG-OPA hydrogel containing amide bonds in Comparative Example 3.

[0126] Example 13

[0127] Tetra-arm polyethylene glycol (MAG) with amino-terminated groups (number average molecular weight 10,000) and tetra-arm polyethylene glycol (MAG) with o-phthalaldehyde-terminated groups containing ester bonds (number average molecular weight 10,000) were dissolved in PBS solution to prepare a 10% (w / w) solution. After complete dissolution, 150 μL of each solution was taken and placed in a 4 mL vial of known weight. 3 mL of PBS solution was added, and the vials were placed in a 37°C constant temperature shaking incubator. At intervals, all solutions were aspirated with a pipette and weighed. Then, 3 mL of fresh PBS solution was added again, and the mass change pattern was statistically analyzed. The mass change pattern is shown in the figure below. Figure 9 As shown. Figure 9 The degradation curves of the 4aPEG-OPA hydrogel containing ester bonds and the 4aPEG-OPA hydrogel containing amide bonds in PBS solution are shown in Examples 13 and 4 Comparative Examples 4.

[0128] Example 14

[0129] Tetra-arm polyethylene glycol (number average molecular weight 10,000) with amino groups at the ends and tetra-arm polyethylene glycol (number average molecular weight 10,000) with o-phthalaldehyde groups containing ester bonds at the ends were dissolved in PBS solution to prepare a 10% (w / w) solution. After complete dissolution, 150 μL of each solution was taken and placed in a 4 mL vial of known weight. 3 mL of PBS solution containing proteinase K (10 U) was added, and the vials were placed in a 37°C constant temperature shaking incubator. Every other day, all the solution was aspirated with a pipette and weighed. Then, 3 mL of fresh PBS solution containing proteinase K was added again, and the mass change pattern was statistically analyzed. The mass change pattern is shown in the figure below. Figure 10 As shown. Figure 10 The degradation curves of the 4aPEG-OPA hydrogel containing ester bonds and the 4aPEG-OPA hydrogel containing amide bonds in PBS solution containing proteinase K are shown in Examples 14 and 5.

[0130] Example 15

[0131] Fresh pigskin was cut into rectangular shapes 2.5 cm wide and 7.5 cm long and glued onto a wooden board. 4aPEG-OPA solution and 4aPEG-NH2 solution containing ester bonds were prepared separately as 10% (w / w) solutions and mixed in equal volumes. 50 μL of each solution was quickly applied to the prepared pigskin pieces, with each application area measuring 2.5 cm × 2.5 cm. After application, the pieces were quickly bonded together and placed in a 37°C water bath for 40 minutes to stabilize. The tensile strength was then measured using a universal testing machine. Figure 11 The chart shows the statistical results of shear and tensile strength tests on pigskin made of different materials in Examples 15, 6, and 7.

[0132] Comparative Example 1

[0133] 1,3-Dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid (15 g) prepared in Example 3 and N-hydroxysuccinimide (16.9 g) were dissolved in 300 mL of anhydrous acetonitrile, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (14.1 g) was added. The reaction was carried out for 12 h. After the reaction was completed, the solution was evaporated to dryness and dissolved in dichloromethane. The solution was washed with water and separated (300 mL × 3). The organic phase was retained and dried over magnesium sulfate and filtered. The crude product was purified by silica gel column chromatography with V as the mobile phase. 石油醚 V 乙酸乙酯 The ratio of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid succinimide ester was 3:1 to obtain a white solid (11 g, 51%). NMR characterization was performed using 500 MHz 1H NMR spectroscopy with deuterated chloroform as the deuteration reagent. Figure 3 The NMR spectrum of 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid succinimide ester prepared for Comparative Example 1.

[0134] Comparative Example 2

[0135] The 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid succinimide ester (11 g) prepared in Comparative Example 1 was dissolved in 300 mL of dichloromethane, followed by the addition of 600 mL of a dichloromethane solution of tetra-arm polyethylene glycol (number average molecular weight 10000, 42.7 g) with amino-terminated groups. The reaction was allowed to proceed for 48 h. After the reaction was completed, the solution was concentrated and evaporated to dryness, and then 500 mL of an aqueous solution of trifluoroacetic acid (V) was added. 三氟乙酸 V 水 The reaction was carried out at a ratio of 1:1 for 1.5 h to remove protection. After the reaction, the sample was transferred to a dialysis bag and dialyzed for 72 h before lyophilization to obtain a white solid (43.2 g, 93%) containing amide bonds. NMR characterization was performed using a 500 M 1H NMR spectrum. The deuteration reagent was D₂O. Figure 5 The NMR spectrum of 4aPEG-OPA with amide bonds at the end is shown in Comparative Example 2.

[0136] Comparative Example 3

[0137] Tetra-arm polyethylene glycol (MAG) with amino groups at the ends and tetra-arm MMG with o-phthalaldehyde groups containing amide bonds at the ends were dissolved in PBS to prepare solutions with mass fractions of 5%, 10%, and 15%. For each mass fraction, 150 μL was taken from each solution, thoroughly mixed in a centrifuge tube, and placed in a 37°C water bath to gel. The gelation time was recorded, and samples were collected at least three times for each mass fraction. The results were statistically analyzed. Figure 8 As shown. By Figure 8 It can be seen that among the three hydrogels with different mass fractions, the 4aPEG-OPA hydrogel containing ester bonds has a significantly faster gelation time than the 4aPEG-OPA hydrogel containing amide bonds.

[0138] Comparative Example 4

[0139] Tetra-arm polyethylene glycol (NUMBER 10,000) with amino groups and tetra-arm polyethylene glycol (NUMBER 10,000) with o-phthalaldehyde groups containing amide bonds were dissolved in PBS solution to prepare a 10% (w / w) solution. After thorough mixing, 150 μL of each solution was taken and placed in a 4 mL vial of known weight. 3 mL of PBS solution was added, and the vials were placed in a 37°C shaking incubator. Every other day, all the solution was removed by pipette and weighed. Then, 3 mL of fresh PBS solution was added again, and the mass change was statistically analyzed. The mass change pattern is shown in the figure below. Figure 9 As shown in the figure, the 4aPEG-OPA hydrogel containing ester bonds has a higher swelling ratio and a faster degradation time compared to the 4aPEG-OPA hydrogel containing amide bonds.

[0140] Comparative Example 5

[0141] Tetra-arm polyethylene glycol (number average molecular weight 10,000) with amino groups and tetra-arm polyethylene glycol (number average molecular weight 10,000) with o-phthalaldehyde groups containing amide bonds were dissolved in PBS solution to prepare a 10% (w / w) solution. After thorough mixing, 150 μL of each solution was taken and placed in a 4 mL vial of known weight. 3 mL of PBS solution containing proteinase K was added, and the vials were placed in a 37°C shaking incubator. Every other day, all the solution was removed by pipette and weighed. Then, 3 mL of fresh PBS solution containing proteinase K was added again, and the mass change was statistically analyzed. The mass change pattern is shown in the figure below. Figure 10 As shown in the figure, the addition of proteinase K accelerates the swelling and degradation rates of 4aPEG-OPA hydrogels containing ester bonds, exhibiting a higher swelling rate and faster degradation time compared to 4aPEG-OPA hydrogels containing amide bonds.

[0142] Comparative Example 6

[0143] Fresh pigskin was cut into rectangular shapes 2.5 cm wide and 7.5 cm long and glued onto a wooden board. 4aPEG-OPA solution and 4aPEG-NH2 solution containing amide bonds were prepared separately as 10% (w / w) solutions and mixed in equal volumes. 50 μL of each solution was quickly applied to the prepared pigskin, covering an area of ​​2.5 cm × 2.5 cm. After application, the surfaces were quickly bonded together and placed in a 37°C water bath for 40 minutes to stabilize. The tensile strength was then measured using a universal testing machine.

[0144] Comparative Example 7

[0145] Fresh pigskin was cut into rectangles 2.5cm wide and 7.5cm long and glued onto a wooden board. 50μL of commercially available fibrin glue solution was quickly applied to each of the prepared pigskin pieces, covering an area of ​​2.5cm x 2.5cm. After application, the pieces were quickly glued together and placed in a 37℃ constant temperature water bath for 40 minutes to stabilize. Afterward, the tensile strength was measured using a universal testing machine. Figure 11 The figures show a comparison of skin stretching between Example 15 and Comparative Examples 6 and 7. As can be seen from the figures, the 4aPEG-OPA hydrogel containing ester bonds has higher tensile strength.

[0146] Verification Example 1

[0147] Compared with the amide-bonded phthalaldehyde-terminated polyethylene glycol prepared in earlier work (application number: 202010455951.3), the ester-bonded phthalaldehyde-terminated polyethylene glycol prepared in this patent has an additional -CH2- vibration peak at 4.38 ppm in the NMR spectrum, while the overall chemical shift remains basically unchanged. Figure 6 The NMR spectra of 4aPEG-OPA containing amide bonds in Comparative Example 2 and 4aPEG-OPA containing ester bonds in Example 11 are shown.

[0148] Verification Example 2

[0149] Through the Figure 1 The grafting rate was calculated using NMR integration, and the calculated grafting rate of 4aPEG-OPA containing ester bonds was 81%. In earlier work (application number: 202010455951.3), the grafting rate of 4aPEG-OPA containing amide bonds and phthalaldehyde-terminated polyethylene glycol containing amide bonds was 69%. To further analyze the grafting rate, three parallel experiments were conducted on each of the two groups of samples, and the grafting rate was statistically analyzed. The results show that the grafting rate of 4aPEG-OPA containing ester bonds is significantly higher than that of 4aPEG-OPA containing amide bonds prepared in the earlier patent (application number: 202010455951.3). This patent improves the grafting rate of phthalaldehyde (by 13%) through process optimization. Figure 7 Statistical chart of grafting rates for 4aPEG-OPA containing ester bonds and 4aPEG-OPA containing amide bonds.

[0150] Verification Example 3

[0151] In the gelation experiment, the gelation time of the two hydrogels at three concentrations of 5%, 10% and 15% was compared. The results showed that the gelation rate of the 4aPEG-OPA hydrogel containing ester bonds was significantly higher than that of the 4aPEG-OPA hydrogel containing amide bonds. Figure 8 This is a comparison chart showing the gelation time of the 4aPEG-OPA hydrogel containing ester bonds in Example 12 and the 4aPEG-OPA hydrogel containing amide bonds in Comparative Example 3.

[0152] Verification Example 4

[0153] In in vitro degradation experiments, two types of gels with a mass fraction of 10% were tested in PBS buffer solution and PBS solution containing proteinase K (concentration of 10 U / mL). The results showed that, compared with the amide-containing 4aPEG-OPA hydrogel, the ester-containing 4aPEG-OPA hydrogel exhibited a higher swelling ratio and a faster degradation rate in both solutions. The addition of proteinase K further accelerated the degradation of the ester-containing 4aPEG-OPA hydrogel. Figure 9 The degradation curves of the two hydrogels in PBS buffer solution are shown for Example 13 and Comparative Example 4. Figure 10 The degradation curves of the two hydrogels in Example 14 and Comparative Example 5 in PBS solution containing proteinase K are shown.

[0154] Verification Example 5

[0155] Using pigskin as the experimental subject, two types of hydrogels, each with a mass fraction of 10%, were subjected to skin-attachment shear tests, with fibrin glue serving as the control group. The results showed that the hydrogel containing ester bonds exhibited higher skin tensile strength. Figure 11 The data compares the skin-attached shear test results of the 4aPEG-OPA hydrogel containing ester bonds in Example 15 and the 4aPEG-OPA hydrogel containing amide bonds in Comparative Example 6, and the fibrin glue in Comparative Example 7.

[0156] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polyethylene glycol hydrogel, characterized in that, It is composed of polyethylene glycol derivatives and polyethylene glycol with amino-terminated groups linked by chemical bonds; The polyethylene glycol derivative comprises a main chain having a structure of formula (IIa) and end groups having a structure of formula (IIb); （Ⅱa）； (IIb); The preparation method of the polyethylene glycol derivative includes the following steps: A) React 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid with polyethylene glycol having a hydroxyl end group to obtain the reaction product; B) Deprotect the reaction product to obtain a polyethylene glycol derivative.

2. The hydrogel according to claim 1, characterized in that, The polyethylene glycol derivative is any one of formula (IIc), formula (IId), formula (IIe), formula (IIf), and formula (IIg): (IIc) (IId) (IIe) (IIf) (IIg) Where a is the degree of polymerization, 1≤a≤1000; b represents the degree of aggregation, where 1 ≤ b ≤ 1000; c represents the degree of aggregation, where 1 ≤ c ≤ 1000; d represents the degree of aggregation, where 1 ≤ d ≤ 1000; e represents the degree of aggregation, where 1 ≤ e ≤ 1000.

3. The hydrogel according to claim 1, characterized in that, The 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid is prepared according to the following method: 1) 3,4-Di(dibromomethyl)benzoic acid was prepared by bromination of 3,4-dimethylbenzoic acid; 2) Hydrolyze 3,4-bis(dibromomethyl)benzoic acid to obtain 3,4-dicarboxybenzoic acid; 3) 3,4-Dicarboxybenzoic acid is reacted with methanol and a catalyst to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid.

4. The polyethylene glycol hydrogel according to claim 1, characterized in that, The end-amino-containing polyethylene glycol comprises a main chain having a structure of formula (IVa) and end groups having any one of the structures of formulas (IVb), (IVc), (IVd). (IVa); (IVb); (IVc); (IVd)。 5. The polyethylene glycol hydrogel according to claim 4, characterized in that, The main chain of the polyethylene glycol with amino-terminal groups is any one of formulas (Va), (Vb), (Vc), (Vd), and (Ve): (And) (Vb) (Vc) (CEO) (Ve) Where a is the degree of polymerization, 1≤a≤1000; b represents the degree of aggregation, where 1 ≤ b ≤ 1000 c represents the degree of aggregation, where 1 ≤ c ≤ 1000 d represents the degree of aggregation, where 1 ≤ d ≤ 1000 e represents the degree of aggregation, where 1 ≤ e ≤ 1000 R is any one of the formulas IVb, IVc, and IVd.

6. A method for preparing a polyethylene glycol hydrogel as described in any one of claims 1 to 5, characterized in that, It is prepared from polyethylene glycol derivatives, polyethylene glycol with amino-terminated ends, and solvent.

7. The preparation method according to claim 6, characterized in that, The solvent is water, physiological saline, or a buffer solution; The mass-volume concentration of the polyethylene glycol derivative is 1~1000 mg / mL; The mass-volume concentration of the amino-terminated polyethylene glycol is 1~1000 mg / mL.

8. The preparation method according to claim 6, characterized in that, The mass ratio of the polyethylene glycol derivative to the end-amino-containing polyethylene glycol is 1:0.01~100.

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